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2024
Zoladek J., Kazzi P. El, Caval V., Vivet-Boudou V., Cannac M., Davies E., Rossi S., Bribes I., Rouilly L., Simonin Y., Jouvenet N., Decroly E., Paillart J. -C., Wilson S. J., Nisole S.
A specific domain within the 3' untranslated region of Usutu virus confers resistance to the exonuclease ISG20 Article de journal
Dans: Nature Communications, vol. 15, p. 8528 , 2024.
Résumé | Liens | BibTeX | Étiquettes: PAILLART, Unité ARN, vivet-boudou
@article{nokey,
title = {A specific domain within the 3' untranslated region of Usutu virus confers resistance to the exonuclease ISG20},
author = {J. Zoladek AND P. El Kazzi AND V. Caval AND V. Vivet-Boudou AND M. Cannac AND E. Davies AND S. Rossi AND I. Bribes AND L. Rouilly AND Y. Simonin AND N. Jouvenet AND E. Decroly AND J.-C. Paillart AND S. J. Wilson AND S. Nisole},
editor = {NPG},
doi = {10.1038/s41467-024-52870-w},
year = {2024},
date = {2024-10-02},
urldate = {2024-10-02},
journal = {Nature Communications},
volume = {15},
pages = {8528 },
abstract = {Usutu virus (USUV) and West Nile virus (WNV) are two closely related emergingmosquito-borneflaviviruses. Their natural hosts are wild birds, but they canalso cause severe neurological disorders in humans. Both viruses are efficientlysuppressed by type I interferon (IFN), which interferes with viral replication,dissemination, pathogenesis and transmission. Here, we show that the repli-cation of USUV and WNV are inhibited through a common set of IFN–inducedgenes (ISGs), with the notable exception of ISG20, which USUV is resistant to.Strikingly, USUV was the only virus among all the other tested mosquito-borneflaviviruses that demonstrated resistance to the 3′–5′exonuclease activity ofISG20. Ourfindings highlight that the intrinsic resistance of the USUV genome,irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a uniquesequence present in its3′untranslated region. Importantly,this genomic region alone can conferISG20 resistance to a susceptibleflavivirus, without compromising its infec-tivity, suggesting that it could be acquired by otherflaviviruses. This studyprovides new insights into the strategy employed by emergingflaviviruses toovercome host defense mechanisms.},
keywords = {PAILLART, Unité ARN, vivet-boudou},
pubstate = {published},
tppubtype = {article}
}
Usutu virus (USUV) and West Nile virus (WNV) are two closely related emergingmosquito-borneflaviviruses. Their natural hosts are wild birds, but they canalso cause severe neurological disorders in humans. Both viruses are efficientlysuppressed by type I interferon (IFN), which interferes with viral replication,dissemination, pathogenesis and transmission. Here, we show that the repli-cation of USUV and WNV are inhibited through a common set of IFN–inducedgenes (ISGs), with the notable exception of ISG20, which USUV is resistant to.Strikingly, USUV was the only virus among all the other tested mosquito-borneflaviviruses that demonstrated resistance to the 3′–5′exonuclease activity ofISG20. Ourfindings highlight that the intrinsic resistance of the USUV genome,irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a uniquesequence present in its3′untranslated region. Importantly,this genomic region alone can conferISG20 resistance to a susceptibleflavivirus, without compromising its infec-tivity, suggesting that it could be acquired by otherflaviviruses. This studyprovides new insights into the strategy employed by emergingflaviviruses toovercome host defense mechanisms.
Tajer L., Paillart J. -C., Dib H., Sabatier J. -M., Fajloun Z., Khattar Z. Abi
Molecular Mechanisms of Bacterial Resistance to Antimicrobial Peptides in the Modern Era: An Updated Review Article de journal
Dans: Microorganisms, vol. 12, p. 1259, 2024.
Résumé | Liens | BibTeX | Étiquettes: marquet Paillart, PAILLART, Unité ARN
@article{nokey,
title = {Molecular Mechanisms of Bacterial Resistance to Antimicrobial Peptides in the Modern Era: An Updated Review},
author = {L. Tajer and J.-C. Paillart and H. Dib and J.-M. Sabatier and Z. Fajloun and Z. Abi Khattar},
url = {https://www.mdpi.com/2076-2607/12/7/1259},
doi = {10.3390/ microorganisms12071259},
year = {2024},
date = {2024-06-21},
urldate = {2024-06-21},
journal = {Microorganisms},
volume = {12},
pages = {1259},
abstract = {Antimicrobial resistance (AMR) poses a serious global health concern, resulting in a
significant number of deaths annually due to infections that are resistant to treatment. Amidst
this crisis, antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional
antibiotics (ATBs). These cationic peptides, naturally produced by all kingdoms of life, play a
crucial role in the innate immune system of multicellular organisms and in bacterial interspecies
competition by exhibiting broad-spectrum activity against bacteria, fungi, viruses, and parasites.
AMPs target bacterial pathogens through multiple mechanisms, most importantly by disrupting
their membranes, leading to cell lysis. However, bacterial resistance to host AMPs has emerged
due to a slow co-evolutionary process between microorganisms and their hosts. Alarmingly, the
development of resistance to last-resort AMPs in the treatment of MDR infections, such as colistin, is
attributed to the misuse of this and the high rate of horizontal genetic transfer of the corresponding
resistance genes. AMP-resistant bacteria employ diverse mechanisms, including but not limited to
proteolytic degradation, extracellular trapping and inactivation, active efflux, as well as complex
modifications in bacterial cell wall and membrane structures. This review comprehensively examines
all constitutive and inducible molecular resistance mechanisms to AMPs supported by experimental
evidence described to date in bacterial pathogens. We also explore the specificity of these mechanisms
toward structurally diverse AMPs to broaden and enhance their potential in developing and applying
them as therapeutics for MDR bacteria. Additionally, we provide insights into the significance of
AMP resistance within the context of host–pathogen interactions.},
keywords = {marquet Paillart, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Antimicrobial resistance (AMR) poses a serious global health concern, resulting in a
significant number of deaths annually due to infections that are resistant to treatment. Amidst
this crisis, antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional
antibiotics (ATBs). These cationic peptides, naturally produced by all kingdoms of life, play a
crucial role in the innate immune system of multicellular organisms and in bacterial interspecies
competition by exhibiting broad-spectrum activity against bacteria, fungi, viruses, and parasites.
