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2015
Borel S, Robert-Hebmann V, Alfaisal J, Jain A, Faure M, Espert L, Chaloin L, Paillart J C, Johansen T, Biard-Piechaczyk M
HIV-1 viral infectivity factor interacts with microtubule-associated protein light chain 3 and inhibits autophagy Journal Article
In: AIDS, vol. 29, no. 3, pp. 275-286, 2015, ISBN: 25490467.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {HIV-1 viral infectivity factor interacts with microtubule-associated protein light chain 3 and inhibits autophagy},
author = {S Borel and V Robert-Hebmann and J Alfaisal and A Jain and M Faure and L Espert and L Chaloin and J C Paillart and T Johansen and M Biard-Piechaczyk},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25490467?dopt=Abstract},
isbn = {25490467},
year = {2015},
date = {2015-01-01},
journal = {AIDS},
volume = {29},
number = {3},
pages = {275-286},
abstract = {Objective: Autophagy, an important anti-viral process triggered during HIV-1 entry by gp41-dependent membrane fusion, is repressed in infected CD4 T cells by an unknown mechanism. The aim of this study was to identify the role of Vif in the autophagy blockade.
Design/methods: To determine the role of Vif in autophagy inhibition, we used cell lines that express CD4 and CXCR4 and primary CD4 T cells. Pull-down experiments, immunoprecipitation assays and computational analyses were performed to analyze the interaction between Vif and LC3B, a major autophagy component, in presence or absence of the anti-viral host factor APOBEC3G, after HIV-1 infection or ectopic expression of Vif. Autophagy was analyzed after infection by viruses expressing Vif (NL4.3) or not (NL4.3ΔVif), or after exogenous Vif expression.
Results: We demonstrate that the C-terminal part of Vif interacts directly with LC3B, independently of the presence of APOBEC3G. Vif binds to pro-LC3 and Atg4-cleaved LC3 forms, and glycine 120, the amino acid conjugated to phosphatidylethanolamine on autophagosomes, is required. Importantly, we evidence that Vif inhibits autophagy during HIV-1 infection. Indeed, autophagy is detected in target cells infected by NL4.3ΔVif, but prevented in cells infected by NL4.3. Furthermore, autophagy triggered in NL4.3ΔVif-infected cells is inhibited when Vif is expressed in trans but is still active when target cells express a mutant of Vif that binds weakly to LC3B.
Conclusions: Our study unveils that Vif inhibits autophagy independently of its action on APOBEC3G and therefore suggest a new function of this viral protein in restricting innate anti-viral mechanisms.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Objective: Autophagy, an important anti-viral process triggered during HIV-1 entry by gp41-dependent membrane fusion, is repressed in infected CD4 T cells by an unknown mechanism. The aim of this study was to identify the role of Vif in the autophagy blockade.
Design/methods: To determine the role of Vif in autophagy inhibition, we used cell lines that express CD4 and CXCR4 and primary CD4 T cells. Pull-down experiments, immunoprecipitation assays and computational analyses were performed to analyze the interaction between Vif and LC3B, a major autophagy component, in presence or absence of the anti-viral host factor APOBEC3G, after HIV-1 infection or ectopic expression of Vif. Autophagy was analyzed after infection by viruses expressing Vif (NL4.3) or not (NL4.3ΔVif), or after exogenous Vif expression.
Results: We demonstrate that the C-terminal part of Vif interacts directly with LC3B, independently of the presence of APOBEC3G. Vif binds to pro-LC3 and Atg4-cleaved LC3 forms, and glycine 120, the amino acid conjugated to phosphatidylethanolamine on autophagosomes, is required. Importantly, we evidence that Vif inhibits autophagy during HIV-1 infection. Indeed, autophagy is detected in target cells infected by NL4.3ΔVif, but prevented in cells infected by NL4.3. Furthermore, autophagy triggered in NL4.3ΔVif-infected cells is inhibited when Vif is expressed in trans but is still active when target cells express a mutant of Vif that binds weakly to LC3B.
Conclusions: Our study unveils that Vif inhibits autophagy independently of its action on APOBEC3G and therefore suggest a new function of this viral protein in restricting innate anti-viral mechanisms.
Design/methods: To determine the role of Vif in autophagy inhibition, we used cell lines that express CD4 and CXCR4 and primary CD4 T cells. Pull-down experiments, immunoprecipitation assays and computational analyses were performed to analyze the interaction between Vif and LC3B, a major autophagy component, in presence or absence of the anti-viral host factor APOBEC3G, after HIV-1 infection or ectopic expression of Vif. Autophagy was analyzed after infection by viruses expressing Vif (NL4.3) or not (NL4.3ΔVif), or after exogenous Vif expression.
Results: We demonstrate that the C-terminal part of Vif interacts directly with LC3B, independently of the presence of APOBEC3G. Vif binds to pro-LC3 and Atg4-cleaved LC3 forms, and glycine 120, the amino acid conjugated to phosphatidylethanolamine on autophagosomes, is required. Importantly, we evidence that Vif inhibits autophagy during HIV-1 infection. Indeed, autophagy is detected in target cells infected by NL4.3ΔVif, but prevented in cells infected by NL4.3. Furthermore, autophagy triggered in NL4.3ΔVif-infected cells is inhibited when Vif is expressed in trans but is still active when target cells express a mutant of Vif that binds weakly to LC3B.
Conclusions: Our study unveils that Vif inhibits autophagy independently of its action on APOBEC3G and therefore suggest a new function of this viral protein in restricting innate anti-viral mechanisms.
2014
Sleiman D, Bernacchi S, Guerrero S X, Brachet F, Larue V, Paillart J C, Tisne C
Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein: Importance of the C-terminal unstructured tail. Journal Article
In: RNA Biol, vol. 11, no. 7, pp. 906-920, 2014, ISBN: 25144404.
Abstract | Links | BibTeX | Tags: HIV RNA chaperone Vif nucleocapsid unstructured domain, MARQUET, PAILLART, Unité ARN
@article{,
title = {Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein: Importance of the C-terminal unstructured tail.},
author = {D Sleiman and S Bernacchi and S X Guerrero and F Brachet and V Larue and J C Paillart and C Tisne},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25144404?dopt=Abstract},
doi = {10.4161/rna.29546},
isbn = {25144404},
year = {2014},
date = {2014-01-01},
journal = {RNA Biol},
volume = {11},
number = {7},
pages = {906-920},
abstract = {The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.},
keywords = {HIV RNA chaperone Vif nucleocapsid unstructured domain, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.
Schlub T E, Grimm A J, Smyth R P, Cromer D, Chopra A, Mallal S, Venturi V, Waugh C, Mak J, Davenport M P
15-20% of HIV substitution mutations are associated with recombination. Journal Article
In: J Virol, vol. 88, no. 7, pp. 3837-3849, 2014, ISBN: 24453357.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {15-20% of HIV substitution mutations are associated with recombination.},
author = {T E Schlub and A J Grimm and R P Smyth and D Cromer and A Chopra and S Mallal and V Venturi and C Waugh and J Mak and M P Davenport},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24453357?dopt=Abstract},
doi = {10.1128/JVI.03136-13},
isbn = {24453357},
year = {2014},
date = {2014-01-01},
journal = {J Virol},
volume = {88},
number = {7},
pages = {3837-3849},
abstract = {HIV undergoes a high rate of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we use a system of silent marker mutations in HIV and a single round of infection in primary T-lymphocytes, combined with a high-throughput sequencing and mathematical modelling approach to directly estimate the viral recombination and mutation rates. From >7 million nt of sequences from HIV infection, we observe 4801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1000 nt respectively). We use experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We find the single cycle virus-induced mutation rate is 4.6 × 10-5 mutations per nt after correction. By sorting our data into recombined and non-recombined sequences, we find a significantly higher mutation rate in recombined regions (p=0.003, Fisher's exact). We use a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination, and is not simply co-located in the genome. By comparing mutation rates in recombined and non-recombined regions we find that recombination-associated mutations account for 15-20% of all mutations occurring during reverse transcription.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
HIV undergoes a high rate of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we use a system of silent marker mutations in HIV and a single round of infection in primary T-lymphocytes, combined with a high-throughput sequencing and mathematical modelling approach to directly estimate the viral recombination and mutation rates. From >7 million nt of sequences from HIV infection, we observe 4801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1000 nt respectively). We use experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We find the single cycle virus-induced mutation rate is 4.6 × 10-5 mutations per nt after correction. By sorting our data into recombined and non-recombined sequences, we find a significantly higher mutation rate in recombined regions (p=0.003, Fisher's exact). We use a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination, and is not simply co-located in the genome. By comparing mutation rates in recombined and non-recombined regions we find that recombination-associated mutations account for 15-20% of all mutations occurring during reverse transcription.
Smyth R P, Schlub T E, Grimm A J, Waugh C, Ellenberg P, Chopra A, Mallal S, Cromer D, Mak J, Davenport M P
Identifying recombination hotspots in the HIV-1 genome. Journal Article
In: J Virol, vol. 88, no. 5, pp. 2891-2902, 2014, ISBN: 24371048.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {Identifying recombination hotspots in the HIV-1 genome.},
author = {R P Smyth and T E Schlub and A J Grimm and C Waugh and P Ellenberg and A Chopra and S Mallal and D Cromer and J Mak and M P Davenport},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24371048?dopt=Abstract},
doi = {10.1128/JVI.03014-13},
isbn = {24371048},
year = {2014},
date = {2014-01-01},
journal = {J Virol},
volume = {88},
number = {5},
pages = {2891-2902},
abstract = {HIV-1 infection is characterised by the rapid generation of genetic diversity that facilitates viral escape from immune selection and antiretroviral therapy. Despite recombination's crucial role in viral diversity and evolution, little is known about the genomic factors that influence recombination between highly similar genomes. In this study, we use a minimally modified full length HIV-1 genome and high throughput sequence analysis to study recombination in gag and pol in T cells. We find that recombination is favoured at a number of recombination hotspots, where recombination occurs six times more frequently than at corresponding coldspots. Interestingly, these hotspots occur near important features of the HIV-1 genome, but do not occur at sites immediately around protease inhibitor or reverse transcriptase inhibitor drug resistance mutations. We show that the recombination hot and cold spots are consistent across five blood donors and are independent of co-receptor mediated entry. Finally, we check common experimental confounders and find that these are not driving the location of recombination hotspots. This is the first study to identify the location of recombination hotspots, between two similar viral genomes with great statistical power and under conditions that closely reflect natural recombination events amongst HIV-1 quasispecies.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
HIV-1 infection is characterised by the rapid generation of genetic diversity that facilitates viral escape from immune selection and antiretroviral therapy. Despite recombination's crucial role in viral diversity and evolution, little is known about the genomic factors that influence recombination between highly similar genomes. In this study, we use a minimally modified full length HIV-1 genome and high throughput sequence analysis to study recombination in gag and pol in T cells. We find that recombination is favoured at a number of recombination hotspots, where recombination occurs six times more frequently than at corresponding coldspots. Interestingly, these hotspots occur near important features of the HIV-1 genome, but do not occur at sites immediately around protease inhibitor or reverse transcriptase inhibitor drug resistance mutations. We show that the recombination hot and cold spots are consistent across five blood donors and are independent of co-receptor mediated entry. Finally, we check common experimental confounders and find that these are not driving the location of recombination hotspots. This is the first study to identify the location of recombination hotspots, between two similar viral genomes with great statistical power and under conditions that closely reflect natural recombination events amongst HIV-1 quasispecies.
El-Wahab E W Abd, Smyth R P, Mailler E, Bernacchi S, Vivet-Boudou V, Hijnen M, Jossinet F, Mak J, Paillart J C, Marquet R
Specific recognition of the HIV-1 genomic RNA by the Gag precursor. Journal Article
In: Nat Commun, vol. 5, pp. 4304, 2014, ISBN: 24986025.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {Specific recognition of the HIV-1 genomic RNA by the Gag precursor.},
author = {E W Abd El-Wahab and R P Smyth and E Mailler and S Bernacchi and V Vivet-Boudou and M Hijnen and F Jossinet and J Mak and J C Paillart and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24986025?dopt=Abstract},
doi = {10.1038/ncomms5304},
isbn = {24986025},
year = {2014},
date = {2014-01-01},
journal = {Nat Commun},
volume = {5},
pages = {4304},
abstract = {During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.
2013
Batisse J, Guerrero S X, Bernacchi S, Richert L, Godet J, Goldschmidt V, Mély Y, Marquet R, de Rocquigny H, Paillart J C
APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells Journal Article
In: Journal of Virology, vol. 87, no. 11, pp. 6492-506, 2013.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{Batisse2013,
title = {APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells},
author = {J Batisse and S X Guerrero and S Bernacchi and L Richert and J Godet and V Goldschmidt and Y Mély and R Marquet and H de Rocquigny and J C Paillart
},
url = {https://pubmed.ncbi.nlm.nih.gov/23576497/},
doi = {10.1128/JVI.03494-12},
year = {2013},
date = {2013-07-01},
journal = {Journal of Virology},
volume = {87},
number = {11},
pages = {6492-506},
abstract = {The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell. },
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.
