Entropic control of particle sizes during viral self-assembly

Morphologic diversity is observed across all families of viruses. Yet these supra-molecular assemblies are produced most of the time in a spontaneous way through complex molecular self-assembly scenarios. The modeling of these phenomena remains a challenging problem within the emerging field of Physical Virology. We present in this work a theoretical analysis aiming at highlighting the particular role of configuration entropy in the control of viral particle size distribution. Specializing this model to retroviruses like HIV-1, we predict a new mechanism of entropic control of both RNA uptake into the viral particle, and of the particle's size distribution. Evidence of this peculiar behavior has been recently reported experimentally

Dual effect of the SR proteins ASF/SF2, SC35 and 9G8 on HIV-1 RNA splicing and virion production

In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication

A role for the tetraspanin CD81 on the late steps of HIV-1 replication in T-lymphoblastic cells

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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

Dual effect of the SR proteins ASF/SF2, SC35 and 9G8 on HIV-1 RNA splicing and virion production

In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication

Specific lipid recruitment by the retroviral gag protein upon HIV-1 assembly : from model membranes to infected cells

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. Here, we addressed the question whether HIV-1 Gag was able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in silico, in vitro on reconstituted membranes and in cellulo at the plasma membrane of the host CD4+ T cells. In silico, we could observe the first PI(4,5)P2 preferential recruitment by HIV-1 MA or Gag while protein docked on artificial membranes. In vitro, using biophysical techniques, we observed the specific trapping of PI(4,5)P2, and, to a lesser extent, cholesterol and the exclusion of sphingomyelin, during HIV-1 myr(-)Gag self-assembly on LUVs and SLBs. Finally, in infected living CD4+ T cells, we measured lipid dynamics within and away from HIV-1 assembly sites using super-resolution stimulated emission depletion (STED) microscopy coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). The analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol but not phosphatidylethanolamine (PE) or sphingomyelin (SM) at the cellular membrane. Furthermore, analyzing CD4+ T cells expressing only HIV-1 Gag protein showed that Gag is the main driving force restricting the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. Our data provide the first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly by selectively recruiting lipids to generate PI(4,5)P2/cholesterol-enriched nanodomains favoring virus assembly, and that HIV-1 does not assemble on pre-existing lipid domains

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

BACKGROUND: The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly. RESULTS: We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. CONCLUSION: These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses

Targeting of MuLV Gag to the plasma membrane is mediated by PI(4,5)P2 and PhosphatidylSerine

Oral presentationInternational audienceMembrane targeting by the modern human immunodeficiency viruses is dependent on the plasma membrane-located phospholipid PI(4,5)P2. In order to determine if evolutionarily distant retroviruses are targeted by a similar mechanism, we generated mutant Gag constructs in the matrix (MA) domain of the Murine Leukemia Virus (MuLV) and examined their binding to membrane models and phenotypes in cell culture. Mutations in the MA polybasic region altered Gag localization, membrane binding and virion production. In addition, we show that MA binds with good affinity to all the phosphatidylinositol phosphates but displays a strong specificity for PI(4,5)P2 only if enhanced by phophatidylserine. Virus production was strongly impaired by PI(4,5)P2 depletion under 5ptaseIV overexpression. Our results suggest that the N-terminal polybasic region of MA is essential for Gag targeting to the plasma membrane and Gag cellular trafficking. The binding of the MA domain to PI(4,5)P2 appears to be a conserved feature among retroviruses, despite the fact that the MuLV-MA domain is structurally different from that of HIV-1 and -2 and lacks a readily identifiable PI(4,5)P2 binding cleft