AMPs target bacterial pathogens through multiple mechanisms, most importantly by disrupting
their membranes, leading to cell lysis. However, bacterial resistance to host AMPs has emerged
due to a slow co-evolutionary process between microorganisms and their hosts. Alarmingly, the
development of resistance to last-resort AMPs in the treatment of MDR infections, such as colistin, is
attributed to the misuse of this and the high rate of horizontal genetic transfer of the corresponding
resistance genes. AMP-resistant bacteria employ diverse mechanisms, including but not limited to
proteolytic degradation, extracellular trapping and inactivation, active efflux, as well as complex
modifications in bacterial cell wall and membrane structures. This review comprehensively examines
all constitutive and inducible molecular resistance mechanisms to AMPs supported by experimental
evidence described to date in bacterial pathogens. We also explore the specificity of these mechanisms
toward structurally diverse AMPs to broaden and enhance their potential in developing and applying
them as therapeutics for MDR bacteria. Additionally, we provide insights into the significance of
AMP resistance within the context of host–pathogen interactions.
significant number of deaths annually due to infections that are resistant to treatment. Amidst
this crisis, antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional
antibiotics (ATBs). These cationic peptides, naturally produced by all kingdoms of life, play a
crucial role in the innate immune system of multicellular organisms and in bacterial interspecies
competition by exhibiting broad-spectrum activity against bacteria, fungi, viruses, and parasites.
AMPs target bacterial pathogens through multiple mechanisms, most importantly by disrupting
their membranes, leading to cell lysis. However, bacterial resistance to host AMPs has emerged
due to a slow co-evolutionary process between microorganisms and their hosts. Alarmingly, the
development of resistance to last-resort AMPs in the treatment of MDR infections, such as colistin, is
attributed to the misuse of this and the high rate of horizontal genetic transfer of the corresponding
resistance genes. AMP-resistant bacteria employ diverse mechanisms, including but not limited to
proteolytic degradation, extracellular trapping and inactivation, active efflux, as well as complex
modifications in bacterial cell wall and membrane structures. This review comprehensively examines
all constitutive and inducible molecular resistance mechanisms to AMPs supported by experimental
evidence described to date in bacterial pathogens. We also explore the specificity of these mechanisms
toward structurally diverse AMPs to broaden and enhance their potential in developing and applying
them as therapeutics for MDR bacteria. Additionally, we provide insights into the significance of
AMP resistance within the context of host–pathogen interactions.
Niedner-Boblenz A., Monecke T., Hennig J., Klostermann M., Hofweber M., Gerber A. P., Anosova I., Mayer W., Müller M., Heym R., Janowski R., Paillart J. -C., Dormann D., Zarnack K., Sattler M., Niessing D.
Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation Article de journal
Dans: bioRxiv, 2024.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{A.2024,
title = {Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation},
author = {A. Niedner-Boblenz and T. Monecke and J. Hennig and M. Klostermann and M. Hofweber and A.P. Gerber and I. Anosova and W. Mayer and M. Müller and R. Heym and R. Janowski and J.-C. Paillart and D. Dormann and K. Zarnack and M. Sattler and D. Niessing},
url = {https://www.biorxiv.org/content/10.1101/2024.03.27.586925v2},
doi = {10.1101/2024.03.27.586925},
year = {2024},
date = {2024-03-29},
urldate = {2024-03-29},
journal = {bioRxiv},
abstract = {RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient “RNA world” PUN-like motifs may have supported the correct folding of early ribozymes.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient “RNA world” PUN-like motifs may have supported the correct folding of early ribozymes.
Krishnan A., Ali L. M., Prabhu S. G., Pillai V. N., Chameettachal A., Vivet-Boudou V., Bernacchi S., Mustafa F., Marquet R., Rizvi T. A.
Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging Article de journal
Dans: RNA Journal, vol. 30, p. 68-88, 2024.
Résumé | Liens | BibTeX | Étiquettes: bernacchi, MARQUET, Unité ARN, vivet-boudou
@article{nokey,
title = {Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging},
author = {A. Krishnan and L.M. Ali and S.G. Prabhu and V.N. Pillai and A. Chameettachal and V. Vivet-Boudou and S. Bernacchi and F. Mustafa and R. Marquet and T.A. Rizvi},
editor = {cold spring harbor laboratory press},
url = {https://rnajournal.cshlp.org/content/30/1/68.long},
doi = {10.1261/rna.079840.123},
year = {2024},
date = {2024-01-10},
urldate = {2024-01-10},
journal = {RNA Journal},
volume = {30},
pages = {68-88},
abstract = {The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to
virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous
regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To
better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ
sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed
that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput
selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences,
with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained
nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our
data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays
performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to
mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating
sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion
assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of
FIV-based vectors for human gene therapy.},
keywords = {bernacchi, MARQUET, Unité ARN, vivet-boudou},
pubstate = {published},
tppubtype = {article}
}
The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to
virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous
regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To
better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ
sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed
that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput
selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences,
with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained
nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our
data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays
performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to
mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating
sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion
assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of
FIV-based vectors for human gene therapy.
virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous
regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To
better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ
sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed
that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput
selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences,
with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained
nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our
data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays
performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to
mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating
sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion
assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of
FIV-based vectors for human gene therapy.
Quignon E., Ferhadian D., Hache A., Vivet-Boudou V., Isel C., Printz-Schweigert A., Donchet A., Crépin T., Marquet R.
Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments Article de journal
Dans: Viruses, vol. 16, no. 3, p. 421, 2024, ISBN: 10.3390/v16030421.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, MARQUET influenza A virus NP nucleoprotein vRNA RNA structure RNA chaperon chemical probing, Unité ARN
@article{nokey,
title = {Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments},
author = {E. Quignon and D. Ferhadian and A. Hache and V. Vivet-Boudou and C. Isel and A. Printz-Schweigert and A. Donchet and T. Crépin and R. Marquet},
url = {https://www.mdpi.com/1999-4915/16/3/421},
isbn = {10.3390/v16030421},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Viruses},
volume = {16},
number = {3},
pages = {421},
abstract = {Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.},
keywords = {MARQUET, MARQUET influenza A virus NP nucleoprotein vRNA RNA structure RNA chaperon chemical probing, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.
2023
Labaronne E., Decimo D., Bertrand L., Guiguettaz L., Sohier T. J. M., Cluet D., Vivet-Boudou V., Dahoui C., François P., Hatin I., Lambotte O., Samri A., Autran B., Etienne L., Goujon C., Paillart J. -C., Namy O., Ramirez B. C., Ohlmann T., Morris A., Ricci E. P.
Dans: (bioRxiv), 2023.
Liens | BibTeX | Étiquettes: PAILLART, Unité ARN
@article{nokey,
title = {Extensive uORF translation from HIV-1 transcripts elicits specific T cell immune responses in infected individuals and conditions DDX3 dependency for expression of main ORFs},
author = {E. Labaronne and D. Decimo and L. Bertrand and L. Guiguettaz and T.J.M. Sohier and D. Cluet and V. Vivet-Boudou and C. Dahoui and P. François and I. Hatin and O. Lambotte and A. Samri and B. Autran and L. Etienne and C. Goujon and J.-C. Paillart and O. Namy and B.C. Ramirez and T. Ohlmann and A. Morris and E.P. Ricci},
doi = {10.1101/2022.04.29.489990},
year = {2023},
date = {2023-09-20},
urldate = {2023-09-20},
journal = {(bioRxiv)},
keywords = {PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Jakob C., Lovate G. L., Desirò D., Gießler L., Smyth R. P., Marquet R., Lamkiewicz K., Marz M., Schwemmle M., Bolte H.
Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging Article de journal
Dans: Nucleic Acids Res, vol. 51, iss. 12, p. 6479-6494, 2023, ISSN: 1362-4962.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, Unité ARN
@article{pmid37224537,
title = {Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging},
author = {C. Jakob and G.L. Lovate and D. Desirò and L. Gießler and R.P. Smyth and R. Marquet and K. Lamkiewicz and M. Marz and M. Schwemmle and H. Bolte},
doi = {10.1093/nar/gkad442},
issn = {1362-4962},
year = {2023},
date = {2023-05-01},
urldate = {2023-05-01},
journal = {Nucleic Acids Res},
volume = {51},
issue = {12},
pages = {6479-6494},
abstract = {A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.},
keywords = {MARQUET, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.
Lista M. J., Jousset A. -C., Cheng M., Saint-André V., Perrot E., Rodrigues M., Primo C. Di, Gadelle D., Toccafondi E., Segeral E., Berlioz-Torrent C., Emiliani S., Mergny J. -L., Lavigne M.
DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence Article de journal
Dans: Retrovirology, vol. 20, no. 1, p. 10, 2023, ISSN: 1742-4690.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, NEGRONI, PAILLART, Unité ARN
@article{pmid37254203,
title = {DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence},
author = {M.J. Lista and A.-C. Jousset and M. Cheng and V. Saint-André and E. Perrot and M. Rodrigues and C. Di Primo and D. Gadelle and E. Toccafondi and E. Segeral and C. Berlioz-Torrent and S. Emiliani and J.-L. Mergny and M. Lavigne},
doi = {10.1186/s12977-023-00627-6},
issn = {1742-4690},
year = {2023},
date = {2023-05-01},
urldate = {2023-05-01},
journal = {Retrovirology},
volume = {20},
number = {1},
pages = {10},
abstract = {BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus.nnRESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes.nnCONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.},
keywords = {MARQUET, NEGRONI, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus.nnRESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes.nnCONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.
2022
Bonaventure B., Rebendenne A., Valadão A. L Chaves, Arnaud-Arnould M., Gracias S., de Gracia F. Garcia, McKellar J., Labaronne E., Tauziet M., Vivet-Boudou V., Bernard E., Briant L., Gros N., Djilli W., Courgnaud V., Parrinello H., Rialle S., Blaise M., Lacroix L., Lavigne M., Paillart J. -C., Ricci E. P, Schulz R., Jouvenet N., Moncorgé O., Goujon C.
The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses Article de journal
Dans: EMBO Rep, p. e54061, 2022, ISSN: 1469-3178.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{pmid36161446,
title = {The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses},
author = {B. Bonaventure and A. Rebendenne and A.L Chaves Valadão and M. Arnaud-Arnould and S. Gracias and F. Garcia de Gracia and J. McKellar and E. Labaronne and M. Tauziet and V. Vivet-Boudou and E. Bernard and L. Briant and N. Gros and W. Djilli and V. Courgnaud and H. Parrinello and S. Rialle and M. Blaise and L. Lacroix and M. Lavigne and J.-C. Paillart and E. P Ricci and R. Schulz and N. Jouvenet and O. Moncorgé and C. Goujon},
url = {https://pubmed.ncbi.nlm.nih.gov/36161446/},
doi = {10.15252/embr.202154061},
issn = {1469-3178},
year = {2022},
date = {2022-09-01},
urldate = {2022-09-01},
journal = {EMBO Rep},
pages = {e54061},
abstract = {Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.
Jakob C., Paul-Stansilaus R., Schwemmle M., Marquet R., Bolte H.
The influenza A virus genome packaging network - complex, flexible and yet unsolved Article de journal
Dans: Nucleic Acids Res, vol. 50, iss. 16, p. 9023-9038, 2022, ISSN: 1362-4962.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, Unité ARN
@article{pmid35993811,
title = {The influenza A virus genome packaging network - complex, flexible and yet unsolved},
author = {C. Jakob and R. Paul-Stansilaus and M. Schwemmle and R. Marquet and H. Bolte},
doi = {10.1093/nar/gkac688},
issn = {1362-4962},
year = {2022},
date = {2022-08-01},
urldate = {2022-08-01},
journal = {Nucleic Acids Res},
volume = {50},
issue = {16},
pages = {9023-9038},
abstract = {The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA-RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA-RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA-RNA interactions. We speculate that identifying such functional intersegmental RNA-RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.},
keywords = {MARQUET, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA-RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA-RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA-RNA interactions. We speculate that identifying such functional intersegmental RNA-RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.
Libre C, Seissler T, Guerrero S, Batisse J, Verriez C, Stupfler B, Gilmer O, Cabrera-Rodriguez R, Weber M, Valenzuela-Fernandez A, Cimarelli A, Etienne L, Marquet R, Paillart J C
A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor Article de journal
Dans: Biomedicines, vol. 10, no. 1, p. 13, 2022.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{Libre2022,
title = {A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor},
author = {C Libre and T Seissler and S Guerrero and J Batisse and C Verriez and B Stupfler and O Gilmer and R Cabrera-Rodriguez and M Weber and A Valenzuela-Fernandez and A Cimarelli and L Etienne and R Marquet and J C Paillart},
url = {https://www.mdpi.com/2227-9059/10/1/13},
doi = {10.1101/2021.01.13.426487},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Biomedicines},
volume = {10},
number = {1},
pages = {13},
abstract = {The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular cytosine deaminases APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutations during reverse transcription. Vif counteracts A3G by several non-redundant mechanisms (transcription, translation and protein degradation) that concur in reducing the levels of A3G in cell and in preventing its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5-untranslated region (5-UTR) of A3G mRNA. Extensive mutagenesis of A3G 5-UTR, combined with an analysis of their translational effect in transfected cells, indicated that the uORF represses A3G translation and that A3G mRNA is translated through a dual leaky-scanning and re-initiation mechanism. Interestingly, the uORF is also mandatory for the Vif-mediated repression of A3G translation. Furthermore, we showed that the redirection of A3G mRNA into stress granules was dependent not only on Vif, but also on the uORF. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific motif to repress its translation.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular cytosine deaminases APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutations during reverse transcription. Vif counteracts A3G by several non-redundant mechanisms (transcription, translation and protein degradation) that concur in reducing the levels of A3G in cell and in preventing its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5-untranslated region (5-UTR) of A3G mRNA. Extensive mutagenesis of A3G 5-UTR, combined with an analysis of their translational effect in transfected cells, indicated that the uORF represses A3G translation and that A3G mRNA is translated through a dual leaky-scanning and re-initiation mechanism. Interestingly, the uORF is also mandatory for the Vif-mediated repression of A3G translation. Furthermore, we showed that the redirection of A3G mRNA into stress granules was dependent not only on Vif, but also on the uORF. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific motif to repress its translation.