Cherrier T, Douce V Le, Eilebrecht S, Riclet R, Marban C, Dequiedt F, Goumon Y, Paillart J C, Mericskay M, Parlakian A, Bausero P, Abbas W, Herbein G, Kurdistani S K, Grana X, Driessche B Van, Schwartz C, Candolfi E, Benecke A G, Lint C Van, Rohr O
CTIP2 is a negative regulator of P-TEFb Journal Article
In: Proc Natl Acad Sci U S A, vol. 110, no. 31, pp. 12655-12660, 2013, ISBN: 23852730.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {CTIP2 is a negative regulator of P-TEFb},
author = {T Cherrier and V Le Douce and S Eilebrecht and R Riclet and C Marban and F Dequiedt and Y Goumon and J C Paillart and M Mericskay and A Parlakian and P Bausero and W Abbas and G Herbein and S K Kurdistani and X Grana and B Van Driessche and C Schwartz and E Candolfi and A G Benecke and C Van Lint and O Rohr},
url = {http://www.ncbi.nlm.nih.gov/pubmed/23852730?dopt=Abstract
http://www.pnas.org/content/110/31/12655.full},
isbn = {23852730},
year = {2013},
date = {2013-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {110},
number = {31},
pages = {12655-12660},
abstract = {The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the β-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the β-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.
2012
Sleiman D, Goldschmidt V, Barraud P, Marquet R, Paillart J C, Tisné C
Initiation of HIV-1 reverse transcription and functional role of nucleocapsid-mediated tRNA/viral genome interactions. Journal Article
In: Virus Res, vol. 169, no. 2, pp. 324-339, 2012, ISBN: 22721779.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Reverse transcription HIV tRNALys3 Nucleocapsid, Unité ARN
@article{,
title = {Initiation of HIV-1 reverse transcription and functional role of nucleocapsid-mediated tRNA/viral genome interactions.},
author = {D Sleiman and V Goldschmidt and P Barraud and R Marquet and J C Paillart and C Tisné},
url = {http://www.sciencedirect.com/science/article/pii/S0168170212002031?v=s5},
doi = {10.1016/j.virusres.2012.06.006},
isbn = {22721779},
year = {2012},
date = {2012-01-01},
journal = {Virus Res},
volume = {169},
number = {2},
pages = {324-339},
abstract = {HIV-1 reverse transcription is initiated from a tRNALys3 molecule annealed to the viral RNA at the primer binding site (PBS). The annealing of tRNALys3 requires the opening of its three-dimensional structure and RNA rearrangements to form an efficient initiation complex recognized by the reverse transcriptase. This annealing is mediated by the nucleocapsid protein (NC). In this paper, we first review the actual knowledge about HIV-1 viral RNA and tRNALys3 structures. Then, we summarize the studies explaining how NC chaperones the formation of the tRNALys3/PBS binary complex. Additional NMR data that investigated the NC interaction with tRNALys3 D-loop are presented. Lastly, we focused on the additional interactions occurring between tRNALys3 and the viral RNA and showed that they are dependent on HIV-1 isolates, ie the sequence and the structure of the viral RNA.},
keywords = {MARQUET, PAILLART, Reverse transcription HIV tRNALys3 Nucleocapsid, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
HIV-1 reverse transcription is initiated from a tRNALys3 molecule annealed to the viral RNA at the primer binding site (PBS). The annealing of tRNALys3 requires the opening of its three-dimensional structure and RNA rearrangements to form an efficient initiation complex recognized by the reverse transcriptase. This annealing is mediated by the nucleocapsid protein (NC). In this paper, we first review the actual knowledge about HIV-1 viral RNA and tRNALys3 structures. Then, we summarize the studies explaining how NC chaperones the formation of the tRNALys3/PBS binary complex. Additional NMR data that investigated the NC interaction with tRNALys3 D-loop are presented. Lastly, we focused on the additional interactions occurring between tRNALys3 and the viral RNA and showed that they are dependent on HIV-1 isolates, ie the sequence and the structure of the viral RNA.
Fournier E, Moules V, Essere B, Paillart J C, Sirbat J D, Isel C, Cavalier A, Rolland J P, Thomas D, Lina B, Marquet R
A supramolecular assembly formed by influenza A virus genomic RNA segments. Journal Article
In: Nucleic Acids Res, vol. 40, no. 5, pp. 2197-2209, 2012, ISBN: 22075989, (First published online: November 10, 2011).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {A supramolecular assembly formed by influenza A virus genomic RNA segments.},
author = {E Fournier and V Moules and B Essere and J C Paillart and J D Sirbat and C Isel and A Cavalier and J P Rolland and D Thomas and B Lina and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22075989},
doi = {10.1093/nar/gkr985},
isbn = {22075989},
year = {2012},
date = {2012-01-01},
journal = {Nucleic Acids Res},
volume = {40},
number = {5},
pages = {2197-2209},
abstract = {The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common 'transition zone' located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.},
note = {First published online: November 10, 2011},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common 'transition zone' located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.
Fournier E, Moules V, Essere B, Paillart J C, Sirbat J D, Cavalier A, Rolland J P, Thomas D, Lina B, Isel C, Marquet R
Interaction network linking the human H3N2 influenza A virus genomic RNA segments. Journal Article
In: Vaccine, vol. 30, no. 51, pp. 7359-7367, 2012, ISSN: ISSN: 0264-410X.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {Interaction network linking the human H3N2 influenza A virus genomic RNA segments.},
author = {E Fournier and V Moules and B Essere and J C Paillart and J D Sirbat and A Cavalier and J P Rolland and D Thomas and B Lina and C Isel and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/pubmed/23063835?dopt=Abstract},
doi = {10.1016/j.vaccine.2012.09.079},
issn = {ISSN: 0264-410X},
year = {2012},
date = {2012-01-01},
journal = {Vaccine},
volume = {30},
number = {51},
pages = {7359-7367},
abstract = {he genome of influenza A viruses is comprised of eight negative-sense viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). In order to be infectious, an influenza A viral particle must encapsidate at least one copy of each of the vRNAs. Thus, even though genome segmentation is evolutionary advantageous, it undeniably complicates viral assembly, which is believed to occur through a selective mechanism that still remains to be understood. Using electron tomography 3D-reconstructions, we show that the eight vRNPs of an influenza A Moscow/10/99 (H3N2) virus are interconnected within a star-like structure as they emerge from a unique "transition zone" at the budding tip of the virions. Notably, this "transition zone" is thick enough to accommodate all described packaging signals. We also report that, in vitro, each vRNA segment is involved in a direct contact with at least one other vRNA partner, in a single network of intermolecular interactions. We show that in several cases, the regions involved in vRNA/vRNA interactions overlap with previously identified packaging signals. Our results thus provide support for the involvement of RNA/RNA interactions in the selection and specific packaging of influenza A genomic RNAs, which appear embedded into an organised supramolecular complex likely held together by direct base-pairings between packaging signals.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
he genome of influenza A viruses is comprised of eight negative-sense viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). In order to be infectious, an influenza A viral particle must encapsidate at least one copy of each of the vRNAs. Thus, even though genome segmentation is evolutionary advantageous, it undeniably complicates viral assembly, which is believed to occur through a selective mechanism that still remains to be understood. Using electron tomography 3D-reconstructions, we show that the eight vRNPs of an influenza A Moscow/10/99 (H3N2) virus are interconnected within a star-like structure as they emerge from a unique "transition zone" at the budding tip of the virions. Notably, this "transition zone" is thick enough to accommodate all described packaging signals. We also report that, in vitro, each vRNA segment is involved in a direct contact with at least one other vRNA partner, in a single network of intermolecular interactions. We show that in several cases, the regions involved in vRNA/vRNA interactions overlap with previously identified packaging signals. Our results thus provide support for the involvement of RNA/RNA interactions in the selection and specific packaging of influenza A genomic RNAs, which appear embedded into an organised supramolecular complex likely held together by direct base-pairings between packaging signals.
Batisse J, Guerrero S X, Bernacchi S, Sleiman D, Gabus C, Darlix J L, Marquet R, Tisné C, Paillart J C
The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication. Journal Article
In: Virus Res, vol. 169, no. 2, pp. 361-376, 2012.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.},
author = {J Batisse and S X Guerrero and S Bernacchi and D Sleiman and C Gabus and J L Darlix and R Marquet and C Tisné and J C Paillart},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22728817?dopt=Abstract},
doi = {22728817},
year = {2012},
date = {2012-01-01},
journal = {Virus Res},
volume = {169},
number = {2},
pages = {361-376},
abstract = {The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.
Smyth R P, Davenport M P, Mak J
The origin of genetic diversity in HIV-1. Journal Article
In: Virus Res, vol. 169, no. 2, pp. 415-429, 2012.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {The origin of genetic diversity in HIV-1.},
author = {R P Smyth and M P Davenport and J Mak},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22728444?dopt=Abstract},
doi = {22728444},
year = {2012},
date = {2012-01-01},
journal = {Virus Res},
volume = {169},
number = {2},
pages = {415-429},
abstract = {One of the hallmarks of HIV infection is the rapid development of a genetically complex population (quasispecies) from an initially limited number of infectious particles. Genetic diversity remains one of the major obstacles to eradication of HIV. The viral quasispecies can respond rapidly to selective pressures, such as that imposed by the immune system and antiretroviral therapy, and frustrates vaccine design efforts. Two unique features of retroviral replication are responsible for the unprecedented variation generated during infection. First, mutations are frequently introduced into the viral genome by the error prone viral reverse transcriptase and through the actions of host cellular factors, such as the APOBEC family of nucleic acid editing enzymes. Second, the HIV reverse transcriptase can utilize both copies of the co-packaged viral genome in a process termed retroviral recombination. When the co-packaged viral genomes are genetically different, retroviral recombination can lead to the shuffling of mutations between viral genomes in the quasispecies. This review outlines the stages of the retroviral life cycle where genetic variation is introduced, focusing on the principal mechanisms of mutation and recombination. Understanding the mechanistic origin of genetic diversity is essential to combating HIV.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
One of the hallmarks of HIV infection is the rapid development of a genetically complex population (quasispecies) from an initially limited number of infectious particles. Genetic diversity remains one of the major obstacles to eradication of HIV. The viral quasispecies can respond rapidly to selective pressures, such as that imposed by the immune system and antiretroviral therapy, and frustrates vaccine design efforts. Two unique features of retroviral replication are responsible for the unprecedented variation generated during infection. First, mutations are frequently introduced into the viral genome by the error prone viral reverse transcriptase and through the actions of host cellular factors, such as the APOBEC family of nucleic acid editing enzymes. Second, the HIV reverse transcriptase can utilize both copies of the co-packaged viral genome in a process termed retroviral recombination. When the co-packaged viral genomes are genetically different, retroviral recombination can lead to the shuffling of mutations between viral genomes in the quasispecies. This review outlines the stages of the retroviral life cycle where genetic variation is introduced, focusing on the principal mechanisms of mutation and recombination. Understanding the mechanistic origin of genetic diversity is essential to combating HIV.
Ducloux C, Mougel M, Goldschmidt V, Didierlaurent L, Marquet R, Isel C
A pyrophosphatase activity associated with purified HIV-1 particles. Journal Article
In: Biochimie, vol. 94, no. 12, pp. 2498-2507, 2012, ISBN: 22766015.
Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {A pyrophosphatase activity associated with purified HIV-1 particles.},
author = {C Ducloux and M Mougel and V Goldschmidt and L Didierlaurent and R Marquet and C Isel},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22766015?dopt=Abstract},
doi = {10.1016/j.biochi.2012.06.025},
isbn = {22766015},
year = {2012},
date = {2012-01-01},
journal = {Biochimie},
volume = {94},
number = {12},
pages = {2498-2507},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
von Kleist M, Metzner P, Marquet R, Schütte C
HIV-1 polymerase inhibition by nucleoside analogs: cellular- and kinetic parameters of efficacy, susceptibility and resistance selection. Journal Article
In: PLoS Comput Biol, vol. 8, no. 1, pp. e1002359, 2012, ISBN: 22275860.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {HIV-1 polymerase inhibition by nucleoside analogs: cellular- and kinetic parameters of efficacy, susceptibility and resistance selection.},
author = {M von Kleist and P Metzner and R Marquet and C Schütte},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22275860},
doi = {10.1371/journal.pcbi.1002359},
isbn = {22275860},
year = {2012},
date = {2012-01-01},
journal = {PLoS Comput Biol},
volume = {8},
number = {1},
pages = {e1002359},
abstract = {Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.
Marquet R, Guerrero S, Bernacchi S, Pernot S, Batisse J, Paillart J C
A transcription factor acts as a HIV-1 accomplice to destroy the cellular defences. Journal Article
In: Med Sci (Paris), vol. 28, no. 4, pp. 356-358, 2012.
Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{marquet2012,
title = {A transcription factor acts as a HIV-1 accomplice to destroy the cellular defences.},
author = {R Marquet and S Guerrero and S Bernacchi and S Pernot and J Batisse and J C Paillart},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22549857?dopt=AbstractPlus},
doi = {10.1051/medsci/2012284007},
year = {2012},
date = {2012-01-01},
journal = {Med Sci (Paris)},
volume = {28},
number = {4},
pages = {356-358},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
2011
Vivet-Boudou V, Isel C, Sleiman M, Smyth R, Gaied N Ben, Barhoum P, Laumond G, Bec G, Götte M, Mak J, Aubertin A M, Burger A, Marquet R
8-Modified-2'-Deoxyadenosine Analogues Induce Delayed Polymerization Arrest during HIV-1 Reverse Transcription. Journal Article
In: PLoS One, vol. 6, no. 11, pp. e27456, 2011, ISBN: PMC3210175, (Published online 2011 November 7).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {8-Modified-2'-Deoxyadenosine Analogues Induce Delayed Polymerization Arrest during HIV-1 Reverse Transcription.},
author = {V Vivet-Boudou and C Isel and M Sleiman and R Smyth and N Ben Gaied and P Barhoum and G Laumond and G Bec and M Götte and J Mak and A M Aubertin and A Burger and R Marquet},
url = {http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0027456},
doi = {doi: 10.1371/journal.pone.0027456},
isbn = {PMC3210175},
year = {2011},
date = {2011-01-01},
journal = {PLoS One},
volume = {6},
number = {11},
pages = {e27456},
abstract = {The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2'-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2'-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.},
note = {Published online 2011 November 7},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2'-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2'-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.
