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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.
Lisa Welker; Jean-Christophe Paillart; Serena Bernacchi. Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses. Viruses 2021, 13, 1559 .
AMA StyleLisa Welker, Jean-Christophe Paillart, Serena Bernacchi. Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses. Viruses. 2021; 13 (8):1559.
Chicago/Turabian StyleLisa Welker; Jean-Christophe Paillart; Serena Bernacchi. 2021. "Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses." Viruses 13, no. 8: 1559.
Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.
Akhil Chameettachal; Valérie Vivet-Boudou; Fathima Nuzra Nagoor Pitchai; Vineeta N Pillai; Lizna Mohamed Ali; Anjana Krishnan; Serena Bernacchi; Farah Mustafa; Roland Marquet; Tahir A Rizvi. A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag. Nucleic Acids Research 2021, 49, 4668 -4688.
AMA StyleAkhil Chameettachal, Valérie Vivet-Boudou, Fathima Nuzra Nagoor Pitchai, Vineeta N Pillai, Lizna Mohamed Ali, Anjana Krishnan, Serena Bernacchi, Farah Mustafa, Roland Marquet, Tahir A Rizvi. A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag. Nucleic Acids Research. 2021; 49 (8):4668-4688.
Chicago/Turabian StyleAkhil Chameettachal; Valérie Vivet-Boudou; Fathima Nuzra Nagoor Pitchai; Vineeta N Pillai; Lizna Mohamed Ali; Anjana Krishnan; Serena Bernacchi; Farah Mustafa; Roland Marquet; Tahir A Rizvi. 2021. "A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag." Nucleic Acids Research 49, no. 8: 4668-4688.
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.
Charlotte Bussienne; Roland Marquet; Jean-Christophe Paillart; Serena Bernacchi. Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role. International Journal of Molecular Sciences 2021, 22, 2871 .
AMA StyleCharlotte Bussienne, Roland Marquet, Jean-Christophe Paillart, Serena Bernacchi. Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role. International Journal of Molecular Sciences. 2021; 22 (6):2871.
Chicago/Turabian StyleCharlotte Bussienne; Roland Marquet; Jean-Christophe Paillart; Serena Bernacchi. 2021. "Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role." International Journal of Molecular Sciences 22, no. 6: 2871.
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). These vRNPs are functional units of the infectious cycle, driving various processes including transcription, nuclear export, translation, and intracellular trafficking pathways for targeting viral components to the assembly sites where the packaging of the viral RNA genetic material into virions occurs. (...)
Serena Bernacchi. Special Issue “Function and Structure of Viral Ribonucleoproteins Complexes”. Viruses 2020, 12, 1355 .
AMA StyleSerena Bernacchi. Special Issue “Function and Structure of Viral Ribonucleoproteins Complexes”. Viruses. 2020; 12 (12):1355.
Chicago/Turabian StyleSerena Bernacchi. 2020. "Special Issue “Function and Structure of Viral Ribonucleoproteins Complexes”." Viruses 12, no. 12: 1355.
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.
Emmanuel Boutant; Jeremy Bonzi; Halina Anton; Maaz Bin Nasim; Raphael Cathagne; Eléonore Réal; Denis Dujardin; Philippe Carl; Pascal Didier; Jean-Christophe Paillart; Roland Marquet; Yves Mély; Hugues de Rocquigny; Serena Bernacchi. Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane. Biophysical Journal 2020, 119, 419 -433.
AMA StyleEmmanuel Boutant, Jeremy Bonzi, Halina Anton, Maaz Bin Nasim, Raphael Cathagne, Eléonore Réal, Denis Dujardin, Philippe Carl, Pascal Didier, Jean-Christophe Paillart, Roland Marquet, Yves Mély, Hugues de Rocquigny, Serena Bernacchi. Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane. Biophysical Journal. 2020; 119 (2):419-433.