Gilmer O., Mailler E., Paillart J. C., Mouhand A., Tisne C., Mak J., Smyth R. P., Marquet R., Vivet-Boudou V.
Structural maturation of the HIV-1 RNA 5' untranslated region by Pr55(Gag) and its maturation products Article de journal
Dans: RNA Biol, vol. 19, no. 1, p. 191-205, 2022, ISBN: 35067194, (1555-8584 (Electronic) 1547-6286 (Linking) Journal Article).
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{nokey,
title = {Structural maturation of the HIV-1 RNA 5' untranslated region by Pr55(Gag) and its maturation products},
author = {O. Gilmer and E. Mailler and J. C. Paillart and A. Mouhand and C. Tisne and J. Mak and R. P. Smyth and R. Marquet and V. Vivet-Boudou},
url = {https://www.tandfonline.com/doi/full/10.1080/15476286.2021.2021677},
isbn = {35067194},
year = {2022},
date = {2022-01-01},
journal = {RNA Biol},
volume = {19},
number = {1},
pages = {191-205},
abstract = {Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55(Gag) precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55(Gag) or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55(Gag) and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55(Gag) maturation products on the gRNA structural maturation.},
note = {1555-8584 (Electronic)
1547-6286 (Linking)
Journal Article},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55(Gag) precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55(Gag) or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55(Gag) and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55(Gag) maturation products on the gRNA structural maturation.
Bernacchi S.
Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview Article de journal
Dans: Viruses, vol. 14, no. 2, 2022, ISBN: 35215917, (1999-4915 (Electronic) 1999-4915 (Linking) Journal Article Review Research Support, Non-U.S. Gov't).
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{nokey,
title = {Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview},
author = {S. Bernacchi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35215917},
doi = {10.3390/v14020324},
isbn = {35215917},
year = {2022},
date = {2022-01-01},
journal = {Viruses},
volume = {14},
number = {2},
abstract = {Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.},
note = {1999-4915 (Electronic)
1999-4915 (Linking)
Journal Article
Review
Research Support, Non-U.S. Gov't},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.
2021
Pitchai F, Chameettachal A, Vivet-Boudou V, Ali L M, Pillai V N, Krishnan A, Bernacchi S, Mustafa F, R Marquet, Rizvi T A
Identification of Pr78Gag binding sites on the Mason-Pfizer monkey virus genomic RNA packaging determinants Article de journal
Dans: J Mol Biol, vol. 433, no. 10, p. 166923, 2021.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, Retroviruses Gag-RNA interactions Purines Footprinting hSHAPE, Unité ARN
@article{F2021,
title = {Identification of Pr78Gag binding sites on the Mason-Pfizer monkey virus genomic RNA packaging determinants},
author = {F Pitchai and A Chameettachal and V Vivet-Boudou and L M Ali and V N Pillai and A Krishnan and S Bernacchi and F Mustafa and Marquet R and T A Rizvi},
url = {https://www.sciencedirect.com/science/article/pii/S0022283621001224?via%3Dihub},
doi = {10.1016/j.jmb.2021.166923},
year = {2021},
date = {2021-01-01},
journal = {J Mol Biol},
volume = {433},
number = {10},
pages = {166923},
abstract = {How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.},
keywords = {MARQUET, Retroviruses Gag-RNA interactions Purines Footprinting hSHAPE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.
Busienne C, Marquet R, Paillart J C, Bernacchi S
Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role Article de journal
Dans: Int. J. Mol. Sci., vol. 22, no. 6, p. 2871, 2021.
Résumé | Liens | BibTeX | Étiquettes: HIV-1, MARQUET, PAILLART, post-translational modifications, Pr55Gag precursor, retroviral Gag precursors, retroviral life cycle, Unité ARN
@article{C2021b,
title = {Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role},
author = {C Busienne and R Marquet and J C Paillart and S Bernacchi},
url = {https://www.mdpi.com/1422-0067/22/6/2871},
doi = {10.3390/ijms22062871},
year = {2021},
date = {2021-01-01},
journal = {Int. J. Mol. Sci.},
volume = {22},
number = {6},
pages = {2871},
abstract = {Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle.},
keywords = {HIV-1, MARQUET, PAILLART, post-translational modifications, Pr55Gag precursor, retroviral Gag precursors, retroviral life cycle, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle.
Chameettachal A, Vivet-Boudou V, Pitchai F N, N Pillai V, Ali L M, Krishnan A, Bernacchi S, Mustafa F, Marquet R, Rizvi T A
A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag Article de journal
Dans: Nucleic Acids Res, vol. 49, no. 8, p. 4668-4688, 2021.
BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{Chameettachal2021,
title = {A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag},
author = {A Chameettachal and V Vivet-Boudou and F N Pitchai and Pillai V N and L M Ali and A Krishnan and S Bernacchi and F Mustafa and R Marquet and T A Rizvi},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nucleic Acids Res},
volume = {49},
number = {8},
pages = {4668-4688},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Stupfler B, Verriez C, Gallois-Montbrun S, Marquet R, Paillart J C
Degradation-independent inhibition of APOBEC3G by HIV-1 Vif protein Article de journal
Dans: Viruses, vol. 13, no. 4, p. 617, 2021.
Résumé | Liens | BibTeX | Étiquettes: APOBEC3G, deamination, encapsidation, HIV, MARQUET, PAILLART, proteasome, RNP granules, Translation, ubiquitin, Unité ARN, vif
@article{Stupfler2021,
title = {Degradation-independent inhibition of APOBEC3G by HIV-1 Vif protein},
author = {B Stupfler and C Verriez and S Gallois-Montbrun and R Marquet and J C Paillart},
url = {https://www.mdpi.com/1999-4915/13/4/617},
doi = {10.3390/v13040617},
year = {2021},
date = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {4},
pages = {617},
abstract = {The ubiquitinproteasome system plays an important role in the cell under normal physiological conditions but also during viral infections. Indeed, many auxiliary proteins from the (HIV-1) divert this system to its own advantage, notably to induce the degradation of cellular restriction factors. For instance, the HIV-1 viral infectivity factor (Vif) has been shown to specifically counteract several cellular deaminases belonging to the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 or A3) family (A3A to A3H) by recruiting an E3-ubiquitin ligase complex and inducing their polyubiquitination and degradation through the proteasome. Although this pathway has been extensively characterized so far, Vif has also been shown to impede A3s through degradation-independent processes, but research on this matter remains limited. In this review, we describe our current knowledge regarding the degradation-independent inhibition of A3s, and A3G in particular, by the HIV-1 Vif protein, the molecular mechanisms involved, and highlight important properties of this small viral protein.},
keywords = {APOBEC3G, deamination, encapsidation, HIV, MARQUET, PAILLART, proteasome, RNP granules, Translation, ubiquitin, Unité ARN, vif},
pubstate = {published},
tppubtype = {article}
}
The ubiquitinproteasome system plays an important role in the cell under normal physiological conditions but also during viral infections. Indeed, many auxiliary proteins from the (HIV-1) divert this system to its own advantage, notably to induce the degradation of cellular restriction factors. For instance, the HIV-1 viral infectivity factor (Vif) has been shown to specifically counteract several cellular deaminases belonging to the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 or A3) family (A3A to A3H) by recruiting an E3-ubiquitin ligase complex and inducing their polyubiquitination and degradation through the proteasome. Although this pathway has been extensively characterized so far, Vif has also been shown to impede A3s through degradation-independent processes, but research on this matter remains limited. In this review, we describe our current knowledge regarding the degradation-independent inhibition of A3s, and A3G in particular, by the HIV-1 Vif protein, the molecular mechanisms involved, and highlight important properties of this small viral protein.
