The HIV-1 Pr55 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 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 specifically associate with the 5' region of the gRNA. Besides, we found that gRNA dimerization significantly impacts Pr55 binding, and we confirmed that the internal loop of stem-loop 1 (SL1) in Psi is crucial for specific interaction with Pr55. 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 recognition. Altogether, our results shed light on the molecular mechanisms allowing the specific selection of gRNA by Pr55 among a variety of svRNAs, all harboring SL1 in their first common exon.
Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55 gag to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55 gag with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.
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.
A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phaseseparated 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 ~2400 Gag and ~120 GagPol by 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.
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.
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.
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