Bernacchi S, Mercenne G, Tournaire C, Marquet R, Paillart J C
Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif Journal Article
In: Nucleic Acids Res, vol. 39, no. 6, pp. 2404-2415, 2011, ISSN: 1362-4962 (Electronic) 0305-1048 (Linking), (Bernacchi, Serena Mercenne, Gaelle Tournaire, Clemence Marquet, Roland Paillart, Jean-Christophe England Nucleic acids research Nucleic Acids Res. 2011 Mar 1;39(6):2404-15. Epub 2010 Nov 13.).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif},
author = {S Bernacchi and G Mercenne and C Tournaire and R Marquet and J C Paillart},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21076154},
doi = {10.1093/nar/gkq979},
issn = {1362-4962 (Electronic) 0305-1048 (Linking)},
year = {2011},
date = {2011-01-01},
journal = {Nucleic Acids Res},
volume = {39},
number = {6},
pages = {2404-2415},
abstract = {The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of beta-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs.},
note = {Bernacchi, Serena
Mercenne, Gaelle
Tournaire, Clemence
Marquet, Roland
Paillart, Jean-Christophe
England
Nucleic acids research
Nucleic Acids Res. 2011 Mar 1;39(6):2404-15. Epub 2010 Nov 13.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of beta-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs.
2010
Mercenne G, Bernacchi S, Richer D, Bec G, Henriet S, Paillart J C, Marquet R
HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation Journal Article
In: Nucleic Acids Res, vol. 38, no. 2, pp. 633-646, 2010, ISBN: 19910370, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: 3' Untranslated Regions 5' Untranslated Regions Binding Sites Cytidine Deaminase/*genetics/metabolism Humans Mutation *Protein Biosynthesis Protein Footprinting RNA, Fluorescence vif Gene Products, Human Immunodeficiency Virus/genetics/*metabolism, MARQUET, Messenger/*metabolism Spectrometry, PAILLART, Unité ARN
@article{,
title = {HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation},
author = {G Mercenne and S Bernacchi and D Richer and G Bec and S Henriet and J C Paillart and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19910370},
isbn = {19910370},
year = {2010},
date = {2010-01-01},
journal = {Nucleic Acids Res},
volume = {38},
number = {2},
pages = {633-646},
abstract = {The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {3' Untranslated Regions 5' Untranslated Regions Binding Sites Cytidine Deaminase/*genetics/metabolism Humans Mutation *Protein Biosynthesis Protein Footprinting RNA, Fluorescence vif Gene Products, Human Immunodeficiency Virus/genetics/*metabolism, MARQUET, Messenger/*metabolism Spectrometry, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.
Safadi Y El, Paillart J C, Laumond G, Aubertin A M, Burger A, Marquet R, Vivet-Boudou V
5-Modified-2'-dU and 2'-dC as mutagenic anti HIV-1 proliferation agents: synthesis and activity Journal Article
In: J Med Chem, vol. 53, no. 4, pp. 1534-1545, 2010, ISBN: 20112915, (1520-4804 (Electronic) 0022-2623 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Anti-HIV Agents/*chemical synthesis/chemistry/pharmacology Cell Line, MARQUET, PAILLART, Tumor Cell Survival/drug effects Genome
@article{,
title = {5-Modified-2'-dU and 2'-dC as mutagenic anti HIV-1 proliferation agents: synthesis and activity},
author = {Y El Safadi and J C Paillart and G Laumond and A M Aubertin and A Burger and R Marquet and V Vivet-Boudou},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20112915},
isbn = {20112915},
year = {2010},
date = {2010-01-01},
journal = {J Med Chem},
volume = {53},
number = {4},
pages = {1534-1545},
abstract = {With the goal of limiting HIV-1 proliferation by increasing the mutation rate of the viral genome, we synthesized a series of pyrimidine nucleoside analogues modified in position 5 of the aglycone moiety but unmodified on the sugar part. The synthetic strategies allow us to prepare the targeted compounds directly from commercially available nucleosides. All compounds were tested for their ability to reduce HIV-1 proliferation in cell culture. Two of them (5-hydroxymethyl-2'-dU (1c) and 5-hydroxymethyl-2'-dC (2c)) displayed a moderate antiviral activity in single passage experiments. The same two compounds plus two additional ones (5-carbamoyl-2'-dU (1a) and 5-carbamoylmethyl-2'-dU (1b)) were potent inhibitors of HIV-1 RT activity in serial passage assays, in which they induced a progressive loss of HIV-1 replication. In addition, viruses collected after seven passages in the presence of 1c and 2c replicated very poorly after withdrawal of these compounds, consistent with the accumulation of deleterious mutations in the HIV-1 genome.},
note = {1520-4804 (Electronic)
0022-2623 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Anti-HIV Agents/*chemical synthesis/chemistry/pharmacology Cell Line, MARQUET, PAILLART, Tumor Cell Survival/drug effects Genome},
pubstate = {published},
tppubtype = {article}
}
With the goal of limiting HIV-1 proliferation by increasing the mutation rate of the viral genome, we synthesized a series of pyrimidine nucleoside analogues modified in position 5 of the aglycone moiety but unmodified on the sugar part. The synthetic strategies allow us to prepare the targeted compounds directly from commercially available nucleosides. All compounds were tested for their ability to reduce HIV-1 proliferation in cell culture. Two of them (5-hydroxymethyl-2'-dU (1c) and 5-hydroxymethyl-2'-dC (2c)) displayed a moderate antiviral activity in single passage experiments. The same two compounds plus two additional ones (5-carbamoyl-2'-dU (1a) and 5-carbamoylmethyl-2'-dU (1b)) were potent inhibitors of HIV-1 RT activity in serial passage assays, in which they induced a progressive loss of HIV-1 replication. In addition, viruses collected after seven passages in the presence of 1c and 2c replicated very poorly after withdrawal of these compounds, consistent with the accumulation of deleterious mutations in the HIV-1 genome.
2009
Henriet S, Mercenne G, Bernacchi S, Paillart J C, Marquet R
In: Microbiol Mol Biol Rev, vol. 73, no. 2, pp. 211-232, 2009, ISBN: 19487726, (1098-5557 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {Tumultuous relationship between the human immunodeficiency virus type 1 viral infectivity factor (Vif) and the human APOBEC-3G and APOBEC-3F restriction factors},
author = {S Henriet and G Mercenne and S Bernacchi and J C Paillart and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19487726},
isbn = {19487726},
year = {2009},
date = {2009-01-01},
journal = {Microbiol Mol Biol Rev},
volume = {73},
number = {2},
pages = {211-232},
abstract = {The viral infectivity factor (Vif) is dispensable for human immunodeficiency virus type 1 (HIV-1) replication in so-called permissive cells but is required for replication in nonpermissive cell lines and for pathogenesis. Virions produced in the absence of Vif have an aberrant morphology and an unstable core and are unable to complete reverse transcription. Recent studies demonstrated that human APOBEC-3G (hA3G) and APOBEC-3F (hA3F), which are selectively expressed in nonpermissive cells, possess strong anti-HIV-1 activity and are sufficient to confer a nonpermissive phenotype. Vif induces the degradation of hA3G and hA3F, suggesting that its main function is to counteract these cellular factors. Most studies focused on the hypermutation induced by the cytidine deaminase activity of hA3G and hA3F and on their Vif-induced degradation by the proteasome. However, recent studies suggested that several mechanisms are involved both in the antiviral activity of hA3G and hA3F and in the way Vif counteracts these antiviral factors. Attempts to reconcile the studies involving Vif in virus assembly and stability with these recent findings suggest that hA3G and hA3F partially exert their antiviral activity independently of their catalytic activity by destabilizing the viral core and the reverse transcription complex, possibly by interfering with the assembly and/or maturation of the viral particles. Vif could then counteract hA3G and hA3F by excluding them from the viral assembly intermediates through competition for the viral genomic RNA, by regulating the proteolytic processing of Pr55(Gag), by enhancing the efficiency of the reverse transcription process, and by inhibiting the enzymatic activities of hA3G and hA3F.},
note = {1098-5557 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The viral infectivity factor (Vif) is dispensable for human immunodeficiency virus type 1 (HIV-1) replication in so-called permissive cells but is required for replication in nonpermissive cell lines and for pathogenesis. Virions produced in the absence of Vif have an aberrant morphology and an unstable core and are unable to complete reverse transcription. Recent studies demonstrated that human APOBEC-3G (hA3G) and APOBEC-3F (hA3F), which are selectively expressed in nonpermissive cells, possess strong anti-HIV-1 activity and are sufficient to confer a nonpermissive phenotype. Vif induces the degradation of hA3G and hA3F, suggesting that its main function is to counteract these cellular factors. Most studies focused on the hypermutation induced by the cytidine deaminase activity of hA3G and hA3F and on their Vif-induced degradation by the proteasome. However, recent studies suggested that several mechanisms are involved both in the antiviral activity of hA3G and hA3F and in the way Vif counteracts these antiviral factors. Attempts to reconcile the studies involving Vif in virus assembly and stability with these recent findings suggest that hA3G and hA3F partially exert their antiviral activity independently of their catalytic activity by destabilizing the viral core and the reverse transcription complex, possibly by interfering with the assembly and/or maturation of the viral particles. Vif could then counteract hA3G and hA3F by excluding them from the viral assembly intermediates through competition for the viral genomic RNA, by regulating the proteolytic processing of Pr55(Gag), by enhancing the efficiency of the reverse transcription process, and by inhibiting the enzymatic activities of hA3G and hA3F.
Vivet-Boudou V, Marquet R, Burger A
Analogues nucleosidiques inhibiteurs de la retrotranscriptase Miscellaneous
2009, ISBN: 09/01787 (fr).
BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@misc{,
title = {Analogues nucleosidiques inhibiteurs de la retrotranscriptase},
author = {V Vivet-Boudou and R Marquet and A Burger},
isbn = {09/01787 (fr)},
year = {2009},
date = {2009-01-01},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {misc}
}
Negroni M, Gallois-Montbrun S, Rossolillo P, Bartolo V Di, Uze G, Simon-Loriere E, Marquet R, Vivet-Boudou V
Lentiviral-based vector and its use in directed evolution of genomic regions, genes and polynucleotides. Miscellaneous
2009, ISBN: EP 09290856.5.
Abstract | BibTeX | Tags: MARQUET, NEGRONI, PAILLART, Unité ARN
@misc{,
title = {Lentiviral-based vector and its use in directed evolution of genomic regions, genes and polynucleotides.},
author = {M Negroni and S Gallois-Montbrun and P Rossolillo and V Di Bartolo and G Uze and E Simon-Loriere and R Marquet and V Vivet-Boudou},
isbn = {EP 09290856.5},
year = {2009},
date = {2009-01-01},
abstract = {The present invention concerns a method of directing evolution of a target polynucleotide of interest for obtaining variants of this target polynucleotide, as well as a method to generate genetic variability by preparing a cell library. This invention also relates to a method to isolate or to screen variants of a polynucleotide or variants of a protein able to impact the phenotype of a cell or to confer a desired phenotype to target cells, and to identify these polynucleotide variants or protein variants responsible for this phenotype.},
keywords = {MARQUET, NEGRONI, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {misc}
}
The present invention concerns a method of directing evolution of a target polynucleotide of interest for obtaining variants of this target polynucleotide, as well as a method to generate genetic variability by preparing a cell library. This invention also relates to a method to isolate or to screen variants of a polynucleotide or variants of a protein able to impact the phenotype of a cell or to confer a desired phenotype to target cells, and to identify these polynucleotide variants or protein variants responsible for this phenotype.
Keating C P, Hill M K, Hawkes D J, Smyth R P, Isel C, Le S Y, Palmenberg A C, Marshall J A, Marquet R, Nabel G J, Mak J
The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA Journal Article
In: Nucleic Acids Res, vol. 37, no. 3, pp. 945-956, 2009, ISBN: 19106143, (1362-4962 (Electronic) Journal Article Research Support, N.I.H., Intramural Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Adenine/analysis Base Sequence Cell Line Codon DNA, Complementary/*biosynthesis DNA, MARQUET, PAILLART, pol HIV-1/*genetics/physiology Humans Nucleic Acid Conformation RNA, Ribonucleic Acid *Reverse Transcription Viral Proteins/metabolism Virion/metabolism Virus Internalization Virus Replication, Unité ARN, Viral/*biosynthesis Dimerization *Genes, Viral/*chemistry *Regulatory Sequences
@article{,
title = {The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA},
author = {C P Keating and M K Hill and D J Hawkes and R P Smyth and C Isel and S Y Le and A C Palmenberg and J A Marshall and R Marquet and G J Nabel and J Mak},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19106143},
isbn = {19106143},
year = {2009},
date = {2009-01-01},
journal = {Nucleic Acids Res},
volume = {37},
number = {3},
pages = {945-956},
abstract = {The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich 'structurally poor' RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5-100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using 'structurally poor' RNA domains in regulating biological process.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't},
keywords = {Adenine/analysis Base Sequence Cell Line Codon DNA, Complementary/*biosynthesis DNA, MARQUET, PAILLART, pol HIV-1/*genetics/physiology Humans Nucleic Acid Conformation RNA, Ribonucleic Acid *Reverse Transcription Viral Proteins/metabolism Virion/metabolism Virus Internalization Virus Replication, Unité ARN, Viral/*biosynthesis Dimerization *Genes, Viral/*chemistry *Regulatory Sequences},
pubstate = {published},
tppubtype = {article}
}
The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich 'structurally poor' RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5-100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using 'structurally poor' RNA domains in regulating biological process.