Goettsch W, Beerenwinkel N, Deng L, Dolken L, Dutilh B E, Erhard F, Kaderali L, von Kleist M, Marquet R, Matthijnssens J, McCallin S, McMahon D, Rattei T, Rij R P Van, Robertson D L, Schwemmle M, Stern-Ginossar N, Marz M
ITN-VIROINF: Understanding (Harmful) Virus-Host Interactions by Linking Virology and Bioinformatics Article de journal
Dans: Viruses, vol. 13, no. 5, 2021, ISBN: 33925452, (1999-4915 (Electronic) 1999-4915 (Linking) Journal Article).
Résumé | Liens | BibTeX | Étiquettes: MARQUET, Unité ARN
@article{Goettsch2021,
title = {ITN-VIROINF: Understanding (Harmful) Virus-Host Interactions by Linking Virology and Bioinformatics},
author = {W Goettsch and N Beerenwinkel and L Deng and L Dolken and B E Dutilh and F Erhard and L Kaderali and M von Kleist and R Marquet and J Matthijnssens and S McCallin and D McMahon and T Rattei and R P Van Rij and D L Robertson and M Schwemmle and N Stern-Ginossar and M Marz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33925452},
doi = {10.3390/v13050766},
isbn = {33925452},
year = {2021},
date = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {5},
abstract = {Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Sklodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals.},
note = {1999-4915 (Electronic)
1999-4915 (Linking)
Journal Article},
keywords = {MARQUET, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Sklodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals.
Gilmer O, Quignon E, Jousset A C, Paillart J C, Marquet R, Vivet-Boudou V
Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond Article de journal
Dans: Viruses, vol. 13, no. 10, p. 1894, 2021.
Résumé | Liens | BibTeX | Étiquettes: Capillary electrophoresis, chemical probe, enzymatic probe, high-throughput sequencing, MARQUET, mutational profiling, PAILLART, RNA, SHAPE, structure, Unité ARN
@article{nokey,
title = {Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond},
author = {O Gilmer and E Quignon and A C Jousset and J C Paillart and R Marquet and V Vivet-Boudou},
url = {https://www.mdpi.com/1999-4915/13/10/1894},
doi = {v13101894},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {10},
pages = {1894},
abstract = {RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.},
keywords = {Capillary electrophoresis, chemical probe, enzymatic probe, high-throughput sequencing, MARQUET, mutational profiling, PAILLART, RNA, SHAPE, structure, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.
Lyonnais S., Sadiq S. K., Lorca-Oro C., Dufau L., Nieto-Marquez S., Escriba T., Gabrielli N., Tan X., Ouizougun-Oubari M., Okoronkwo J., Reboud-Ravaux M., Gatell J. M., Marquet R., Paillart J. C., Meyerhans A., Tisne C., Gorelick R. J., Mirambeau G.
The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation Article de journal
Dans: Viruses, vol. 13, no. 11, 2021, ISBN: 34835118, (1999-4915 (Electronic) 1999-4915 (Linking) Journal Article).
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{nokey,
title = {The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation},
author = {S. Lyonnais and S. K. Sadiq and C. Lorca-Oro and L. Dufau and S. Nieto-Marquez and T. Escriba and N. Gabrielli and X. Tan and M. Ouizougun-Oubari and J. Okoronkwo and M. Reboud-Ravaux and J. M. Gatell and R. Marquet and J. C. Paillart and A. Meyerhans and C. Tisne and R. J. Gorelick and G. Mirambeau},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34835118},
doi = {10.3390/v13112312},
isbn = {34835118},
year = {2021},
date = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {11},
abstract = {A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of approximately 2400 Gag and approximately 120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.},
note = {1999-4915 (Electronic)
1999-4915 (Linking)
Journal Article},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of approximately 2400 Gag and approximately 120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.
Welker L, Paillart J C, Bernacchi S
Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses Article de journal
Dans: Viruses, vol. 13, no. 8, p. 1559, 2021, ISBN: 34452424, (1999-4915 (Electronic) 1999-4915 (Linking) Journal Article Review).
Résumé | Liens | BibTeX | Étiquettes: PAILLART, Unité ARN
@article{Welker2021,
title = {Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses},
author = {L Welker and J C Paillart and S Bernacchi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34452424},
doi = {10.3390/v13081559},
isbn = {34452424},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {8},
pages = {1559},
abstract = {Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.},
note = {1999-4915 (Electronic)
1999-4915 (Linking)
Journal Article
Review},
keywords = {PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.
2020
Bernacchi S
Special Issue "Function and Structure of Viral Ribonucleoproteins Complexes" Article de journal
Dans: Viruses, vol. 12, no. 12, p. E1355, 2020.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{S.2020,
title = {Special Issue "Function and Structure of Viral Ribonucleoproteins Complexes"},
author = {S Bernacchi},
url = {https://www.mdpi.com/1999-4915/12/12/1355},
doi = { 10.3390/v12121355 },
year = {2020},
date = {2020-11-26},
journal = {Viruses},
volume = {12},
number = {12},
pages = {E1355},
abstract = {RNA viruses are extraordinary evolution machines that efficiently ensure their replication by taking advantage of the association with viral and cellular components to form ribonucleic complexes (vRNPs) [...]. },
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA viruses are extraordinary evolution machines that efficiently ensure their replication by taking advantage of the association with viral and cellular components to form ribonucleic complexes (vRNPs) [...].
Ali L M, Pitchai F N, Vivet-Boudou V, Chameettachal A, Jabeen A, Pillai V N, Mustafa F, Marquet R, Rizvi T A
Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation Article de journal
Dans: Frontiers in Microbiology, no. 11, p. 595410, 2020.