Chicago/Turabian StyleEmmanuel Boutant; Jeremy Bonzi; Halina Anton; Maaz Bin Nasim; Raphael Cathagne; Eléonore Réal; Denis Dujardin; Philippe Carl; Pascal Didier; Jean-Christophe Paillart; Roland Marquet; Yves Mély; Hugues de Rocquigny; Serena Bernacchi. 2020. "Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane." Biophysical Journal 119, no. 2: 419-433.
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.
Serena Bernacchi. Dynamic Light Scattering Analysis on RNA Associated to Proteins. Methods in Molecular Biology 2020, 2113, 31 -39.
AMA StyleSerena Bernacchi. Dynamic Light Scattering Analysis on RNA Associated to Proteins. Methods in Molecular Biology. 2020; 2113 ():31-39.
Chicago/Turabian StyleSerena Bernacchi. 2020. "Dynamic Light Scattering Analysis on RNA Associated to Proteins." Methods in Molecular Biology 2113, no. : 31-39.
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.
Serena Bernacchi; Eric Ennifar. Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry. Methods in Molecular Biology 2020, 2113, 237 -250.
AMA StyleSerena Bernacchi, Eric Ennifar. Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry. Methods in Molecular Biology. 2020; 2113 ():237-250.
Chicago/Turabian StyleSerena Bernacchi; Eric Ennifar. 2020. "Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry." Methods in Molecular Biology 2113, no. : 237-250.
The HIV-1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAsviaits 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 (WT) Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs), or a non-myristoylated 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 MP, was found to be dispensable for the association of Gag with the gRNA in the cytosol. STATEMENT of SIGNIFICANCE Formation of HIV-1 retroviral particles relies on specific interactions between the retroviral Gag precursor and the unspliced genomic RNA (gRNA). During the late phase of replication, Gag orchestrates the assembly of newly formed viruses at the plasma membrane (PM). It has been shown that the intracellular HIV-1 gRNA recognition is governed by the two-zinc finger (ZF) motifs of the nucleocapsid (NC) domain in Gag. Here we provided a clear picture of the role of ZFs in the cellular trafficking of Gag-gRNA complexes to the PM by showing that either ZF was sufficient to efficiently promote these interactions in the cytoplasm, while interestingly, ZF2 played a more prominent role in the relocation of these ribonucleoprotein complexes at the PM assembly sites.
Emmanuel Boutant; Jeremy Bonzi; Halina Anton; Maaz Bin Nasim; Raphael Cathagne; Eleonor Real; Denis Dujardin; Philippe Carl; Pascal Didier; Jean-Cristophe Paillart; Roland Marquet; Yves Mely; Hugues De Rocquigny; Serena Bernacchi. The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane. 2020, 1 .
AMA StyleEmmanuel Boutant, Jeremy Bonzi, Halina Anton, Maaz Bin Nasim, Raphael Cathagne, Eleonor Real, Denis Dujardin, Philippe Carl, Pascal Didier, Jean-Cristophe Paillart, Roland Marquet, Yves Mely, Hugues De Rocquigny, Serena Bernacchi. The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane. . 2020; ():1.
Chicago/Turabian StyleEmmanuel Boutant; Jeremy Bonzi; Halina Anton; Maaz Bin Nasim; Raphael Cathagne; Eleonor Real; Denis Dujardin; Philippe Carl; Pascal Didier; Jean-Cristophe Paillart; Roland Marquet; Yves Mely; Hugues De Rocquigny; Serena Bernacchi. 2020. "The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane." , no. : 1.
The Pr55Gag precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55Gag interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gag∆p6 is a truncated form of Pr55Gag lacking the p6 domain usually used as a default surrogate for wild type Pr55Gag for in vitro analysis. With recent advance in production of full-length recombinant Pr55Gag, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55Gag by systematically comparing binding of Pr55Gag and Gag∆p6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55Gag to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gag∆p6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55Gag and Gag∆p6 uniformly increased the Kd values of the RNA-protein complexes by ~ 2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55Gag-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation.