2008
Barraud P, Paillart J C, Marquet R, Tisne C
Advances in the structural understanding of Vif proteins Journal Article
In: Curr HIV Res, vol. 6, no. 2, pp. 91-99, 2008, ISBN: 18336256, (1873-4251 (Electronic) Journal Article Research Support, Non-U.S. Gov't Review).
Abstract | Links | BibTeX | Tags: Amino Acid Motifs Gene Products, MARQUET, PAILLART, Tertiary Simian immunodeficiency virus/*genetics, Unité ARN, vif/*genetics/*metabolism HIV-1/*genetics HIV-2/*genetics Humans Protein Binding Protein Structure
@article{,
title = {Advances in the structural understanding of Vif proteins},
author = {P Barraud and J C Paillart and R Marquet and C Tisne},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18336256},
isbn = {18336256},
year = {2008},
date = {2008-01-01},
journal = {Curr HIV Res},
volume = {6},
number = {2},
pages = {91-99},
abstract = {The multidomain HIV-1 Vif protein recruits several cellular partners to achieve neutralization of the antiviral activity of APOBEC3 proteins. Vif neutralizes APOBEC3G and APOBEC3F predominantly by forming an E3 ubiquitin ligase with Cullin5, ElonginB and ElonginC that targets these proteins for degradation by the ubiquitin-proteasome pathway. Vif associates with the Cullin5-ElonginB-ElonginC complex by binding directly to ElonginC via its SOCS-box motif and to Cullin5 via hydrophobic residues within a zinc-binding region formed by a conserved HCCH motif. The HIV-1 Vif-Cullin5-ElonginBC complex is then able to ubiquitinate the APOBEC3G factor bound to Vif by its N-terminal domain. In this review, we summarize the current knowledge about the structural determinants of Vif that allow it to interact with cellular and viral partners.},
note = {1873-4251 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't
Review},
keywords = {Amino Acid Motifs Gene Products, MARQUET, PAILLART, Tertiary Simian immunodeficiency virus/*genetics, Unité ARN, vif/*genetics/*metabolism HIV-1/*genetics HIV-2/*genetics Humans Protein Binding Protein Structure},
pubstate = {published},
tppubtype = {article}
}
The multidomain HIV-1 Vif protein recruits several cellular partners to achieve neutralization of the antiviral activity of APOBEC3 proteins. Vif neutralizes APOBEC3G and APOBEC3F predominantly by forming an E3 ubiquitin ligase with Cullin5, ElonginB and ElonginC that targets these proteins for degradation by the ubiquitin-proteasome pathway. Vif associates with the Cullin5-ElonginB-ElonginC complex by binding directly to ElonginC via its SOCS-box motif and to Cullin5 via hydrophobic residues within a zinc-binding region formed by a conserved HCCH motif. The HIV-1 Vif-Cullin5-ElonginBC complex is then able to ubiquitinate the APOBEC3G factor bound to Vif by its N-terminal domain. In this review, we summarize the current knowledge about the structural determinants of Vif that allow it to interact with cellular and viral partners.
2007
Vivet-Boudou V, Paillart J C, Burger A, Marquet R
In: Nucleosides Nucleotides Nucleic Acids, vol. 26, no. 6-7, pp. 743-746, 2007, ISBN: 18066893, (1525-7770 (Print) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Antiviral Agents/*chemical synthesis/chemistry/*pharmacology HIV-1/*drug effects/*physiology Imidazoles/*chemical synthesis/chemistry/*pharmacology Mutagenesis/*drug effects Virus Replication/*drug effects, MARQUET, PAILLART, Unité ARN
@article{,
title = {In search of new inhibitors of HIV-1 replication: synthesis and study of 1-(2'-Deoxy-beta-D-Ribofuranosyl)-1,2,4-triazole-3-carboxamide as a selective viral mutagenic agent},
author = {V Vivet-Boudou and J C Paillart and A Burger and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18066893},
isbn = {18066893},
year = {2007},
date = {2007-01-01},
journal = {Nucleosides Nucleotides Nucleic Acids},
volume = {26},
number = {6-7},
pages = {743-746},
abstract = {With the emergence of HIV strains resistant or cross-resistant to nearly all antiretroviral regimen, novel therapy approaches have to be considered. As a part of our current work on viral mutagenic compounds, we prepared 1-(2' -deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (2' -deoxy-ribavirin) and its 5' -triphosphate derivative. The nucleoside mutagenic activity was evaluated on HIV-1 NL4-3 in CEMx174 cell culture. After 2.5 months, no reduction on HIV-1 viability was observed. On the other hand, in vitro experiments with purified HIV-1 RT demonstrated that the triphosphate analog can be incorporated opposite to several natural nucleosides.},
note = {1525-7770 (Print)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Antiviral Agents/*chemical synthesis/chemistry/*pharmacology HIV-1/*drug effects/*physiology Imidazoles/*chemical synthesis/chemistry/*pharmacology Mutagenesis/*drug effects Virus Replication/*drug effects, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
With the emergence of HIV strains resistant or cross-resistant to nearly all antiretroviral regimen, novel therapy approaches have to be considered. As a part of our current work on viral mutagenic compounds, we prepared 1-(2' -deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (2' -deoxy-ribavirin) and its 5' -triphosphate derivative. The nucleoside mutagenic activity was evaluated on HIV-1 NL4-3 in CEMx174 cell culture. After 2.5 months, no reduction on HIV-1 viability was observed. On the other hand, in vitro experiments with purified HIV-1 RT demonstrated that the triphosphate analog can be incorporated opposite to several natural nucleosides.
Sinck L, Richer D, Howard J, Alexander M, Purcell D F, Marquet R, Paillart J C
In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs Journal Article
In: RNA, vol. 13, no. 12, pp. 2141-2150, 2007, ISSN: 1469-9001 (Electronic) 1355-8382 (Linking), (Sinck, Lucile Richer, Delphine Howard, Jane Alexander, Marina Purcell, Damian F J Marquet, Roland Paillart, Jean-Christophe Research Support, Non-U.S. Gov't United States RNA (New York, N.Y.) RNA. 2007 Dec;13(12):2141-50. Epub 2007 Oct 9.).
Abstract | Links | BibTeX | Tags: Dimerization Drug Stability Genes, env Genes, MARQUET, Messenger/genetics RNA, nef Genes, PAILLART, tat HIV-1/*genetics Humans *RNA Splicing RNA, Unité ARN, Viral Genes, Viral/*genetics
@article{,
title = {In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs},
author = {L Sinck and D Richer and J Howard and M Alexander and D F Purcell and R Marquet and J C Paillart},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17925344},
doi = {rna.678307 [pii] 10.1261/rna.678307},
issn = {1469-9001 (Electronic) 1355-8382 (Linking)},
year = {2007},
date = {2007-01-01},
journal = {RNA},
volume = {13},
number = {12},
pages = {2141-2150},
abstract = {The human immunodeficiency virus type 1 (HIV-1) packages its genomic RNA as a dimer of homologous RNA molecules that has to be selected among a multitude of cellular and viral RNAs. Interestingly, spliced viral mRNAs are packaged into viral particles with a relatively low efficiency despite the fact that they contain most of the extended packaging signal found in the 5' untranslated region of the genomic RNA, including the dimerization initiation site (DIS). As a consequence, HIV-1 spliced viral RNAs can theoretically homodimerize and heterodimerize with the genomic RNA, and thus they should directly compete with genomic RNA for packaging. To shed light on this issue, we investigated for the first time the in vitro dimerization properties of spliced HIV-1 RNAs. We found that singly spliced (env, vpr) and multispliced (tat, rev, and nef) RNA fragments are able to dimerize in vitro, and to efficiently form heterodimers with genomic RNA. Chemical probing experiments and inhibition of RNA dimerization by an antisense oligonucleotide directed against the DIS indicated that the DIS is structurally functional in spliced HIV-1 RNA, and that RNA dimerization occurs through a loop-loop interaction. In addition, by combining in vitro transcription and dimerization assays, we show that heterodimers can be efficiently formed only when the two RNA fragments are synthesized simultaneously, in the same environment. Together, our results support a model in which RNA dimerization would occur during transcription in the nucleus and could thus play a major role in splicing, transport, and localization of HIV-1 RNA.},
note = {Sinck, Lucile
Richer, Delphine
Howard, Jane
Alexander, Marina
Purcell, Damian F J
Marquet, Roland
Paillart, Jean-Christophe
Research Support, Non-U.S. Gov't
United States
RNA (New York, N.Y.)
RNA. 2007 Dec;13(12):2141-50. Epub 2007 Oct 9.},
keywords = {Dimerization Drug Stability Genes, env Genes, MARQUET, Messenger/genetics RNA, nef Genes, PAILLART, tat HIV-1/*genetics Humans *RNA Splicing RNA, Unité ARN, Viral Genes, Viral/*genetics},
pubstate = {published},
tppubtype = {article}
}
The human immunodeficiency virus type 1 (HIV-1) packages its genomic RNA as a dimer of homologous RNA molecules that has to be selected among a multitude of cellular and viral RNAs. Interestingly, spliced viral mRNAs are packaged into viral particles with a relatively low efficiency despite the fact that they contain most of the extended packaging signal found in the 5' untranslated region of the genomic RNA, including the dimerization initiation site (DIS). As a consequence, HIV-1 spliced viral RNAs can theoretically homodimerize and heterodimerize with the genomic RNA, and thus they should directly compete with genomic RNA for packaging. To shed light on this issue, we investigated for the first time the in vitro dimerization properties of spliced HIV-1 RNAs. We found that singly spliced (env, vpr) and multispliced (tat, rev, and nef) RNA fragments are able to dimerize in vitro, and to efficiently form heterodimers with genomic RNA. Chemical probing experiments and inhibition of RNA dimerization by an antisense oligonucleotide directed against the DIS indicated that the DIS is structurally functional in spliced HIV-1 RNA, and that RNA dimerization occurs through a loop-loop interaction. In addition, by combining in vitro transcription and dimerization assays, we show that heterodimers can be efficiently formed only when the two RNA fragments are synthesized simultaneously, in the same environment. Together, our results support a model in which RNA dimerization would occur during transcription in the nucleus and could thus play a major role in splicing, transport, and localization of HIV-1 RNA.
Ennifar E, Paillart J C, Bernacchi S, Walter P, Pale P, Decout J L, Marquet R, Dumas P
A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site Journal Article
In: Biochimie, vol. 89, no. 10, pp. 1195-1203, 2007, ISBN: 17434658.
Abstract | Links | BibTeX | Tags: ENNIFAR, MARQUET, PAILLART, Unité ARN
@article{,
title = {A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site},
author = {E Ennifar and J C Paillart and S Bernacchi and P Walter and P Pale and J L Decout and R Marquet and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17434658},
isbn = {17434658},
year = {2007},
date = {2007-01-01},
journal = {Biochimie},
volume = {89},
number = {10},
pages = {1195-1203},
abstract = {Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop-loop complex between two DIS hairpins. Crystal structures of the DIS loop-loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop-loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity.},
keywords = {ENNIFAR, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop-loop complex between two DIS hairpins. Crystal structures of the DIS loop-loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop-loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity.
Henriet S, Sinck L, Bec G, Gorelick R J, Marquet R, Paillart J C
Vif is a RNA chaperone that could temporally regulate RNA dimerization and the early steps of HIV-1 reverse transcription Journal Article
In: Nucleic Acids Res, vol. 35, no. 15, pp. 5141-5153, 2007, ISBN: 17660191, (1362-4962 (Electronic) Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Amino Acyl/metabolism RNA, Capsid Proteins/metabolism DNA, gag/metabolism Gene Products, Human Immunodeficiency Virus, Human Immunodeficiency Virus vif Gene Products, MARQUET, PAILLART, Single-Stranded/biosynthesis Dimerization Gene Products, Transfer, Unité ARN, vif/*metabolism HIV-1/*genetics Molecular Chaperones/*metabolism RNA, Viral/*metabolism *Reverse Transcription Viral Proteins/metabolism gag Gene Products
@article{,
title = {Vif is a RNA chaperone that could temporally regulate RNA dimerization and the early steps of HIV-1 reverse transcription},
author = {S Henriet and L Sinck and G Bec and R J Gorelick and R Marquet and J C Paillart},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17660191},
isbn = {17660191},
year = {2007},
date = {2007-01-01},
journal = {Nucleic Acids Res},
volume = {35},
number = {15},
pages = {5141-5153},
abstract = {HIV-1 Vif (viral infectivity factor) is associated with the assembly complexes and packaged at low level into the viral particles, and is essential for viral replication in non-permissive cells. Viral particles produced in the absence of Vif exhibit structural defects and are defective in the early steps of reverse transcription. Here, we show that Vif is able to anneal primer tRNA(Lys3) to the viral RNA, to decrease pausing of reverse transcriptase during (-) strand strong-stop DNA synthesis, and to promote the first strand transfer. Vif also stimulates formation of loose HIV-1 genomic RNA dimers. These results indicate that Vif is a bona fide RNA chaperone. We next studied the effects of Vif in the presence of HIV-1 NCp, which is a well-established RNA chaperone. Vif inhibits NCp-mediated formation of tight RNA dimers and hybridization of tRNA(Lys3), while it has little effects on NCp-mediated strand transfer and it collaborates with nucleocapsid (NC) to increase RT processivity. Thus, Vif might negatively regulate NC-assisted maturation of the RNA dimer and early steps of reverse transcription in the assembly complexes, but these inhibitory effects would be relieved after viral budding, thanks to the limited packaging of Vif in the virions.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't},
keywords = {Amino Acyl/metabolism RNA, Capsid Proteins/metabolism DNA, gag/metabolism Gene Products, Human Immunodeficiency Virus, Human Immunodeficiency Virus vif Gene Products, MARQUET, PAILLART, Single-Stranded/biosynthesis Dimerization Gene Products, Transfer, Unité ARN, vif/*metabolism HIV-1/*genetics Molecular Chaperones/*metabolism RNA, Viral/*metabolism *Reverse Transcription Viral Proteins/metabolism gag Gene Products},
pubstate = {published},
tppubtype = {article}
}
HIV-1 Vif (viral infectivity factor) is associated with the assembly complexes and packaged at low level into the viral particles, and is essential for viral replication in non-permissive cells. Viral particles produced in the absence of Vif exhibit structural defects and are defective in the early steps of reverse transcription. Here, we show that Vif is able to anneal primer tRNA(Lys3) to the viral RNA, to decrease pausing of reverse transcriptase during (-) strand strong-stop DNA synthesis, and to promote the first strand transfer. Vif also stimulates formation of loose HIV-1 genomic RNA dimers. These results indicate that Vif is a bona fide RNA chaperone. We next studied the effects of Vif in the presence of HIV-1 NCp, which is a well-established RNA chaperone. Vif inhibits NCp-mediated formation of tight RNA dimers and hybridization of tRNA(Lys3), while it has little effects on NCp-mediated strand transfer and it collaborates with nucleocapsid (NC) to increase RT processivity. Thus, Vif might negatively regulate NC-assisted maturation of the RNA dimer and early steps of reverse transcription in the assembly complexes, but these inhibitory effects would be relieved after viral budding, thanks to the limited packaging of Vif in the virions.