Résumé | Liens | BibTeX | Étiquettes: base paired purines, MARQUET, Mason-Pfizer monkey virus, PAILLART, retroviruses, RNA packaging, RNA secondary structure, RNA-Gag interaction, SHAPE, single-stranded purines, Unité ARN
@article{L.2020,
title = {Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation },
author = {L M Ali and F N Pitchai and V Vivet-Boudou and A Chameettachal and A Jabeen and V N Pillai and F Mustafa and R Marquet and T A Rizvi
},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2020.595410/full},
doi = { 10.3389/fmicb.2020.595410 },
year = {2020},
date = {2020-11-05},
journal = {Frontiers in Microbiology},
number = {11},
pages = {595410},
abstract = {A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop (Pal SL) that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified. },
keywords = {base paired purines, MARQUET, Mason-Pfizer monkey virus, PAILLART, retroviruses, RNA packaging, RNA secondary structure, RNA-Gag interaction, SHAPE, single-stranded purines, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop (Pal SL) that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified.
Boutant E, Bonzi J, Anton H, Nasim M Bin, Cathagne R, Real E, Dujardin D, Carl P, Didier P, Paillart J C, Marquet R, Mely Y, de Rocquigny H, Bernacchi S
Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane Article de journal
Dans: Biophys J, vol. 119, no. 2, p. 419-433, 2020, ISBN: 32574557.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{,
title = {Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane},
author = {E Boutant and J Bonzi and H Anton and M Bin Nasim and R Cathagne and E Real and D Dujardin and P Carl and P Didier and J C Paillart and R Marquet and Y Mely and H de Rocquigny and S Bernacchi},
url = {https://pubmed.ncbi.nlm.nih.gov/32574557/},
doi = {10.1016/j.bpj.2020.05.035},
isbn = {32574557},
year = {2020},
date = {2020-01-01},
journal = {Biophys J},
volume = {119},
number = {2},
pages = {419-433},
abstract = {The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.
R Marquet C Verriez J-C Paillart, Stupfler B
Les APOBEC : histoire d’une famille de protéines antivirales et mutagènes. Article de journal
Dans: Virologie, vol. 24, no. 6, p. 381-418, 2020.
Résumé | BibTeX | Étiquettes: APOBEC3, cancer, Facteurs de restriction, MARQUET, PAILLART, Unité ARN, vif, VIH-1
@article{Verriez2020,
title = {Les APOBEC : histoire d’une famille de protéines antivirales et mutagènes.},
author = {C Verriez, R Marquet, J-C Paillart and B Stupfler},
year = {2020},
date = {2020-01-01},
journal = {Virologie},
volume = {24},
number = {6},
pages = {381-418},
abstract = {La réponse immunitaire innée est une réponse non spécifique qui constitue la première ligne de défense en cas d’infection, notamment en permettant l’élimination des pathogènes par phagocytose ou apoptose. Au sein des cellules immunitaires, cette réponse innée se caractérise entre autres par la synthèse de protéines nommées facteurs de restriction dont le rôle est d’inhiber la réplication virale. Parmi ces facteurs, les protéines de la famille APOBEC3 (Apolipoprotein B mRNA-editing Enzyme Catalytic polypeptide-like 3 ou A3) constituent des facteurs antiviraux majeurs qui ciblent de nombreux types de virus. L’une des cibles des A3 est le virus de l’immunodéficience humaine de type 1 (VIH-1) : l’activité désaminase de certaines A3 convertit une fraction des cytidines du génome viral en uridines et perturbe son expression. Néanmoins, le VIH-1 contrecarre les A3 en exprimant la protéine Vif qui les inhibe en détournant divers mécanismes cellulaires. Par ailleurs, les APOBEC3 participent au maintien de l’intégrité génétique par l’inhibition des rétroéléments mais contribuent également à la cancérogenèse, à l’image d’A3A et A3B, deux facteurs majeurs dans ce processus. L’éventail de leurs activités, combiné aux récentes études montrant leur implication dans la régulation de virus émergents (Zika, SARS-CoV-2), permettent d’envisager les A3 ainsi que leurs partenaires viraux comme axes thérapeutiques.},
keywords = {APOBEC3, cancer, Facteurs de restriction, MARQUET, PAILLART, Unité ARN, vif, VIH-1},
pubstate = {published},
tppubtype = {article}
}
La réponse immunitaire innée est une réponse non spécifique qui constitue la première ligne de défense en cas d’infection, notamment en permettant l’élimination des pathogènes par phagocytose ou apoptose. Au sein des cellules immunitaires, cette réponse innée se caractérise entre autres par la synthèse de protéines nommées facteurs de restriction dont le rôle est d’inhiber la réplication virale. Parmi ces facteurs, les protéines de la famille APOBEC3 (Apolipoprotein B mRNA-editing Enzyme Catalytic polypeptide-like 3 ou A3) constituent des facteurs antiviraux majeurs qui ciblent de nombreux types de virus. L’une des cibles des A3 est le virus de l’immunodéficience humaine de type 1 (VIH-1) : l’activité désaminase de certaines A3 convertit une fraction des cytidines du génome viral en uridines et perturbe son expression. Néanmoins, le VIH-1 contrecarre les A3 en exprimant la protéine Vif qui les inhibe en détournant divers mécanismes cellulaires. Par ailleurs, les APOBEC3 participent au maintien de l’intégrité génétique par l’inhibition des rétroéléments mais contribuent également à la cancérogenèse, à l’image d’A3A et A3B, deux facteurs majeurs dans ce processus. L’éventail de leurs activités, combiné aux récentes études montrant leur implication dans la régulation de virus émergents (Zika, SARS-CoV-2), permettent d’envisager les A3 ainsi que leurs partenaires viraux comme axes thérapeutiques.
Bernacchi S, Ennifar E
Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry Chapitre d'ouvrage
Dans: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, p. 237-250, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006318.
Résumé | Liens | BibTeX | Étiquettes: Aminoglycosides, dimerization, drug interaction, ENNIFAR, HIV-1, initiation site, ITC, MARQUET, PAILLART, RNA, Thermodynamics RNA, Unité ARN, Viral
@inbook{,
title = {Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry},
author = {S Bernacchi and E Ennifar},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006318},
doi = {10.1007/978-1-0716-0278-2_16},
isbn = {32006318},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {237-250},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity.},
keywords = {Aminoglycosides, dimerization, drug interaction, ENNIFAR, HIV-1, initiation site, ITC, MARQUET, PAILLART, RNA, Thermodynamics RNA, Unité ARN, Viral},
pubstate = {published},
tppubtype = {inbook}
}
Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity.
Bernacchi S
Dynamic Light Scattering Analysis on RNA Associated to Proteins Chapitre d'ouvrage
Dans: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, p. 31-39, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006306.