Noé Dubois; Keith K. Khoo; Shannon Ghossein; Tanja Seissler; Philippe Wolff; William J. McKinstry; Johnson Mak; Jean-Christophe Paillart; Roland Marquet; Serena Bernacchi. The C-terminal p6 domain of the HIV-1 Pr55Gag precursor is required for specific binding to the genomic RNA. RNA Biology 2018, 15, 923 -936.
AMA StyleNoé Dubois, Keith K. Khoo, Shannon Ghossein, Tanja Seissler, Philippe Wolff, William J. McKinstry, Johnson Mak, Jean-Christophe Paillart, Roland Marquet, Serena Bernacchi. The C-terminal p6 domain of the HIV-1 Pr55Gag precursor is required for specific binding to the genomic RNA. RNA Biology. 2018; 15 (7):923-936.
Chicago/Turabian StyleNoé Dubois; Keith K. Khoo; Shannon Ghossein; Tanja Seissler; Philippe Wolff; William J. McKinstry; Johnson Mak; Jean-Christophe Paillart; Roland Marquet; Serena Bernacchi. 2018. "The C-terminal p6 domain of the HIV-1 Pr55Gag precursor is required for specific binding to the genomic RNA." RNA Biology 15, no. 7: 923-936.
The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA) molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…), the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.
Noé Dubois; Roland Marquet; Jean-Christophe Paillart; Serena Bernacchi. Retroviral RNA Dimerization: From Structure to Functions. Frontiers in Microbiology 2018, 9, 527 .
AMA StyleNoé Dubois, Roland Marquet, Jean-Christophe Paillart, Serena Bernacchi. Retroviral RNA Dimerization: From Structure to Functions. Frontiers in Microbiology. 2018; 9 ():527.
Chicago/Turabian StyleNoé Dubois; Roland Marquet; Jean-Christophe Paillart; Serena Bernacchi. 2018. "Retroviral RNA Dimerization: From Structure to Functions." Frontiers in Microbiology 9, no. : 527.
The HIV-1 Pr55(Gag) precursor specifically selects genomic RNA (gRNA) from a large variety of cellular and spliced viral RNAs (svRNAs), however the molecular mechanisms of this selective recognition remains poorly understood. To gain better understanding of this process, we analyzed the interactions between Pr55(Gag) and a large panel of viral RNA (vRNA) fragments encompassing the main packaging signal (Psi) and its flanking regions by fluorescence spectroscopy. We showed that the gRNA harbors a high affinity binding site which is absent from svRNA species, suggesting that this site might be crucial for selecting the HIV-1 genome. Our stoichiometry analysis of protein/RNA complexes revealed that few copies of Pr55(Gag) specifically associate with the 5\u27 region of the gRNA. Besides, we found that gRNA dimerization significantly impacts Pr55(Gag) binding, and we confirmed that the internal loop of stem-loop 1 (SL1) in Psi is crucial for specific interaction with Pr55(Gag). Our analysis of gRNA fragments of different length supports the existence of a long-range tertiary interaction involving sequences upstream and downstream of the Psi region. This long-range interaction might promote optimal exposure of SL1 for efficient Pr55(Gag) recognition. Altogether, our results shed light on the molecular mechanisms allowing the specific selection of gRNA by Pr55(Gag) among a variety of svRNAs, all harboring SL1 in their first common exon
Serena Bernacchi; Ekram Abd El-Wahab; Noé Dubois; Marcel Hijnen; Redmond P. Smyth; Johnson Mak; Roland Marquet; Jean-Christophe Paillart. HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry. RNA Biology 2016, 14, 90 -103.
AMA StyleSerena Bernacchi, Ekram Abd El-Wahab, Noé Dubois, Marcel Hijnen, Redmond P. Smyth, Johnson Mak, Roland Marquet, Jean-Christophe Paillart. HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry. RNA Biology. 2016; 14 (1):90-103.