Houzet L, Paillart J C, Smagulova F, Maurel S, Morichaud Z, Marquet R, Mougel M
HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms Journal Article
In: Nucleic Acids Res, vol. 35, no. 8, pp. 2695-2704, 2007, ISBN: 17426127, (1362-4962 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms},
author = {L Houzet and J C Paillart and F Smagulova and S Maurel and Z Morichaud and R Marquet and M Mougel},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17426127},
isbn = {17426127},
year = {2007},
date = {2007-01-01},
journal = {Nucleic Acids Res},
volume = {35},
number = {8},
pages = {2695-2704},
abstract = {In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. In order to determine the encapsidation mechanisms of these RNAs, we determined the packaging efficiencies and specificities of genomic RNA, singly and fully spliced HIV mRNAs and different host RNAs species: 7SL RNA, U6 snRNA and GAPDH mRNA using RT-QPCR. Except GAPDH mRNA, all RNAs are selectively encapsidated. Singly spliced RNAs, harboring the Rev-responsible element, and fully spliced viral RNAs, which do not contain this motif, are enriched in virions to similar levels, even though they are exported from the nucleus by different routes. Deletions of key motifs (SL1 and/or SL3) of the packaging signal of genomic RNA indicate that HIV and host RNAs are encapsidated through independent mechanisms, while genomic and spliced viral RNA compete for the same trans-acting factor due to the presence of the 5' common exon containing the TAR, poly(A) and U5-PBS hairpins. Surprisingly, the RNA dimerization initiation site (DIS/SL1) appears to be the main packaging determinant of genomic RNA, but is not involved in packaging of spliced viral RNAs, suggesting a functional interaction with intronic sequences. Active and selective packaging of host and spliced viral RNAs provide new potential functions to these RNAs in the early stages of the virus life cycle.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. In order to determine the encapsidation mechanisms of these RNAs, we determined the packaging efficiencies and specificities of genomic RNA, singly and fully spliced HIV mRNAs and different host RNAs species: 7SL RNA, U6 snRNA and GAPDH mRNA using RT-QPCR. Except GAPDH mRNA, all RNAs are selectively encapsidated. Singly spliced RNAs, harboring the Rev-responsible element, and fully spliced viral RNAs, which do not contain this motif, are enriched in virions to similar levels, even though they are exported from the nucleus by different routes. Deletions of key motifs (SL1 and/or SL3) of the packaging signal of genomic RNA indicate that HIV and host RNAs are encapsidated through independent mechanisms, while genomic and spliced viral RNA compete for the same trans-acting factor due to the presence of the 5' common exon containing the TAR, poly(A) and U5-PBS hairpins. Surprisingly, the RNA dimerization initiation site (DIS/SL1) appears to be the main packaging determinant of genomic RNA, but is not involved in packaging of spliced viral RNAs, suggesting a functional interaction with intronic sequences. Active and selective packaging of host and spliced viral RNAs provide new potential functions to these RNAs in the early stages of the virus life cycle.
Bernacchi S, Henriet S, Dumas P, Paillart J C, Marquet R
RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study Journal Article
In: J Biol Chem, vol. 282, no. 36, pp. 26361-26368, 2007, ISBN: 17609216.
Abstract | Links | BibTeX | Tags: 5' Untranslated Regions/genetics/immunology/metabolism Binding Sites/physiology Cytidine Deaminase Cytosine Deaminase/immunology/metabolism DNA, gag/genetics/immunology/metabolism Gene Products, Human Immunodeficiency Virus, MARQUET, Natural/physiology Nucleoside Deaminases/immunology/metabolism Oligonucleotides/genetics/immunology/metabolism Protein Binding/physiology Protein Biosynthesis/physiology RNA, PAILLART, Unité ARN, vif/genetics/immunology/*metabolism Genome, Viral/genetics/immunology/*metabolism DNA-Binding Proteins/genetics/immunology/*metabolism Gene Products, Viral/genetics/immunology/*metabolism RNA-Binding Proteins/genetics/immunology/*metabolism Repressor Proteins/genetics/immunology/*metabolism vif Gene Products, Viral/physiology HIV Long Terminal Repeat/physiology HIV-1/genetics/immunology/*metabolism/pathogenicity Humans Immunity
@article{,
title = {RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study},
author = {S Bernacchi and S Henriet and P Dumas and J C Paillart and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17609216},
isbn = {17609216},
year = {2007},
date = {2007-01-01},
journal = {J Biol Chem},
volume = {282},
number = {36},
pages = {26361-26368},
abstract = {The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.},
keywords = {5' Untranslated Regions/genetics/immunology/metabolism Binding Sites/physiology Cytidine Deaminase Cytosine Deaminase/immunology/metabolism DNA, gag/genetics/immunology/metabolism Gene Products, Human Immunodeficiency Virus, MARQUET, Natural/physiology Nucleoside Deaminases/immunology/metabolism Oligonucleotides/genetics/immunology/metabolism Protein Binding/physiology Protein Biosynthesis/physiology RNA, PAILLART, Unité ARN, vif/genetics/immunology/*metabolism Genome, Viral/genetics/immunology/*metabolism DNA-Binding Proteins/genetics/immunology/*metabolism Gene Products, Viral/genetics/immunology/*metabolism RNA-Binding Proteins/genetics/immunology/*metabolism Repressor Proteins/genetics/immunology/*metabolism vif Gene Products, Viral/physiology HIV Long Terminal Repeat/physiology HIV-1/genetics/immunology/*metabolism/pathogenicity Humans Immunity},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.
Bodlenner A, Alix A, Weibel J M, Pale P, Ennifar E, Paillart J C, Walter P, Marquet R, Dumas P
Synthesis of a neamine dimer targeting the dimerization initiation site of HIV-1 RNA Journal Article
In: Org Lett, vol. 9, no. 22, pp. 4415-4418, 2007, ISBN: 17915882.
Abstract | Links | BibTeX | Tags: ENNIFAR, MARQUET, PAILLART, Unité ARN
@article{,
title = {Synthesis of a neamine dimer targeting the dimerization initiation site of HIV-1 RNA},
author = {A Bodlenner and A Alix and J M Weibel and P Pale and E Ennifar and J C Paillart and P Walter and R Marquet and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17915882},
isbn = {17915882},
year = {2007},
date = {2007-01-01},
journal = {Org Lett},
volume = {9},
number = {22},
pages = {4415-4418},
abstract = {A neamine dimer designed to bind to a specific sequence of HIV-1 RNA has been synthesized. Starting from neomycin B (1), a five-step synthesis efficiently provided a key protected neamine monomer 6 (28%). From the latter, coupling reactions with activated diacids gave dimers. After deprotection, a neamine dimer was obtained as the hexachlorohydrate salt 15 with 13% overall yield over nine steps.},
keywords = {ENNIFAR, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A neamine dimer designed to bind to a specific sequence of HIV-1 RNA has been synthesized. Starting from neomycin B (1), a five-step synthesis efficiently provided a key protected neamine monomer 6 (28%). From the latter, coupling reactions with activated diacids gave dimers. After deprotection, a neamine dimer was obtained as the hexachlorohydrate salt 15 with 13% overall yield over nine steps.
2006
Goldschmidt V, Didierjean J, Ehresmann B, Ehresmann C, Isel C, Marquet R
Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance Journal Article
In: Nucleic Acids Res, vol. 34, no. 1, pp. 42-52, 2006, ISBN: 16394022, (1362-4962 (Electronic) Journal Article).
Abstract | Links | BibTeX | Tags: Adenosine Triphosphate/pharmacology Anti-HIV Agents/*pharmacology DNA/biosynthesis DNA Primers DNA, Calf Thymus/metabolism Zidovudine/pharmacology, MARQUET, Non-U.S. Gov't Reverse Transcriptase Inhibitors/*pharmacology *Reverse Transcription/drug effects Ribonuclease H, PAILLART, Single-Stranded/biosynthesis Drug Resistance, Unité ARN, Viral HIV-1 Reverse Transcriptase/*metabolism Magnesium/*pharmacology Nucleosides/pharmacology Research Support
@article{,
title = {Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance},
author = {V Goldschmidt and J Didierjean and B Ehresmann and C Ehresmann and C Isel and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16394022},
isbn = {16394022},
year = {2006},
date = {2006-01-01},
journal = {Nucleic Acids Res},
volume = {34},
number = {1},
pages = {42-52},
abstract = {Metal ions are essential for DNA polymerase and RNase H activities of HIV-1 reverse transcriptase (RT). RT studies are routinely performed at 6-8 mM Mg2+, despite the fact that the in vivo concentration might be as low as 0.2 mM. We studied the influence of MgCl2 and ATP, which likely binds a significant fraction of the magnesium pool in vivo, on the DNA polymerase and RNase H activities of HIV-1 RT, its inhibition by nucleoside RT inhibitors (NRTIs) and primer unblocking by AZT-resistant RT. At low Mg2+ concentration, reverse transcription of a natural template strongly increased despite a dramatically reduced intrinsic polymerase activity under such conditions. Low Mg2+ concentrations affected the RNA stability and indirectly decreased its degradation by the RNase H activity. The reduced RNA degradation prevented premature dissociation of the template and primer strands that otherwise generated dead-end DNA products. In addition, low Mg2+ dramatically decreased the incorporation of NRTIs into DNA and increased nucleotide excision by AZT-resistant RT. The latter effect is also most likely owing to the diminished cleavage of the RNA template. Thus, differences in the free Mg2+ concentration between different cell types or during the cell cycle might strongly affect HIV-1 replication and its inhibition.},
note = {1362-4962 (Electronic)
Journal Article},
keywords = {Adenosine Triphosphate/pharmacology Anti-HIV Agents/*pharmacology DNA/biosynthesis DNA Primers DNA, Calf Thymus/metabolism Zidovudine/pharmacology, MARQUET, Non-U.S. Gov't Reverse Transcriptase Inhibitors/*pharmacology *Reverse Transcription/drug effects Ribonuclease H, PAILLART, Single-Stranded/biosynthesis Drug Resistance, Unité ARN, Viral HIV-1 Reverse Transcriptase/*metabolism Magnesium/*pharmacology Nucleosides/pharmacology Research Support},
pubstate = {published},
tppubtype = {article}
}
Metal ions are essential for DNA polymerase and RNase H activities of HIV-1 reverse transcriptase (RT). RT studies are routinely performed at 6-8 mM Mg2+, despite the fact that the in vivo concentration might be as low as 0.2 mM. We studied the influence of MgCl2 and ATP, which likely binds a significant fraction of the magnesium pool in vivo, on the DNA polymerase and RNase H activities of HIV-1 RT, its inhibition by nucleoside RT inhibitors (NRTIs) and primer unblocking by AZT-resistant RT. At low Mg2+ concentration, reverse transcription of a natural template strongly increased despite a dramatically reduced intrinsic polymerase activity under such conditions. Low Mg2+ concentrations affected the RNA stability and indirectly decreased its degradation by the RNase H activity. The reduced RNA degradation prevented premature dissociation of the template and primer strands that otherwise generated dead-end DNA products. In addition, low Mg2+ dramatically decreased the incorporation of NRTIs into DNA and increased nucleotide excision by AZT-resistant RT. The latter effect is also most likely owing to the diminished cleavage of the RNA template. Thus, differences in the free Mg2+ concentration between different cell types or during the cell cycle might strongly affect HIV-1 replication and its inhibition.
Vivet-Boudou V, Didierjean J, Isel C, Marquet R
Nucleoside and nucleotide inhibitors of HIV-1 replication Journal Article
In: Cell Mol Life Sci, vol. 63, no. 2, pp. 163-186, 2006, ISBN: 16389458, (1420-682X (Print) Journal Article Review).