Résumé | Liens | BibTeX | Étiquettes: Dynamic light scattering, Hydrodynamic radius Diffusion coefficient, MARQUET, PAILLART, Pr55Gag, precursor, Protein-RNA interactions, Unité ARN, Viral assembly, Viral genomic RNA Viral spliced RNA
@inbook{,
title = {Dynamic Light Scattering Analysis on RNA Associated to Proteins},
author = {S Bernacchi},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006306},
doi = {10.1007/978-1-0716-0278-2_4},
isbn = {32006306},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {31-39},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {Dynamic light scattering represents an accurate, robust, and reliable technique to analyze molecule size in solution and monitor their interactions in real time. Here, we describe how to analyze by DLS an RNA-protein interaction. In our frame, we studied complexes formed between RNA fragments derived from the genome of HIV-1 in association with the viral precursor Pr55Gag. These interactions are crucial for the specific selection of the viral genomic RNA (gRNA) from the bulk of the viral spliced and cellular RNAs. This chapter displays how DLS allows to characterize the interactions that regulate the early steps of viral assembly.},
keywords = {Dynamic light scattering, Hydrodynamic radius Diffusion coefficient, MARQUET, PAILLART, Pr55Gag, precursor, Protein-RNA interactions, Unité ARN, Viral assembly, Viral genomic RNA Viral spliced RNA},
pubstate = {published},
tppubtype = {inbook}
}
Dynamic light scattering represents an accurate, robust, and reliable technique to analyze molecule size in solution and monitor their interactions in real time. Here, we describe how to analyze by DLS an RNA-protein interaction. In our frame, we studied complexes formed between RNA fragments derived from the genome of HIV-1 in association with the viral precursor Pr55Gag. These interactions are crucial for the specific selection of the viral genomic RNA (gRNA) from the bulk of the viral spliced and cellular RNAs. This chapter displays how DLS allows to characterize the interactions that regulate the early steps of viral assembly.
Quang N Nguyen, Goudey S, Ségéral E, Mohammad A, Lemoine S, Blugeon C, Versapuech M, Paillart J C, Berlioz-Torrent C, Emiliani S, Gallois-Montbrun S
Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection Article de journal
Dans: Retrovirology, vol. 17, no. 1, p. 25, 2020, ISBN: 32807178.
Résumé | Liens | BibTeX | Étiquettes: HIV RNA Alternative splicing Viral transcriptome ONT long-read sequencing, MARQUET, PAILLART, Unité ARN
@article{,
title = {Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection},
author = {N Nguyen Quang and S Goudey and E Ségéral and A Mohammad and S Lemoine and C Blugeon and M Versapuech and J C Paillart and C Berlioz-Torrent and S Emiliani and S Gallois-Montbrun},
url = {https://pubmed.ncbi.nlm.nih.gov/32807178/},
doi = {10.1186/s12977-020-00533-1},
isbn = {32807178},
year = {2020},
date = {2020-01-01},
journal = {Retrovirology},
volume = {17},
number = {1},
pages = {25},
abstract = {Background
Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells.
Results
ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build ﾓsplice treesﾔ, a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation.
Conclusion
ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.
Background},
keywords = {HIV RNA Alternative splicing Viral transcriptome ONT long-read sequencing, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Background
Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells.
Results
ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build ﾓsplice treesﾔ, a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation.
Conclusion
ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.
Background
Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells.
Results
ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build ﾓsplice treesﾔ, a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation.
Conclusion
ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.
Background
2019
Mailler E, Paillart J C, Marquet R, Smyth R P, Vivet-Boudou V
The evolution of RNA structural probing methods: From gels to next-generation sequencing Article de journal
Dans: Wiley Interdiscip Rev RNA, vol. 10, no. 2, p. e1518, 2019, ISBN: 30485688.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, NGS RNA probing structure, PAILLART, Unité ARN
@article{,
title = {The evolution of RNA structural probing methods: From gels to next-generation sequencing},
author = {E Mailler and J C Paillart and R Marquet and R P Smyth and V Vivet-Boudou},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30485688?dopt=Abstract},
doi = {10.1002/wrna.1518},
isbn = {30485688},
year = {2019},
date = {2019-01-01},
journal = {Wiley Interdiscip Rev RNA},
volume = {10},
number = {2},
pages = {e1518},
abstract = {RNA molecules are important players in all domains of life and the study of the relationship between their multiple flexible states and the associated biological roles has increased in recent years. For several decades, chemical and enzymatic structural probing experiments have been used to determine RNA structure. During this time, there has been a steady improvement in probing reagents and experimental methods, and today the structural biologist community has a large range of tools at its disposal to probe the secondary structure of RNAs in vitro and in cells. Early experiments used radioactive labeling and polyacrylamide gel electrophoresis as read-out methods. This was superseded by capillary electrophoresis, and more recently by next-generation sequencing. Today, powerful structural probing methods can characterize RNA structure on a genome-wide scale. In this review, we will provide an overview of RNA structural probing methodologies from a historical and technical perspective. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Methods > RNA Analyses in vitro and In Silico RNA Methods > RNA Analyses in Cells.},
keywords = {MARQUET, NGS RNA probing structure, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA molecules are important players in all domains of life and the study of the relationship between their multiple flexible states and the associated biological roles has increased in recent years. For several decades, chemical and enzymatic structural probing experiments have been used to determine RNA structure. During this time, there has been a steady improvement in probing reagents and experimental methods, and today the structural biologist community has a large range of tools at its disposal to probe the secondary structure of RNAs in vitro and in cells. Early experiments used radioactive labeling and polyacrylamide gel electrophoresis as read-out methods. This was superseded by capillary electrophoresis, and more recently by next-generation sequencing. Today, powerful structural probing methods can characterize RNA structure on a genome-wide scale. In this review, we will provide an overview of RNA structural probing methodologies from a historical and technical perspective. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Methods > RNA Analyses in vitro and In Silico RNA Methods > RNA Analyses in Cells.
Krishnan A, Pillai V, Chameettachal A, Ali L M, Pitchai F Nuzra Nagoor, Tariq S, Mustafa F, Marquet R, Rizvi T A
Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag. Article de journal
Dans: Viruses, vol. 11, no. 8, p. 689, 2019, ISBN: 31357656.
Résumé | Liens | BibTeX | Étiquettes: Gag protein purification His-tag fusion protein Pr50Gag protein expression feline immunodeficiency virus (FIV) retroviral RNA packaging viral assembly, MARQUET, PAILLART, Unité ARN
@article{,
title = {Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50^{Gag}.},
author = {A Krishnan and V Pillai and A Chameettachal and L M Ali and F Nuzra Nagoor Pitchai and S Tariq and F Mustafa and R Marquet and T A Rizvi},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31357656?report=&dispmax=200&tool=PubCrawler_2.23},
doi = {10.3390/v11080689},
isbn = {31357656},
year = {2019},
date = {2019-01-01},
journal = {Viruses},
volume = {11},
number = {8},
pages = {689},
abstract = {The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.},
keywords = {Gag protein purification His-tag fusion protein Pr50Gag protein expression feline immunodeficiency virus (FIV) retroviral RNA packaging viral assembly, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.