Chicago/Turabian StyleSerena Bernacchi; Ekram Abd El-Wahab; Noé Dubois; Marcel Hijnen; Redmond P. Smyth; Johnson Mak; Roland Marquet; Jean-Christophe Paillart. 2016. "HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry." RNA Biology 14, no. 1: 90-103.
Human immunodeficiency virus type 1 (HIV-1) replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA) by the viral Pr55Gag precursor polyprotein, and the processes leading to its incorporation into viral particles.
Elodie Mailler; Serena Bernacchi; Roland Marquet; Jean-Christophe Paillart; Valérie Vivet-Boudou; Redmond P. Smyth. The Life-Cycle of the HIV-1 Gag–RNA Complex. Viruses 2016, 8, 248 .
AMA StyleElodie Mailler, Serena Bernacchi, Roland Marquet, Jean-Christophe Paillart, Valérie Vivet-Boudou, Redmond P. Smyth. The Life-Cycle of the HIV-1 Gag–RNA Complex. Viruses. 2016; 8 (9):248.
Chicago/Turabian StyleElodie Mailler; Serena Bernacchi; Roland Marquet; Jean-Christophe Paillart; Valérie Vivet-Boudou; Redmond P. Smyth. 2016. "The Life-Cycle of the HIV-1 Gag–RNA Complex." Viruses 8, no. 9: 248.
HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT.
Pierre-Jean Racine; Celia Chamontin; Hugues De Rocquigny; Serena Bernacchi; Jean-Christophe Paillart; Marylène Mougel. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly. Scientific Reports 2016, 6, 27536 .
AMA StylePierre-Jean Racine, Celia Chamontin, Hugues De Rocquigny, Serena Bernacchi, Jean-Christophe Paillart, Marylène Mougel. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly. Scientific Reports. 2016; 6 (1):27536.
Chicago/Turabian StylePierre-Jean Racine; Celia Chamontin; Hugues De Rocquigny; Serena Bernacchi; Jean-Christophe Paillart; Marylène Mougel. 2016. "Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly." Scientific Reports 6, no. 1: 27536.
RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55Gag protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding. (a) The specific recognition of the 5′ region of the HIV-1 genomic RNA by the structural protein Pr55Gag as a model of protein-RNA interaction. The 5′ region of the HIV-1 genomic RNA folds into several independent structural domains. From 5′ to 3′, these are the transactivation response (TAR) element (which mediates efficient transcription), polyadenylation signal (PolyA), primer-binding site (PBS) domain and four closely spaced stem-loop structures termed SL1–SL4. SL1 promotes the dimerization of genomic RNA, SL2 contains the major splice donor (SD) site, SL3 has historically been considered the major packaging signal (Psi) and SL4 forms an unstable stem loop containing the gag AUG translation initiation codon. The HIV-1 structural protein Pr55Gag is comprised of matrix (MA), capsid (CA) and nucleocapsid (NC) domains, as well as the p6 terminal domain. (b–d) MIME relies on the random introduction of mutations into an RNA target, the physical separation of functional from nonfunctional RNA and the quantification of RNA mutations in each population using next-generation sequencing. (b) Mutations (red circles) are introduced by error-prone PCR into the 5′ region of the HIV-1 genomic RNA (nucleotides 1–532). Inclusion of a T7 initiation site in the 5′ primer (red solid line) allows RNA (black dashed line) to be generated directly from the PCR product. (c) Functional RNA is selected from nonfunctional RNA using a His-tagged protein and magnetic beads. (d) RNA is reverse transcribed into DNA (black solid line) and randomly fragmented into 100- to 500-bp fragments. Illumina-specific adaptors (green solid line) and sample-specific barcodes to be used in multiplexing are added to the fragmented DNA, and the fragments are sequenced on an Illumina HiSeq 2000 instrument in paired-end 100 mode. Full size image View in article (a) At low Pr55Gag concentrations, low-affinity mutant RNA (red circles) may not be bound in sufficient amounts, meaning that mutations that further impair binding