RNA Control of HIV-1 Particle Size Polydispersity

HIV-1, an enveloped RNA virus, produces viral particles that are known to be much more heterogeneous in size than is typical of non-enveloped viruses. We present here a novel strategy to study HIV-1 Viral Like Particles (VLP) assembly by measuring the size distribution of these purified VLPs and subsequent viral cores thanks to Atomic Force Microscopy imaging and statistical analysis. This strategy allowed us to identify whether the presence of viral RNA acts as a modulator for VLPs and cores size heterogeneity in a large population of particles. These results are analyzed in the light of a recently proposed statistical physics model for the self-assembly process. In particular, our results reveal that the modulation of size distribution by the presence of viral RNA is qualitatively reproduced, suggesting therefore an entropic origin for the modulation of RNA uptake by the nascent VLP

Identification and characterization of a new family of restriction factors - IFITMs

Le VIH, comme tous les virus, nécessite la coopération de nombreuses protéines cellulaires pour réaliser son cycle viral. Néanmoins, un panel de protéines est spécifiquement dédié à la protection des cellules et ce, de façon autonome. Ces protéines sont nommées facteurs de restriction. Jusqu’à présent, un grand nombre de protéines ayant une action antivirale plus ou moins importante contre le VIH a été identifié. Seules quelques-Unes sont bien étudiées telles que APOBEC3G, Tétherine, TRIM5α ou SamHD1. Toutefois, ces facteurs de restriction ne permettent pas à eux seuls d’expliquer la permissivité de tous les types cellulaires. Notre étude a donc pour objet la découverte de nouvelles protéines agissant contre le VIH. Parmi un grand nombre de candidats, nous avons identifié une famille de protéines agissant contre le VIH : les IFITMs. Il a été précédemment décrit que l’expression des IFITMs protège les cellules de l’infection par le VIH mais aussi par de nombreux autres virus, filovirus, coronavirus, flavivirus ou encore paramyxovirus. Nous décrivons ici un nouveau mécanisme antiviral mis en place par les IFITMs. L’expression des IFITMs dans les cellules productrices de virus affecte la production de particules infectieuses. D’une part, moins de particules sont produites et d’autre part les particules sont moins infectieuses. Nous nous sommes focalisés sur le défaut d’infection et celui-Ci corrèle avec l’incorporation des IFITMs à la membrane des virions. Les virus produits et portant les IFITMs présentent un défaut lors du processus d’entrée dans la cellule cible au cours de l’étape de fusion. Ceci n’est pas dû à un défaut d’incorporation de l’enveloppe virale, mais le mécanisme précis n’est pas encore compris. Il apparaît donc que les IFITMs peuvent jouer un double rôle restrictif, protégeant les cellules où ils sont exprimés et diminuant la production de particules infectieuses.HIV, as all viruses, hijacks the cellular machinery to its benefits. Nevertheless, a range of proteins protects cells by an autonomous way, they are called restriction factor and belong to a system called “the intrinsic immunity”. Up to now, a certain number of cellular proteins, most of which belong to this defense system, have been described to be more or less detrimental for HIV replication, as for example, APOBEC3G, Tetherin, TRIM5α and SamHD1. Given that a large number of cellular restriction factors are likely to be discovered, we started to screen to look for new ones. Among a lot of candidates, we have identified a family of proteins interfering with HIV: the IFITMs. Previously, studies described IFITMs as proteins protecting cell against viral entry and fighting a lot of viruses, including HIV. In this thesis, we described a new antiviral mechanism mediated by IFITMs against retroviruses. When IFITMs are expressed in cells producing new virions, there is a high defect of infectious particle release due to a defect of particle release and a defect of the infectivity of particles. We focus on the intrinsic defect of infectivity and we show that particles cannot anymore enter into target cells and this observation is correlated with the incorporation of IFITMs in viral membranes. The defect is located at the fusion step and is not explained by a defect of envelope incorporation.To conclude, IFITMs have a double restrictive effect, protecting cells from incoming viruses and decreasing the production of infectious particles

IFITM, a common barrier to many viruses

International audienceDuring evolution, organisms developed adaptative mechanisms to survive continuous aggressions from a variety of pathogens. Among these lines of defence, many cellular proteins have been described to modulate viral replication and are the subject of intense study. This review will focus on IFITM (interferon induced transmembrane protein), a family of proteins that act against a particularly wide range of viruses. We will summarize our knowledge of the antiviral mechanisms used by IFITM to interfere with the replication of several viruses, and more specifically HIV (human immunodeficiency virus)

A Novel Entry/Uncoating Assay Reveals the Presence of at Least Two Species of Viral Capsids During Synchronized HIV-1 Infection

International audienceTo better characterize the behavior of HIV-1 capsids we developed EURT, for Entry/Uncoating assay based on core-packaged RNA availability and Translation. EURT is an alternative to Blam-Vpr, but as reporter RNA translation relies on core opening, it can be used to study viral capsids behavior. Our study reveals the existence of two major capsid species, a dead end one in which the viral genome is readily exposed to the cytoplasm and a functional one in which such exposure requires artificial core destabilization. Although reverse transcription drives a faster loss of susceptibility of viral cores to high doses of PF74, it does not lead to higher exposure of the viral genome, implying that viral cores protect the genome irrespectively of reverse transcription. Lastly, IFNα drifts cores from functional to non-functional species, revealing a novel core-destabilizing activity. This assay sheds new light on the behavior of viral cores inside target cells

Contribution of reverse transcription to the susceptibility of viral cores to high doses of PF74.