Abstract | Links | BibTeX | Tags: Biological Mutation Nucleosides/chemistry/pharmacology Nucleotides/chemistry/pharmacology Protein Structure, Drug Resistance, MARQUET, PAILLART, Tertiary Reverse Transcriptase Inhibitors/*chemistry/*pharmacology Virus Replication, Unité ARN, Viral/*genetics HIV Infections/drug therapy HIV-1/drug effects/*enzymology/genetics Humans Models
@article{,
title = {Nucleoside and nucleotide inhibitors of HIV-1 replication},
author = {V Vivet-Boudou and J Didierjean and C Isel and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16389458},
isbn = {16389458},
year = {2006},
date = {2006-01-01},
journal = {Cell Mol Life Sci},
volume = {63},
number = {2},
pages = {163-186},
abstract = {HIV-1 reverse transcriptase (RT) is one of the main targets for antiviral therapy. Two classes of RT inhibitors can be distinguished: those that are nucleoside or nucleotide analogues (sharing the common NRTIs abbreviation) and those that are not. This review focuses on the NRTIs, which are highly efficient in slowing down viral replication and are used in combination regimens. Unfortunately, the current inhibitors do not completely suppress viral replication and due to the high capacity of adaptation of HIV, allow the selection of drug-resistant viruses. Resistance mechanisms to NRTIs have been extensively investigated and can be divided into two types: improved discrimination of a nucleotide analogue relative to the natural substrate or increased phosphorolytic cleavage of an analogue-blocked primer. This knowledge is important both for the development of new drugs designed to target resistant strains and for the development of new antiviral strategies. The NRTIs currently in clinical trials and new developments in this area are also reviewed.},
note = {1420-682X (Print)
Journal Article
Review},
keywords = {Biological Mutation Nucleosides/chemistry/pharmacology Nucleotides/chemistry/pharmacology Protein Structure, Drug Resistance, MARQUET, PAILLART, Tertiary Reverse Transcriptase Inhibitors/*chemistry/*pharmacology Virus Replication, Unité ARN, Viral/*genetics HIV Infections/drug therapy HIV-1/drug effects/*enzymology/genetics Humans Models},
pubstate = {published},
tppubtype = {article}
}
HIV-1 reverse transcriptase (RT) is one of the main targets for antiviral therapy. Two classes of RT inhibitors can be distinguished: those that are nucleoside or nucleotide analogues (sharing the common NRTIs abbreviation) and those that are not. This review focuses on the NRTIs, which are highly efficient in slowing down viral replication and are used in combination regimens. Unfortunately, the current inhibitors do not completely suppress viral replication and due to the high capacity of adaptation of HIV, allow the selection of drug-resistant viruses. Resistance mechanisms to NRTIs have been extensively investigated and can be divided into two types: improved discrimination of a nucleotide analogue relative to the natural substrate or increased phosphorolytic cleavage of an analogue-blocked primer. This knowledge is important both for the development of new drugs designed to target resistant strains and for the development of new antiviral strategies. The NRTIs currently in clinical trials and new developments in this area are also reviewed.
Ennifar E, Paillart J C, Bodlenner A, Walter P, Weibel J M, Aubertin A M, Pale P, Dumas P, Marquet R
Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell Journal Article
In: Nucleic Acids Research, vol. 34, no. 8, pp. 2328-39, 2006.
Abstract | Links | BibTeX | Tags: Aminoglycosides/*chemistry Anti-HIV Agents/*chemistry Binding Sites Cell Line Crystallography, ENNIFAR, MARQUET, Molecular RNA, Non-U.S. Gov't Virion/chemistry, PAILLART, Unité ARN, Viral/*chemistry Research Support, X-Ray Dimerization Drug Delivery Systems HIV-1/*genetics Humans Models
@article{Ennifar2006,
title = {Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell},
author = {E Ennifar and J C Paillart and A Bodlenner and P Walter and J M Weibel and A M Aubertin and P Pale and P Dumas and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16679451},
doi = {10.1093/nar/gkl317 },
year = {2006},
date = {2006-01-01},
journal = {Nucleic Acids Research},
volume = {34},
number = {8},
pages = {2328-39},
abstract = {The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility.},
keywords = {Aminoglycosides/*chemistry Anti-HIV Agents/*chemistry Binding Sites Cell Line Crystallography, ENNIFAR, MARQUET, Molecular RNA, Non-U.S. Gov't Virion/chemistry, PAILLART, Unité ARN, Viral/*chemistry Research Support, X-Ray Dimerization Drug Delivery Systems HIV-1/*genetics Humans Models},
pubstate = {published},
tppubtype = {article}
}
The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility.
2005
Henriet S, Richer D, Bernacchi S, Decroly E, Vigne R, Ehresmann B, Ehresmann C, Paillart J C, Marquet R
Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA Journal Article
In: J Mol Biol, vol. 354, no. 1, pp. 55-72, 2005, ISBN: 16236319, (0022-2836 (Print) Journal Article).
Abstract | Links | BibTeX | Tags: 5' Untranslated Regions/chemistry/*metabolism Base Sequence Electrophoretic Mobility Shift Assay Gene Products, MARQUET, Non-U.S. Gov't, PAILLART, Unité ARN, vif/*metabolism HIV Long Terminal Repeat HIV-1/*genetics/*metabolism Hela Cells Humans Molecular Sequence Data Nucleic Acid Conformation Protein Binding RNA, Viral/chemistry/*metabolism RNA-Binding Proteins/metabolism Recombinant Proteins/metabolism Research Support
@article{,
title = {Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA},
author = {S Henriet and D Richer and S Bernacchi and E Decroly and R Vigne and B Ehresmann and C Ehresmann and J C Paillart and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16236319},
isbn = {16236319},
year = {2005},
date = {2005-01-01},
journal = {J Mol Biol},
volume = {354},
number = {1},
pages = {55-72},
abstract = {The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.},
note = {0022-2836 (Print)
Journal Article},
keywords = {5' Untranslated Regions/chemistry/*metabolism Base Sequence Electrophoretic Mobility Shift Assay Gene Products, MARQUET, Non-U.S. Gov't, PAILLART, Unité ARN, vif/*metabolism HIV Long Terminal Repeat HIV-1/*genetics/*metabolism Hela Cells Humans Molecular Sequence Data Nucleic Acid Conformation Protein Binding RNA, Viral/chemistry/*metabolism RNA-Binding Proteins/metabolism Recombinant Proteins/metabolism Research Support},
pubstate = {published},
tppubtype = {article}
}
The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.
Gaied N Ben, Glasser N, Ramalanjaona N, Beltz H, Wolff P, Marquet R, Burger A, Mely Y
8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties Journal Article
In: Nucleic Acids Res, vol. 33, no. 3, pp. 1031-1039, 2005, ISBN: 15718302, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, MARQUET, PAILLART, Unité ARN
@article{,
title = {8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties},
author = {N Ben Gaied and N Glasser and N Ramalanjaona and H Beltz and P Wolff and R Marquet and A Burger and Y Mely},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15718302},
isbn = {15718302},
year = {2005},
date = {2005-01-01},
journal = {Nucleic Acids Res},
volume = {33},
number = {3},
pages = {1031-1039},
abstract = {We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.},
note = {1362-4962
Journal Article},
keywords = {ENNIFAR, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.
Bernacchi S, Ennifar E, Toth K, Walter P, Langowski J, Dumas P
Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site Journal Article
In: J Biol Chem, vol. 280, no. 48, pp. 40112-40121, 2005, ISBN: 16169845, (0021-9258 (Print) Journal Article).
Abstract | Links | BibTeX | Tags: Base Sequence Binding Sites Crystallography, Drug Electrophoresis Genome, ENNIFAR, Fluorescence Spectrophotometry Temperature Thermodynamics Time Factors Ultraviolet Rays Virus Replication, MARQUET, Non-U.S. Gov't Spectrometry, PAILLART, Unité ARN, Viral HIV-1/*chemistry Kinetics Molecular Sequence Data *Nucleic Acid Conformation Protein Binding RNA/chemistry RNA, Viral/*chemistry Research Support, X-Ray Dimerization Dose-Response Relationship
@article{,
title = {Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site},
author = {S Bernacchi and E Ennifar and K Toth and P Walter and J Langowski and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16169845},
isbn = {16169845},
year = {2005},
date = {2005-01-01},
journal = {J Biol Chem},
volume = {280},
number = {48},
pages = {40112-40121},
abstract = {We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.},
note = {0021-9258 (Print)
Journal Article},
keywords = {Base Sequence Binding Sites Crystallography, Drug Electrophoresis Genome, ENNIFAR, Fluorescence Spectrophotometry Temperature Thermodynamics Time Factors Ultraviolet Rays Virus Replication, MARQUET, Non-U.S. Gov't Spectrometry, PAILLART, Unité ARN, Viral HIV-1/*chemistry Kinetics Molecular Sequence Data *Nucleic Acid Conformation Protein Binding RNA/chemistry RNA, Viral/*chemistry Research Support, X-Ray Dimerization Dose-Response Relationship},
pubstate = {published},
tppubtype = {article}
}
We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.
2004
Paillart J C, Shehu-Xhilaga M, Marquet R, Mak J
Dimerization of retroviral RNA genomes: an inseparable pair Journal Article
In: Nat Rev Microbiol, vol. 2, no. 6, pp. 461-472, 2004, ISBN: 15152202, (1740-1526 Journal Article Review Review, Tutorial).
Links | BibTeX | Tags: Base Sequence Dimerization *Genome, MARQUET, Molecular Molecular Sequence Data Nucleic Acid Conformation Virus Replication/*genetics, PAILLART, Unité ARN, Viral HIV-1/*genetics/growth & development Human Models
@article{,
title = {Dimerization of retroviral RNA genomes: an inseparable pair},
author = {J C Paillart and M Shehu-Xhilaga and R Marquet and J Mak},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15152202},
isbn = {15152202},
year = {2004},
date = {2004-01-01},
journal = {Nat Rev Microbiol},
volume = {2},
number = {6},
pages = {461-472},
note = {1740-1526
Journal Article
Review
Review, Tutorial},
keywords = {Base Sequence Dimerization *Genome, MARQUET, Molecular Molecular Sequence Data Nucleic Acid Conformation Virus Replication/*genetics, PAILLART, Unité ARN, Viral HIV-1/*genetics/growth & development Human Models},
pubstate = {published},
tppubtype = {article}
}
Paillart J C, Dettenhofer M, Yu X F, Ehresmann C, Ehresmann B, Marquet R
First snapshots of the HIV-1 RNA structure in infected cells and in virions Journal Article
In: J Biol Chem, vol. 279, no. 46, pp. 48397-48403, 2004, ISBN: 15355993, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {First snapshots of the HIV-1 RNA structure in infected cells and in virions},
author = {J C Paillart and M Dettenhofer and X F Yu and C Ehresmann and B Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15355993},
isbn = {15355993},
year = {2004},
date = {2004-01-01},
journal = {J Biol Chem},
volume = {279},
number = {46},
pages = {48397-48403},
abstract = {With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5'-untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5'-untranslated region in infected cells points to the existence of the various stem-loop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA(3) (Lys) annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.},
note = {0021-9258
Journal Article},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5'-untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5'-untranslated region in infected cells points to the existence of the various stem-loop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA(3) (Lys) annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.
Goldschmidt V, Paillart J C, Rigourd M, Ehresmann B, Aubertin A M, Ehresmann C, Marquet R
Structural variability of the initiation complex of HIV-1 reverse transcription Journal Article
In: J Biol Chem, vol. 279, no. 34, pp. 35923-35931, 2004, ISBN: 15194685, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{,
title = {Structural variability of the initiation complex of HIV-1 reverse transcription},
author = {V Goldschmidt and J C Paillart and M Rigourd and B Ehresmann and A M Aubertin and C Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15194685},
isbn = {15194685},
year = {2004},
date = {2004-01-01},
journal = {J Biol Chem},
volume = {279},
number = {34},
pages = {35923-35931},
abstract = {HIV-1 reverse transcription is initiated from a tRNA(3)(Lys) molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA(3)(Lys) are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA(3)(Lys) annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA(3)(Lys) are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA(3)(Lys) annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA(His)). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.},
note = {0021-9258
Journal Article},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
HIV-1 reverse transcription is initiated from a tRNA(3)(Lys) molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA(3)(Lys) are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA(3)(Lys) annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA(3)(Lys) are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA(3)(Lys) annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA(His)). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.
2003
Ennifar E, Paillart J C, Marquet R, Ehresmann B, Ehresmann C, Dumas P, Walter P
HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics Journal Article
In: J Biol Chem, vol. 278, no. 4, pp. 2723-2730, 2003, ISBN: 12435744, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: Anti-Bacterial Agents/*pharmacology Binding Sites Dimerization HIV-1/*metabolism Models, ENNIFAR, MARQUET, Molecular Neomycin/pharmacology Nucleic Acid Conformation Paromomycin/pharmacology Protein Binding RNA/metabolism *RNA, Non-U.S. Gov't Temperature Ultraviolet Rays, PAILLART, Unité ARN, Viral Ribosomes/*metabolism Support
@article{,
title = {HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics},
author = {E Ennifar and J C Paillart and R Marquet and B Ehresmann and C Ehresmann and P Dumas and P Walter},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12435744},
isbn = {12435744},
year = {2003},
date = {2003-01-01},
journal = {J Biol Chem},
volume = {278},
number = {4},
pages = {2723-2730},
abstract = {Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.},
note = {0021-9258
Journal Article},
keywords = {Anti-Bacterial Agents/*pharmacology Binding Sites Dimerization HIV-1/*metabolism Models, ENNIFAR, MARQUET, Molecular Neomycin/pharmacology Nucleic Acid Conformation Paromomycin/pharmacology Protein Binding RNA/metabolism *RNA, Non-U.S. Gov't Temperature Ultraviolet Rays, PAILLART, Unité ARN, Viral Ribosomes/*metabolism Support},
pubstate = {published},
tppubtype = {article}
}
Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.