Kalloush R M, Vivet-Boudou V, Ali L M, Pillai V, Mustafa F, Marquet R, Rizvi T A
Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging Article de journal
Dans: RNA Biol, vol. 16, no. 5, p. 612-625, 2019, ISBN: 30773097.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, MARQUET PAILLART Mason-Pfizer monkey virus (MPMV) RNA packaging RNA secondary structure Retroviruses U5/Gag LRIs gag, Mason-Pfizer monkey virus (MPMV) RNA packaging RNA secondary structure Retroviruses U5/Gag LRIs gag, PAILLART, Unité ARN
@article{,
title = {Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging},
author = {R M Kalloush and V Vivet-Boudou and L M Ali and V Pillai and F Mustafa and R Marquet and T A Rizvi},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30773097?dopt=Abstract},
doi = {10.1080/15476286.2019.1572424},
isbn = {30773097},
year = {2019},
date = {2019-01-01},
journal = {RNA Biol},
volume = {16},
number = {5},
pages = {612-625},
abstract = {The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5' UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.},
keywords = {MARQUET, MARQUET PAILLART Mason-Pfizer monkey virus (MPMV) RNA packaging RNA secondary structure Retroviruses U5/Gag LRIs gag, Mason-Pfizer monkey virus (MPMV) RNA packaging RNA secondary structure Retroviruses U5/Gag LRIs gag, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5' UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.
2018
Smyth R P, Smith M R, Jousset A C, Despons L, Laumond G, Decoville T, Cattenoz P, Moog C, Jossinet F, Mougel M, Paillart J C, von Kleist M, Marquet R
Dans: Nucleic Acids Res, vol. 46, no. 9, p. e57, 2018, ISBN: 29514260.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{,
title = {In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging},
author = {R P Smyth and M R Smith and A C Jousset and L Despons and G Laumond and T Decoville and P Cattenoz and C Moog and F Jossinet and M Mougel and J C Paillart and M von Kleist and R Marquet},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29514260?dopt=Abstract},
doi = {10.1093/nar/gky152},
isbn = {29514260},
year = {2018},
date = {2018-01-01},
journal = {Nucleic Acids Res},
volume = {46},
number = {9},
pages = {e57},
abstract = {Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5′ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5′ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5′ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5′ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.
Pitchai F N N, Ali L, Pillai V N, Chameettachal A, Ashraf S S, Mustafa F, Marquet R, Rizvi T A
Expression, purification, and characterization of biologically active full-length Mason-Pfizer monkey virus (MPMV) Pr78Gag Article de journal
Dans: Sci Rep, vol. 8, no. 1, p. 11793, 2018, ISBN: 30087395.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{,
title = {Expression, purification, and characterization of biologically active full-length Mason-Pfizer monkey virus (MPMV) Pr78^{Gag}},
author = {F N N Pitchai and L Ali and V N Pillai and A Chameettachal and S S Ashraf and F Mustafa and R Marquet and T A Rizvi},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30087395},
doi = {10.1038/s41598-018-30142-0},
isbn = {30087395},
year = {2018},
date = {2018-01-01},
journal = {Sci Rep},
volume = {8},
number = {1},
pages = {11793},
abstract = {MPMV precursor polypeptide Pr78Gag orchestrates assembly and packaging of genomic RNA (gRNA) into virus particles. Therefore, we have expressed recombinant full-length Pr78Gag either with or without His6-tag in bacterial as well as eukaryotic cultures and purified the recombinant protein from soluble fractions of the bacterial cultures. The recombinant Pr78Gag protein has the intrinsic ability to assemble in vitro to form virus like particles (VLPs). Consistent with this observation, the recombinant protein could form VLPs in both prokaryotes and eukaryotes. VLPs formed in eukaryotic cells by recombinant Pr78Gag with or without His6-tag can encapsidate MPMV transfer vector RNA, suggesting that the inclusion of the His6-tag to the full-length Pr78Gag did not interfere with its expression or biological function. This study demonstrates the expression and purification of a biologically active, recombinant Pr78Gag, which should pave the way to study RNA-protein interactions involved in the MPMV gRNA packaging process.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
MPMV precursor polypeptide Pr78Gag orchestrates assembly and packaging of genomic RNA (gRNA) into virus particles. Therefore, we have expressed recombinant full-length Pr78Gag either with or without His6-tag in bacterial as well as eukaryotic cultures and purified the recombinant protein from soluble fractions of the bacterial cultures. The recombinant Pr78Gag protein has the intrinsic ability to assemble in vitro to form virus like particles (VLPs). Consistent with this observation, the recombinant protein could form VLPs in both prokaryotes and eukaryotes. VLPs formed in eukaryotic cells by recombinant Pr78Gag with or without His6-tag can encapsidate MPMV transfer vector RNA, suggesting that the inclusion of the His6-tag to the full-length Pr78Gag did not interfere with its expression or biological function. This study demonstrates the expression and purification of a biologically active, recombinant Pr78Gag, which should pave the way to study RNA-protein interactions involved in the MPMV gRNA packaging process.
Mustafa F, Vivet-Boudou V, Jabeen A, Ali L M, Kalloush R M, Marquet R, Rizvi T A
The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA Article de journal
Dans: RNA Biol, vol. 15, no. 8, p. 1047-1059, 2018, ISBN: 29929424.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, Mouse mammary tumor virus RNA packaging and dimerization RNA secondary structure Retroviruses palindrome, PAILLART, Unité ARN
@article{,
title = {The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA},
author = {F Mustafa and V Vivet-Boudou and A Jabeen and L M Ali and R M Kalloush and R Marquet and T A Rizvi},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29929424?dopt=Abstract},
doi = {10.1080/15476286.2018.1486661},
isbn = {29929424},
year = {2018},
date = {2018-01-01},
journal = {RNA Biol},
volume = {15},
number = {8},
pages = {1047-1059},
abstract = {Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.},
keywords = {MARQUET, Mouse mammary tumor virus RNA packaging and dimerization RNA secondary structure Retroviruses palindrome, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.
Ferhadian D, Contrant M, Printz-Schweigert A, Smyth R P, Paillart J C, Marquet R
Structural and functional motifs in influenza virus RNAs Article de journal
Dans: Front Microbiol, vol. 9, p. 599, 2018, ISBN: 29651275.
Résumé | Liens | BibTeX | Étiquettes: CRNA RNA RNA structure influenza influenza A virus promoter vRN, MARQUET, PAILLART, Unité ARN
@article{,
title = {Structural and functional motifs in influenza virus RNAs},
author = {D Ferhadian and M Contrant and A Printz-Schweigert and R P Smyth and J C Paillart and R Marquet},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29651275?dopt=Abstract},
doi = {10.3389/fmicb.2018.00559},
isbn = {29651275},
year = {2018},
date = {2018-01-01},
journal = {Front Microbiol},
volume = {9},
pages = {599},
abstract = {Influenza A viruses (IAV) are responsible for recurrent influenza epidemics and occasional devastating pandemics in humans and animals. They belong to the Orthomyxoviridae family and their genome consists of eight (-) sense viral RNA (vRNA) segments of different lengths coding for a