may not be detected in the protein-bound fraction. Conversely, at high Pr55Gag concentrations, high-affinity mutant RNA (blue triangles) may be completely protein bound. (b) The ratio between detected mutation frequencies in the unbound and bound fractions for three different Pr55Gag/RNA ratios. (c) RNAs with distinct mutations compete for Pr55Gag binding according to the law of mass action. The relative binding affinity of mutated (indicated with stars) versus wild-type (no stars) RNA (Kdm(i)/Kdw(i)) can be computed from the ratio-of-ratios (bound versus unbound and wild-type versus mutant RNA, see equations (2)–(4)). (d) Statistical attributes of relative binding affinities Kdm(i)/Kdw(i) are obtained through a resampling procedure: the effect of mutation m at position i is recomputed for all positions j, where position j is wild-type w (see equation (6)). Since the number of sequence reads that span position i and j decreases as the distance between i and j increases, this is analogous to a jackknife resampling procedure. For robustness, we only calculated Kd values for pairs of positions that have at least 50% coverage relative to i. (e) The relative binding affinity of the mutations 'm(max)' that maximally affect binding at nucleotide position i, expressed as log2(Kdm(max),w(i,*)/Kdw,w(i,*)) are assessed, where * indicates that all eligible wild-type positions j are considered. Medians of the maximally affecting mutations are depicted as red lines. The 5th–95th percentiles are in light gray and the 25th–75th percentiles are in dark gray. (f) P values computed from the resampling procedure (equations (7) and (8)) after correction by the false-discovery rate method of Benjamini and Hochberg (BHFDR) show positions where Pr55Gag binding is significantly decreased upon mutation m(max) at position i in the viral RNA (P > 0.05, small black dots; P < 0.05, open circles; P < 0.01, closed circles; P < 0.001, large black dots). Data was obtained from the use of two independent libraries. A detailed explanation of the procedure is described in Supplementary Note 2. Full size image View in article (a–c) Single-variation analysis can detect nucleotide positions involved in Watson-Crick and wobble base pairing, and this may distinguish between various proposed structural models of the HIV-1 RNA genome. Green and yellow circles denote A and C nucleotides in which structure-affecting mutations significantly impair Pr55Gag binding and structure-preserving mutations impair Pr55Gag binding to an extent markedly less than all (green) or one (yellow) of the other structure-affecting mutations. Blue circles denote G and U nucleotides where structure-affecting mutations significantly impair Pr55Gag binding and in which structure-preserving mutations preserve or improve Pr55Gag binding. Numerical results for the statistical tests can be found in Supplementary Data 3. (a) Model showing an extended SL1, a short SL2 helix and a long-range interaction...
Redmond Smyth; Laurence Despons; Gong Huili; Serena Bernacchi; Marcel Hijnen; Johnson Mak; Fabrice Jossinet; Li Weixi; Jean-Christophe Paillart; Max von Kleist; Roland Marquet. Mutational interference mapping experiment (MIME) for studying RNA structure and function. Nature Methods 2015, 12, 866 -872.
AMA StyleRedmond Smyth, Laurence Despons, Gong Huili, Serena Bernacchi, Marcel Hijnen, Johnson Mak, Fabrice Jossinet, Li Weixi, Jean-Christophe Paillart, Max von Kleist, Roland Marquet. Mutational interference mapping experiment (MIME) for studying RNA structure and function. Nature Methods. 2015; 12 (9):866-872.
Chicago/Turabian StyleRedmond Smyth; Laurence Despons; Gong Huili; Serena Bernacchi; Marcel Hijnen; Johnson Mak; Fabrice Jossinet; Li Weixi; Jean-Christophe Paillart; Max von Kleist; Roland Marquet. 2015. "Mutational interference mapping experiment (MIME) for studying RNA structure and function." Nature Methods 12, no. 9: 866-872.
Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication.
Santiago Guerrero; Julien Batisse; Camille Libre; Serena Bernacchi; Roland Marquet; Jean-Christophe Paillart. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus. Viruses 2015, 7, 199 -218.
AMA StyleSantiago Guerrero, Julien Batisse, Camille Libre, Serena Bernacchi, Roland Marquet, Jean-Christophe Paillart. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus. Viruses. 2015; 7 (1):199-218.
Chicago/Turabian StyleSantiago Guerrero; Julien Batisse; Camille Libre; Serena Bernacchi; Roland Marquet; Jean-Christophe Paillart. 2015. "HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus." Viruses 7, no. 1: 199-218.
The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.
Dona Sleiman; Serena Bernacchi; Santiago Guerrero; Franck Brachet; Valéry Larue; Jean-Christophe Paillart; Carine Tisné. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. RNA Biology 2014, 11, 906 -920.
AMA StyleDona Sleiman, Serena Bernacchi, Santiago Guerrero, Franck Brachet, Valéry Larue, Jean-Christophe Paillart, Carine Tisné. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. RNA Biology. 2014; 11 (7):906-920.
Chicago/Turabian StyleDona Sleiman; Serena Bernacchi; Santiago Guerrero; Franck Brachet; Valéry Larue; Jean-Christophe Paillart; Carine Tisné. 2014. "Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein." RNA Biology 11, no. 7: 906-920.
During assembly of HIV-1 particles in infected cells, the viral Pr55Gag protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55Gag achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55Gag binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55Gag to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.
Ekram Abd El-Wahab; Redmond Smyth; Elodie Mailler; Serena Bernacchi; Valérie Vivet-Boudou; Marcel Hijnen; Fabrice Jossinet; Johnson Mak; Jean-Christophe Paillart; Roland Marquet. Specific recognition of the HIV-1 genomic RNA by the Gag precursor. Nature Communications 2014, 5, 4304 .
AMA StyleEkram Abd El-Wahab, Redmond Smyth, Elodie Mailler, Serena Bernacchi, Valérie Vivet-Boudou, Marcel Hijnen, Fabrice Jossinet, Johnson Mak, Jean-Christophe Paillart, Roland Marquet. Specific recognition of the HIV-1 genomic RNA by the Gag precursor. Nature Communications. 2014; 5 (1):4304.
Chicago/Turabian StyleEkram Abd El-Wahab; Redmond Smyth; Elodie Mailler; Serena Bernacchi; Valérie Vivet-Boudou; Marcel Hijnen; Fabrice Jossinet; Johnson Mak; Jean-Christophe Paillart; Roland Marquet. 2014. "Specific recognition of the HIV-1 genomic RNA by the Gag precursor." Nature Communications 5, no. 1: 4304.
Mannoside glycolipid conjugates are able to inhibit human immunodeficiency virus type 1 (HIV-1) trans-infection mediated by human dendritic cells (DCs). The conjugates are formed by three building blocks: a linear or branched mannose head, a hydrophilic linker, and a 24-carbon lipid chain. We have shown that, even as single molecules, these compounds efficiently target mannose-binding lectins, such as DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) important for HIV-1 transmission. With the goal to optimize their inhibitory activity by supramolecular structure formation, we have compared saturated and unsaturated conjugates, as single molecules, self-assemblies of dynamic micelles, and photopolymerized cross-linked polymers. Surface plasmon resonance showed that, unexpectedly, polymers of trivalent conjugates did not display a higher binding affinity for DC-SIGN than single molecules. Interactions on a chip or in solution were independent of calcium; however, binding to DCs was inhibited by a calcium chelator. Moreover, HIV-1 trans-infection was mostly inhibited by dynamic micelles and not by rigid polymers. The inhibition data revealed a clear correlation between the structure and molecular assembly of a conjugate and its biological antiviral activity. We present an interaction model between DC-SIGN and conjugates—either single molecules, micelles, or polymers—that highlights that the most effective interactions by dynamic micelles involve both mannose heads and lipid chains. Our data reveal that trivalent glycolipid conjugates display the highest microbicide potential for HIV prophylaxis, as dynamic micelles conjugates and not as rigid polymers.