2002
Paillart J C, Skripkin E, Ehresmann B, Ehresmann C, Marquet R
In vitro evidence for a long range pseudoknot in the 5'-untranslated and matrix coding regions of HIV-1 genomic RNA Journal Article
In: J Biol Chem, vol. 277, no. 8, pp. 5995-6004, 2002, ISBN: 11744696, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: 5' Untranslated Regions/*genetics Base Sequence Dimerization HIV-1/*genetics Human Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Oligodeoxyribonucleotides, Antisense/pharmacology Poly A/chemistry/genetics RNA, MARQUET, Non-U.S. Gov't Virus Replication/genetics, Nucleic Acid Support, PAILLART, Unité ARN, Viral/chemistry/drug effects/*genetics Sequence Alignment Sequence Homology
@article{,
title = {In vitro evidence for a long range pseudoknot in the 5'-untranslated and matrix coding regions of HIV-1 genomic RNA},
author = {J C Paillart and E Skripkin and B Ehresmann and C Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11744696},
isbn = {11744696},
year = {2002},
date = {2002-01-01},
journal = {J Biol Chem},
volume = {277},
number = {8},
pages = {5995-6004},
abstract = {The 5'-untranslated leader region of human immunodeficiency virus type 1 (HIV-1) RNA contains multiple signals that control distinct steps of the viral replication cycle such as transcription, reverse transcription, genomic RNA dimerization, splicing, and packaging. It is likely that fine tuned coordinated regulation of these functions is achieved through specific RNA-protein and RNA-RNA interactions. In a search for cis-acting elements important for the tertiary structure of the 5'-untranslated region of HIV-1 genomic RNA, we identified, by ladder selection experiments, a short stretch of nucleotides directly downstream of the poly(A) signal that interacts with a nucleotide sequence located in the matrix region. Confirmation of the sequence of the interacting sites was obtained by partial or complete inhibition of this interaction by antisense oligonucleotides and by nucleotide substitutions. In the wild type RNA, this long range interaction was intramolecular, since no intermolecular RNA association was detected by gel electrophoresis with an RNA mutated in the dimerization initiation site and containing both sequences involved in the tertiary interaction. Moreover, the functional importance of this interaction is supported by its conservation in all HIV-1 isolates as well as in HIV-2 and simian immunodeficiency virus. Our results raise the possibility that this long range RNA-RNA interaction might be involved in the full-length genomic RNA selection during packaging, repression of the 5' polyadenylation signal, and/or splicing regulation.},
note = {0021-9258
Journal Article},
keywords = {5' Untranslated Regions/*genetics Base Sequence Dimerization HIV-1/*genetics Human Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Oligodeoxyribonucleotides, Antisense/pharmacology Poly A/chemistry/genetics RNA, MARQUET, Non-U.S. Gov't Virus Replication/genetics, Nucleic Acid Support, PAILLART, Unité ARN, Viral/chemistry/drug effects/*genetics Sequence Alignment Sequence Homology},
pubstate = {published},
tppubtype = {article}
}
The 5'-untranslated leader region of human immunodeficiency virus type 1 (HIV-1) RNA contains multiple signals that control distinct steps of the viral replication cycle such as transcription, reverse transcription, genomic RNA dimerization, splicing, and packaging. It is likely that fine tuned coordinated regulation of these functions is achieved through specific RNA-protein and RNA-RNA interactions. In a search for cis-acting elements important for the tertiary structure of the 5'-untranslated region of HIV-1 genomic RNA, we identified, by ladder selection experiments, a short stretch of nucleotides directly downstream of the poly(A) signal that interacts with a nucleotide sequence located in the matrix region. Confirmation of the sequence of the interacting sites was obtained by partial or complete inhibition of this interaction by antisense oligonucleotides and by nucleotide substitutions. In the wild type RNA, this long range interaction was intramolecular, since no intermolecular RNA association was detected by gel electrophoresis with an RNA mutated in the dimerization initiation site and containing both sequences involved in the tertiary interaction. Moreover, the functional importance of this interaction is supported by its conservation in all HIV-1 isolates as well as in HIV-2 and simian immunodeficiency virus. Our results raise the possibility that this long range RNA-RNA interaction might be involved in the full-length genomic RNA selection during packaging, repression of the 5' polyadenylation signal, and/or splicing regulation.
1997
Paillart J C, Westhof E, Ehresmann C, Ehresmann B, Marquet R
Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA Journal Article
In: J Mol Biol, vol. 270, no. 1, pp. 36-49, 1997, ISBN: 9231899, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Dimerization HIV-1/*genetics Kinetics Models, MARQUET, Molecular Mutagenesis, Non-U.S. Gov't, PAILLART, Site-Directed Nucleic Acid Conformation Purines/chemistry RNA, Unité ARN, Viral/*chemistry/genetics/*metabolism Support
@article{,
title = {Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA},
author = {J C Paillart and E Westhof and C Ehresmann and B Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9231899},
isbn = {9231899},
year = {1997},
date = {1997-01-01},
journal = {J Mol Biol},
volume = {270},
number = {1},
pages = {36-49},
abstract = {Retroviruses encapsidate two molecules of genomic RNA that are noncovalently linked close to their 5' ends in a region called the dimer linkage structure (DLS). The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) constitutes the essential part of the DLS in vitro and is crucial for efficient HIV-1 replication in cell culture. We previously identified the DIS as a hairpin structure, located upstream of the major splice donor site, that contains in the loop a six-nucleotide self-complementary sequence preceded and followed by two and one purines, respectively. Two RNA monomers form a kissing loop complex via intermolecular interactions of the six nucleotide self-complementary sequence. Here, we introduced compensatory mutations in the self-complementary sequence and/or a mutation in the flanking purines. We determined the kinetics of dimerization, the thermal stabilities and the apparent equilibrium dissociation constants of wild-type and mutant dimers and used chemical probing to obtain structural information. Our results demonstrate the importance of the 5'-flanking purine and of the two central bases of the self-complementary sequence in the dimerization process. The experimental data are rationalized by triple interactions between these residues in the deep groove of the kissing helix and are incorporated into a three-dimensional model of the kissing loop dimer. In addition, chemical probing and molecular modeling favor the existence of a non-canonical interaction between the conserved adenine residues at the first and last positions in the DIS loop. Furthermore, we show that destabilization of the kissing loop complex at the DIS can be compensated by interactions involving sequences located downstream of the splice donor site of the HIV-1 genomic RNA.},
note = {0022-2836
Journal Article},
keywords = {Dimerization HIV-1/*genetics Kinetics Models, MARQUET, Molecular Mutagenesis, Non-U.S. Gov't, PAILLART, Site-Directed Nucleic Acid Conformation Purines/chemistry RNA, Unité ARN, Viral/*chemistry/genetics/*metabolism Support},
pubstate = {published},
tppubtype = {article}
}
Retroviruses encapsidate two molecules of genomic RNA that are noncovalently linked close to their 5' ends in a region called the dimer linkage structure (DLS). The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) constitutes the essential part of the DLS in vitro and is crucial for efficient HIV-1 replication in cell culture. We previously identified the DIS as a hairpin structure, located upstream of the major splice donor site, that contains in the loop a six-nucleotide self-complementary sequence preceded and followed by two and one purines, respectively. Two RNA monomers form a kissing loop complex via intermolecular interactions of the six nucleotide self-complementary sequence. Here, we introduced compensatory mutations in the self-complementary sequence and/or a mutation in the flanking purines. We determined the kinetics of dimerization, the thermal stabilities and the apparent equilibrium dissociation constants of wild-type and mutant dimers and used chemical probing to obtain structural information. Our results demonstrate the importance of the 5'-flanking purine and of the two central bases of the self-complementary sequence in the dimerization process. The experimental data are rationalized by triple interactions between these residues in the deep groove of the kissing helix and are incorporated into a three-dimensional model of the kissing loop dimer. In addition, chemical probing and molecular modeling favor the existence of a non-canonical interaction between the conserved adenine residues at the first and last positions in the DIS loop. Furthermore, we show that destabilization of the kissing loop complex at the DIS can be compensated by interactions involving sequences located downstream of the splice donor site of the HIV-1 genomic RNA.
1996
Skripkin E, Paillart J C, Marquet R, Blumenfeld M, Ehresmann B, Ehresmann C
Mechanisms of inhibition of in vitro dimerization of HIV type I RNA by sense and antisense oligonucleotides Journal Article
In: J Biol Chem, vol. 271, no. 46, pp. 28812-28817, 1996, ISBN: 8910525, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: Antisense/*pharmacology RNA, Biopolymers HIV-1/*genetics Nucleic Acid Conformation Oligonucleotides/*pharmacology Oligonucleotides, MARQUET, Non-U.S. Gov't, PAILLART, Unité ARN, Viral/*antagonists & inhibitors/chemistry Support
@article{,
title = {Mechanisms of inhibition of in vitro dimerization of HIV type I RNA by sense and antisense oligonucleotides},
author = {E Skripkin and J C Paillart and R Marquet and M Blumenfeld and B Ehresmann and C Ehresmann},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8910525},
isbn = {8910525},
year = {1996},
date = {1996-01-01},
journal = {J Biol Chem},
volume = {271},
number = {46},
pages = {28812-28817},
abstract = {Retroviruses display a strong selective pressure to maintain the dimeric nature of their genomic RNAs, suggesting that dimerization is essential for viral replication. Recently, we identified the cis-element required for initiation of human immunodeficiency virus type I (HIV-I) RNA dimerization in vitro. The dimerization initiation site (DIS) is a hairpin structure containing a self-complementary sequence in the loop. We proposed that dimerization is initiated by a loop-loop kissing interaction involving the self-complementary sequence present in each monomer. We tested the ability of sense and antisense oligonucleotides targeted against the DIS to interfere with a preformed viral RNA dimer. Self-dimerization and inhibition properties of the tested oligonucleotides are dictated by the nature of the loop. An RNA loop is absolutely required in the case of sense oligonucleotides, whereas the nature and the sequence of the stem is not important. They form reversible loop-loop interactions and act as competitive inhibitors. Antisense oligonucleotides are less efficient in self-dimerization and are more potent inhibitors than sense oligonucleotides. They are less sensitive to the nature of the loop than the antisense oligonucleotides. Antisense hairpins with either RNA or DNA stems are able to form highly stable and irreversible complexes with viral RNA, resulting from complete extension of base pairing initiated by loop-loop interaction.},
note = {0021-9258
Journal Article},
keywords = {Antisense/*pharmacology RNA, Biopolymers HIV-1/*genetics Nucleic Acid Conformation Oligonucleotides/*pharmacology Oligonucleotides, MARQUET, Non-U.S. Gov't, PAILLART, Unité ARN, Viral/*antagonists & inhibitors/chemistry Support},
pubstate = {published},
tppubtype = {article}
}
Retroviruses display a strong selective pressure to maintain the dimeric nature of their genomic RNAs, suggesting that dimerization is essential for viral replication. Recently, we identified the cis-element required for initiation of human immunodeficiency virus type I (HIV-I) RNA dimerization in vitro. The dimerization initiation site (DIS) is a hairpin structure containing a self-complementary sequence in the loop. We proposed that dimerization is initiated by a loop-loop kissing interaction involving the self-complementary sequence present in each monomer. We tested the ability of sense and antisense oligonucleotides targeted against the DIS to interfere with a preformed viral RNA dimer. Self-dimerization and inhibition properties of the tested oligonucleotides are dictated by the nature of the loop. An RNA loop is absolutely required in the case of sense oligonucleotides, whereas the nature and the sequence of the stem is not important. They form reversible loop-loop interactions and act as competitive inhibitors. Antisense oligonucleotides are less efficient in self-dimerization and are more potent inhibitors than sense oligonucleotides. They are less sensitive to the nature of the loop than the antisense oligonucleotides. Antisense hairpins with either RNA or DNA stems are able to form highly stable and irreversible complexes with viral RNA, resulting from complete extension of base pairing initiated by loop-loop interaction.
Paillart J C, Skripkin E, Ehresmann B, Ehresmann C, Marquet R
A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA Journal Article
In: Proc Natl Acad Sci U S A, vol. 93, no. 11, pp. 5572-5577, 1996, ISBN: 8643617, (0027-8424 Journal Article).
Abstract | Links | BibTeX | Tags: Base Composition Base Sequence HIV-1/*genetics Heat Human Kinetics Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Nucleic Acid Denaturation Plasmids RNA, Genetic, MARQUET, Non-U.S. Gov't Thermodynamics Transcription, PAILLART, Unité ARN, Viral/biosynthesis/*chemistry/*metabolism Support
@article{,
title = {A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA},
author = {J C Paillart and E Skripkin and B Ehresmann and C Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8643617},
isbn = {8643617},
year = {1996},
date = {1996-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {93},
number = {11},
pages = {5572-5577},
abstract = {RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.},
note = {0027-8424
Journal Article},
keywords = {Base Composition Base Sequence HIV-1/*genetics Heat Human Kinetics Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Nucleic Acid Denaturation Plasmids RNA, Genetic, MARQUET, Non-U.S. Gov't Thermodynamics Transcription, PAILLART, Unité ARN, Viral/biosynthesis/*chemistry/*metabolism Support},
pubstate = {published},
tppubtype = {article}
}
RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.