<p>A) Dual reporter viral particles were produced by co-transfection of HEK293T cells with DNAs coding Gag-Pol, Env along with the two genomes: EU-repRNA and pRRL-GFP that contains all the elements required for its mobilization in target cells. To favor co-packaging of EU-repRNA in reverse transcription competent viral particles, a ratio of 9 to 1 was used between pRRL and EU-repRNA. <i>CMV</i>, cytomegalovirus promoter; <i>LTR</i>, long terminal repeats; <i>Ψ</i>, packaging sequence; <i>PBS</i>, primer binding sequence; <i>RRE</i>, Rev-responsive element; <i>cPPT-CTS</i>, central polypurine tract-central termination sequence; <i>PPT</i>, polypurine tract. B) Dual reporter viral particles containing a wild type or a mutated RT (Q151N) were used to challenge HeLaP4 cells in the presence or absence of Nevirapine (10 μM). PF74 was added at 23 μM at the indicated time post infection and the overall amount of luciferase activity was measured at ten hours post infection. Values are normalized for the ones observed in the absence of PF74. The graphs present AVG and SEM obtained from 4 to 9 independent experiments. *, statistically significant differences following a Student t test (p≤0.05). C) The graph presents the decrease in the amount of PF74-induced luciferase activity in the individual experiments that compose Fig 8B at t3 and t4. D) The graph presents the percentage of PF74-induced luciferase activity lost in reverse transcription competent viruses over reverse transcription incompetent ones (data of Fig 8B, pooling t3 and t4 data points).</p

Time dependent accumulation of Luciferase over time and influence of Nevirapine and different concentrations of PF74.

<p>A) To determine the extent to which reverse transcription could expose the viral genome and therefore increase the accumulation of Luciferase activity, dual reporter and reverse transcription competent viral particles were used to challenge HeLaP4 cells according to the scheme provided in A. B) For each condition, the amount of Luciferase that accumulated over time is provided after normalization to the activity measured 1 hour post infection. The graph presents AVG and SEM of 8 independent experiments.</p

PF74 induces increased Eu-repRNA availability to translation in purified HIV-1 cores <i>in vitro</i>.

<p>A) Cartoon presenting the experimental setup used to purify viral cores. Briefly, virion particles are passed by ultracentrifugation first through a 10% sucrose cushion containing detergent that deprives the virus of its membrane, and then through a 25% sucrose cushion through which viral cores pellet. Due to their fragility, viral cores are then either immediately analyzed by WB (B) or resuspended in rabbit reticulocyte lysate (RRL) in the presence of PF74 for 30 minutes prior to Luciferase measurement (C). The panel and the graph present data obtained from 3 independent experiments. As control for possible effects of PF74 on RRL activity, a control mRNA coding Luc (ctl mRNA) was used in parallel. *statistically significant differences following a Student t test (p≤0.05).</p

Effects of time dependent addition of high doses of PF74 on EURT and viral infectivity.

<p>A) Experimental scheme used here. Depending on the input virus (containing EU-repRNA, or a miniviral genome competent for a single round of infection and coding GFP), cells were either lysed at 10 hours post infection for EURT (B), or analyzed three days afterwards by flow cytometry (C). The graphs present averages and SEM of four independent experiments.</p

IFNα-treatment of MDDCs decreases the proportion of PF74-dependent luciferase activity that can be measured following EURT assay.

<p>A) HIV-1 viral particles bearing X4- or R5-tropic envelopes were used to challenge either HeLa P4 or primary MDDCs treated for 24 hours prior to viral challenge with the indicated concentration of IFNα. Infections were carried out in the presence or absence of PF74 (10 μg/mL, 23 μM). Luciferase accumulation was then measured as described before. Averages and SEM are presented here after normalization to the values obtained in untreated cells. The pie charts above the graph present the relative proportions of PF74-dependent and independent activities measured in the presence or absence of IFNα. B) The graph present individual donors (n = 8 for MDDCs and 4 independent experiments for HeLaP4 cells) that were used in the left panel, with a focus on the decrease in the percentage of PF74-dependent activity measured following incubation with IFNα. *statistically significant differences following a Student t test (p≤0.05).</p

Comparison of the EURT assay with the gold standard in HIV entry, the Blam-Vpr assay.

<p>To compare the EURT assay with the widely used Blam-Vpr assay, virions were produced by transfection of HEK293T cells using either reporters. A) Viruses were produced with an R5- or an X4- tropic Env (JR-FL and NL4-3, respectively), then used to challenge SupT1 and monocyte-derived dendritic cells (MDDCs, derived upon incubation of blood monocytes with GM-CSF and IL4 for 4 days). Envelope-receptor specificity was then measured according to the two viral entry assays in lymphocytes (SupT1) and in myeloid cells (MDDCs) either after cell lysis and luciferase activity measurement, or following the incubation of target cells with the fluorescent dye CCF2 and flow cytometry. B) SupT1 were used as target cells to compare the inhibitory rates that could be retrieved upon usage of Vpr-Blam- or EU-repRNA bearing HIV-1 X4 particles in the presence of the HIV-1 fusion inhibitor T20. The graphs present averages and SEM obtained with 3 independent experiments. * p≤0.05 after a Student t test between WT and the respective indicated conditions.</p