Evelyne Schaeffer; Laure Dehuyser; David Sigwalt; Vincent Flacher; Serena Bernacchi; Olivier Chaloin; Jean-Serge Remy; Christopher G. Mueller; Rachid Baati; Alain Wagner. Dynamic Micelles of Mannoside Glycolipids are more Efficient than Polymers for Inhibiting HIV-1 trans-Infection. Bioconjugate Chemistry 2013, 24, 1813 -1823.
AMA StyleEvelyne Schaeffer, Laure Dehuyser, David Sigwalt, Vincent Flacher, Serena Bernacchi, Olivier Chaloin, Jean-Serge Remy, Christopher G. Mueller, Rachid Baati, Alain Wagner. Dynamic Micelles of Mannoside Glycolipids are more Efficient than Polymers for Inhibiting HIV-1 trans-Infection. Bioconjugate Chemistry. 2013; 24 (11):1813-1823.
Chicago/Turabian StyleEvelyne Schaeffer; Laure Dehuyser; David Sigwalt; Vincent Flacher; Serena Bernacchi; Olivier Chaloin; Jean-Serge Remy; Christopher G. Mueller; Rachid Baati; Alain Wagner. 2013. "Dynamic Micelles of Mannoside Glycolipids are more Efficient than Polymers for Inhibiting HIV-1 trans-Infection." Bioconjugate Chemistry 24, no. 11: 1813-1823.
The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55Gag. Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain (161PPLP164) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55Gag, Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.
Julien Batisse; Santiago Guerrero; Serena Bernacchi; Ludovic Richert; Julien Godet; Valérie Goldschmidt; Yves Mély; Roland Marquet; Hugues De Rocquigny; Jean-Christophe Paillart. APOBEC3G Impairs the Multimerization of the HIV-1 Vif Protein in Living Cells. Journal of Virology 2013, 87, 6492 -6506.
AMA StyleJulien Batisse, Santiago Guerrero, Serena Bernacchi, Ludovic Richert, Julien Godet, Valérie Goldschmidt, Yves Mély, Roland Marquet, Hugues De Rocquigny, Jean-Christophe Paillart. APOBEC3G Impairs the Multimerization of the HIV-1 Vif Protein in Living Cells. Journal of Virology. 2013; 87 (11):6492-6506.
Chicago/Turabian StyleJulien Batisse; Santiago Guerrero; Serena Bernacchi; Ludovic Richert; Julien Godet; Valérie Goldschmidt; Yves Mély; Roland Marquet; Hugues De Rocquigny; Jean-Christophe Paillart. 2013. "APOBEC3G Impairs the Multimerization of the HIV-1 Vif Protein in Living Cells." Journal of Virology 87, no. 11: 6492-6506.
The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.
Julien Batisse; Santiago Guerrero; Serena Bernacchi; Dona Sleiman; Caroline Gabus; Jean-Luc Darlix; Roland Marquet; Carine Tisne; Jean-Christophe Paillart. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication. Virus Research 2012, 169, 361 -376.
AMA StyleJulien Batisse, Santiago Guerrero, Serena Bernacchi, Dona Sleiman, Caroline Gabus, Jean-Luc Darlix, Roland Marquet, Carine Tisne, Jean-Christophe Paillart. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication. Virus Research. 2012; 169 (2):361-376.
Chicago/Turabian StyleJulien Batisse; Santiago Guerrero; Serena Bernacchi; Dona Sleiman; Caroline Gabus; Jean-Luc Darlix; Roland Marquet; Carine Tisne; Jean-Christophe Paillart. 2012. "The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication." Virus Research 169, no. 2: 361-376.