Paillart J C, Skripkin E, Ehresmann B, Ehresmann C, Marquet R
The use of chemical modification interference and inverse PCR mutagenesis to identify the dimerization initiation site of HIV-1 genomic RNA Journal Article
In: Pharm Acta Helv, vol. 71, no. 1, pp. 21-28, 1996, ISBN: 8786995, (0031-6865 Journal Article).
Abstract | Links | BibTeX | Tags: Base Sequence *Genome, MARQUET, Non-U.S. Gov't, PAILLART, Site-Directed Polymerase Chain Reaction RNA, Unité ARN, Viral HIV-1/*chemistry Human Molecular Sequence Data Mutagenesis, Viral/*chemistry Support
@article{,
title = {The use of chemical modification interference and inverse PCR mutagenesis to identify the dimerization initiation site of HIV-1 genomic RNA},
author = {J C Paillart and E Skripkin and B Ehresmann and C Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8786995},
isbn = {8786995},
year = {1996},
date = {1996-01-01},
journal = {Pharm Acta Helv},
volume = {71},
number = {1},
pages = {21-28},
abstract = {The retroviral genome consists of two identical RNA molecules physically linked together close to their 5' end, in a region called the Dimer Linkage Structure (DLS). Recent findings suggest that dimerization is involved in encapsidation, regulation of translation and reverse transcription. Previous in vitro studies localized the DLS of HIV-1 in a region downstream of the splice donor (SD) site. More recently, we showed that dimerization of HIV-1 RNA also involves sequences upstream of the SD site. Modification interference experiments and site-directed mutagenesis were used to identify the nucleotides required in the dimerization process of HIV-1 RNA. Our results point out a self-complementary sequence located in a hairpin loop, between the Primer Binding Site (PBS) and the SD site, as the Dimerization Initiation Site.},
note = {0031-6865
Journal Article},
keywords = {Base Sequence *Genome, MARQUET, Non-U.S. Gov't, PAILLART, Site-Directed Polymerase Chain Reaction RNA, Unité ARN, Viral HIV-1/*chemistry Human Molecular Sequence Data Mutagenesis, Viral/*chemistry Support},
pubstate = {published},
tppubtype = {article}
}
The retroviral genome consists of two identical RNA molecules physically linked together close to their 5' end, in a region called the Dimer Linkage Structure (DLS). Recent findings suggest that dimerization is involved in encapsidation, regulation of translation and reverse transcription. Previous in vitro studies localized the DLS of HIV-1 in a region downstream of the splice donor (SD) site. More recently, we showed that dimerization of HIV-1 RNA also involves sequences upstream of the SD site. Modification interference experiments and site-directed mutagenesis were used to identify the nucleotides required in the dimerization process of HIV-1 RNA. Our results point out a self-complementary sequence located in a hairpin loop, between the Primer Binding Site (PBS) and the SD site, as the Dimerization Initiation Site.
Paillart J C, Marquet R, Skripkin E, Ehresmann C, Ehresmann B
Dimerization of retroviral genomic RNAs: structural and functional implications Journal Article
In: Biochimie, vol. 78, no. 7, pp. 639-653, 1996, ISBN: 8955907, (0300-9084 Journal Article Review Review, Academic).
Abstract | Links | BibTeX | Tags: Animals Base Sequence HIV-1/genetics Human Microscopy, DNA Support, Electron Molecular Sequence Data *Nucleic Acid Conformation RNA, MARQUET, Non-U.S. Gov't, PAILLART, Unité ARN, Viral/*chemistry/metabolism Rats Retroviridae/*genetics Sequence Analysis
@article{,
title = {Dimerization of retroviral genomic RNAs: structural and functional implications},
author = {J C Paillart and R Marquet and E Skripkin and C Ehresmann and B Ehresmann},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8955907},
isbn = {8955907},
year = {1996},
date = {1996-01-01},
journal = {Biochimie},
volume = {78},
number = {7},
pages = {639-653},
abstract = {Retroviruses are a family of widespread small animal viruses at the origin of a diversity of diseases. They share common structural and functional properties such as reverse transcription of their RNA genome and integration of the proviral DNA into the host genome, and have the particularity of packaging a diploid genome. The genome of all retroviruses is composed of two homologous RNA molecules that are non-covalently linked near their 5' end in a region called the dimer linkage structure (DLS). There is now considerable evidence that a specific site (or sites) in the 5' leader region of all retroviruses, located either upstream or/and downstream of the major splice donor site, is involved in the dimer linkage. For MoMuLV and especially HIV-1, it was shown that dimerization is initiated at a stem-loop structure named the dimerization initiation site (DIS). The DIS of HIV-1 and related regions in other retroviruses corresponds to a highly conserved structure with a self-complementary loop sequence, that is involved in a typical loop-loop 'kissing' complex which can be further stabilized by long distance interactions or by conformational rearrangements. RNA interactions involved in the viral RNA dimer were postulated to regulate several key steps in retroviral cycle, such as: i) translation and encapsidation: the arrest of gag translation imposed by the highly structured DLS-encapsidation signal would leave the RNA genome available for the encapsidation machinery; and ii) recombination during reverse transcription: the presence of two RNA molecules in particles would be necessary for variability and viability of virus progeny and the ordered structure imposed by the DLS would be required for efficient reverse transcription.},
note = {0300-9084
Journal Article
Review
Review, Academic},
keywords = {Animals Base Sequence HIV-1/genetics Human Microscopy, DNA Support, Electron Molecular Sequence Data *Nucleic Acid Conformation RNA, MARQUET, Non-U.S. Gov't, PAILLART, Unité ARN, Viral/*chemistry/metabolism Rats Retroviridae/*genetics Sequence Analysis},
pubstate = {published},
tppubtype = {article}
}
Retroviruses are a family of widespread small animal viruses at the origin of a diversity of diseases. They share common structural and functional properties such as reverse transcription of their RNA genome and integration of the proviral DNA into the host genome, and have the particularity of packaging a diploid genome. The genome of all retroviruses is composed of two homologous RNA molecules that are non-covalently linked near their 5' end in a region called the dimer linkage structure (DLS). There is now considerable evidence that a specific site (or sites) in the 5' leader region of all retroviruses, located either upstream or/and downstream of the major splice donor site, is involved in the dimer linkage. For MoMuLV and especially HIV-1, it was shown that dimerization is initiated at a stem-loop structure named the dimerization initiation site (DIS). The DIS of HIV-1 and related regions in other retroviruses corresponds to a highly conserved structure with a self-complementary loop sequence, that is involved in a typical loop-loop 'kissing' complex which can be further stabilized by long distance interactions or by conformational rearrangements. RNA interactions involved in the viral RNA dimer were postulated to regulate several key steps in retroviral cycle, such as: i) translation and encapsidation: the arrest of gag translation imposed by the highly structured DLS-encapsidation signal would leave the RNA genome available for the encapsidation machinery; and ii) recombination during reverse transcription: the presence of two RNA molecules in particles would be necessary for variability and viability of virus progeny and the ordered structure imposed by the DLS would be required for efficient reverse transcription.
Paillart J C, Berthoux L, Ottmann M, Darlix J L, Marquet R, Ehresmann B, Ehresmann C
In: J Virol, vol. 70, no. 12, pp. 8348-8354, 1996, ISBN: 8970954, (0022-538x Journal Article).
Abstract | Links | BibTeX | Tags: Animals COS Cells DNA, Genetic Virion *Virus Assembly, MARQUET, Non-U.S. Gov't Transcription, Nucleic Acid Support, PAILLART, Post-Translational Proviruses/genetics *RNA, Unité ARN, Viral *Regulatory Sequences, Viral HIV-1/*genetics/physiology Human Mutagenesis Protein Processing, Viral/*biosynthesis Gene Expression Genome
@article{,
title = {A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis},
author = {J C Paillart and L Berthoux and M Ottmann and J L Darlix and R Marquet and B Ehresmann and C Ehresmann},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8970954},
isbn = {8970954},
year = {1996},
date = {1996-01-01},
journal = {J Virol},
volume = {70},
number = {12},
pages = {8348-8354},
abstract = {In retroviruses, the genomic RNA is in the form of a 60S-70S complex composed of two identical genome-length RNA molecules tightly associated through numerous interactions. A major interaction, called the dimer linkage structure, has been found near the RNA 5' end and is probably involved in the control of translation, packaging, and recombination during proviral DNA synthesis. Recently, a small sequence corresponding to a stem-loop structure located in the 5' leader of human immunodeficiency virus type 1 (HIV-1) RNA was found to be required for the initiation of HIV-1 RNA dimerization in vitro and named the dimerization initiation site (E. Skripkin, J.-C. Paillart, R. Marquet, B. Ehresmann, and C. Ehresmann, Proc. Natl. Acad. Sci. USA 91: 4945-4949, 1994). To investigate the possible role of this 5' stem-loop in HIV-1 virion formation and infectivity, four mutant viruses were generated and analyzed in vivo. Results show that deletion of the stem-loop structure reduces infectivity by a factor of 10(3) whereas loop substitutions cause a decrease of 10- to 100-fold. The level of genomic RNA packaging was found to be decreased fivefold in mutants virions containing the stem-loop deletion and only twofold in the loop-substituted virions. Surprisingly, the second DNA strand transfer during reverse transcription was found to be severely impaired upon stem-loop deletion. Taken together, these results indicate that the stem-loop structure called the dimerization initiation site is a cis element acting on both genomic RNA packaging and synthesis of proviral DNA.},
note = {0022-538x
Journal Article},
keywords = {Animals COS Cells DNA, Genetic Virion *Virus Assembly, MARQUET, Non-U.S. Gov't Transcription, Nucleic Acid Support, PAILLART, Post-Translational Proviruses/genetics *RNA, Unité ARN, Viral *Regulatory Sequences, Viral HIV-1/*genetics/physiology Human Mutagenesis Protein Processing, Viral/*biosynthesis Gene Expression Genome},
pubstate = {published},
tppubtype = {article}
}
In retroviruses, the genomic RNA is in the form of a 60S-70S complex composed of two identical genome-length RNA molecules tightly associated through numerous interactions. A major interaction, called the dimer linkage structure, has been found near the RNA 5' end and is probably involved in the control of translation, packaging, and recombination during proviral DNA synthesis. Recently, a small sequence corresponding to a stem-loop structure located in the 5' leader of human immunodeficiency virus type 1 (HIV-1) RNA was found to be required for the initiation of HIV-1 RNA dimerization in vitro and named the dimerization initiation site (E. Skripkin, J.-C. Paillart, R. Marquet, B. Ehresmann, and C. Ehresmann, Proc. Natl. Acad. Sci. USA 91: 4945-4949, 1994). To investigate the possible role of this 5' stem-loop in HIV-1 virion formation and infectivity, four mutant viruses were generated and analyzed in vivo. Results show that deletion of the stem-loop structure reduces infectivity by a factor of 10(3) whereas loop substitutions cause a decrease of 10- to 100-fold. The level of genomic RNA packaging was found to be decreased fivefold in mutants virions containing the stem-loop deletion and only twofold in the loop-substituted virions. Surprisingly, the second DNA strand transfer during reverse transcription was found to be severely impaired upon stem-loop deletion. Taken together, these results indicate that the stem-loop structure called the dimerization initiation site is a cis element acting on both genomic RNA packaging and synthesis of proviral DNA.
Marquet R, Paillart J C, Skripkin E, Ehresmann C, Ehresmann B
Localization of the dimerization initiation site of HIV-1 genomic RNA and mechanism of dimerization. Book Chapter
In: Sarma, R H; Sarma, M H (Ed.): Biological Structure and Dynamics: Proceedings of the Ninth Conversation in the Discipline Biomolecular Stereodynamics, held at the State University of New York at Albany, June 20-24, 1995, vol. 2, pp. 61-72, Adenine Press, 1996.
Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@inbook{,
title = {Localization of the dimerization initiation site of HIV-1 genomic RNA and mechanism of dimerization.},
author = {R Marquet and J C Paillart and E Skripkin and C Ehresmann and B Ehresmann},
editor = {R H Sarma and M H Sarma},
url = {http://books.google.fr/books?hl=fr&id=SOpqAAAAMAAJ&q=Marquet#search_anchor},
year = {1996},
date = {1996-01-01},
booktitle = {Biological Structure and Dynamics: Proceedings of the Ninth Conversation in the Discipline Biomolecular Stereodynamics, held at the State University of New York at Albany, June 20-24, 1995},
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publisher = {Adenine Press},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
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}
Paillart J C, Skripkin E, Ehresmann B, Ehresmann C, Marquet R
A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA Journal Article
In: Proc Natl Acad Sci U S A, vol. 93, no. 11, pp. 5572-5577, 1996, ISBN: 8643617, (0027-8424 Journal Article).
Abstract | Links | BibTeX | Tags: Base Composition Base Sequence HIV-1/*genetics Heat Human Kinetics Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Nucleic Acid Denaturation Plasmids RNA, Genetic, MARQUET, Non-U.S. Gov't Thermodynamics Transcription, PAILLART, Unité ARN, Viral/biosynthesis/*chemistry/*metabolism Support
@article{,
title = {A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA},
author = {J C Paillart and E Skripkin and B Ehresmann and C Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8643617},
isbn = {8643617},
year = {1996},
date = {1996-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {93},
number = {11},
pages = {5572-5577},
abstract = {RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.},
note = {0027-8424
Journal Article},
keywords = {Base Composition Base Sequence HIV-1/*genetics Heat Human Kinetics Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Nucleic Acid Denaturation Plasmids RNA, Genetic, MARQUET, Non-U.S. Gov't Thermodynamics Transcription, PAILLART, Unité ARN, Viral/biosynthesis/*chemistry/*metabolism Support},
pubstate = {published},
tppubtype = {article}
}
RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.
