Molecular mechanisms behind the generation of pro-oncogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s), characterized by amino acid insertions at the COOH-terminal region of the viral protein, have been recently identified and studied for their biological activity. Different from their wild-type counterpart (refp17), vp17s display a potent B cell growth and clonogenic activity. Recent data have highlighted the higher prevalence of vp17s in people living with HIV-1 (PLWH) with lymphoma compared with those without lymphoma, suggesting that vp17s may play a key role in lymphomagenesis. Molecular mechanisms involved in vp17 development are still unknown. Here we assessed the efficiency of HIV-1 Reverse Transcriptase (RT) in processing this genomic region and highlighted the existence of hot spots of mutation in Gag, at the end of the matrix protein and close to the matrix-capsid junction. This is possibly due to the presence of inverted repeats and palindromic sequences together with a high content of Adenine in the 322-342 nucleotide portion, which constrain HIV-1 RT to pause on the template. To define the recombinogenic properties of hot spots of mutation in the matrix gene, we developed plasmid vectors expressing Gag and a minimally modified Gag variant, and measured homologous recombination following cell co-nucleofection by next-generation sequencing. Data obtained allowed us to show that a wide range of recombination events occur in concomitance with the identified hot spots of mutation and that imperfect events may account for vp17s generation.

VLPs generated by the fusion of RSV-F or hMPV-F glycoprotein to HIV-Gag show improved immunogenicity and neutralizing response in mice.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccines have been long overdue. Structure-based vaccine design created a new momentum in the last decade, and the first RSV vaccines have finally been approved in older adults and pregnant individuals. These vaccines are based on recombinant stabilized pre-fusion F glycoproteins administered as soluble proteins. Multimeric antigenic display could markedly improve immunogenicity and should be evaluated in the next generations of vaccines. Here we tested a new virus like particles-based vaccine platform which utilizes the direct fusion of an immunogen of interest to the structural human immunodeficient virus (HIV) protein Gag to increase its surface density and immunogenicity. We compared, in mice, the immunogenicity of RSV-F or hMPV-F based immunogens delivered either as soluble proteins or displayed on the surface of our VLPs. VLP associated F-proteins showed better immunogenicity and induced superior neutralizing responses. Moreover, when combining both VLP associated and soluble immunogens in a heterologous regimen, VLP-associated immunogens provided added benefits when administered as the prime immunization.

Intracellular trafficking of HIV-1 Gag via Syntaxin 6-positive compartments/vesicles: Involvement in tumor necrosis factor secretion.

HIV-1 Gag protein is synthesized in the cytosol and is transported to the plasma membrane, where viral particle assembly and budding occur. Endosomes are alternative sites of Gag accumulation. However, the intracellular transport pathways and carriers for Gag have not been clarified. We show here that Syntaxin6 (Syx6), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane fusion in post-Golgi networks, is a molecule responsible for Gag trafficking and also for tumor necrosis factor-α (TNFα) secretion and that Gag and TNFα are cotransported via Syx6-positive compartments/vesicles. Confocal and live-cell imaging revealed that Gag colocalized and cotrafficked with Syx6, a fraction of which localizes in early and recycling endosomes. Syx6 knockdown reduced HIV-1 particle production, with Gag distributed diffusely throughout the cytoplasm. Coimmunoprecipitation and pulldown show that Gag binds to Syx6, but not its SNARE partners or their assembly complexes, suggesting that Gag preferentially binds free Syx6. The Gag matrix domain and the Syx6 SNARE domain are responsible for the interaction and cotrafficking. In immune cells, Syx6 knockdown/knockout similarly impaired HIV-1 production. Interestingly, HIV-1 infection facilitated TNFα secretion, and this enhancement did not occur in Syx6-depleted cells. Confocal and live-cell imaging revealed that TNFα and Gag partially colocalized and were cotransported via Syx6-positive compartments/vesicles. Biochemical analyses indicate that TNFα directly binds the C-terminal domain of Syx6. Altogether, our data provide evidence that both Gag and TNFα make use of Syx6-mediated trafficking machinery and suggest that Gag expression does not inhibit but rather facilitates TNFα secretion in HIV-1 infection.

Intrinsic resistance of HIV-2 and SIV to the maturation inhibitor GSK2838232.

GSK2838232 (GSK232) is a novel maturation inhibitor that blocks the proteolytic cleavage of HIV-1 Gag at the junction of capsid and spacer peptide 1 (CA/SP1), rendering newly-formed virions non-infectious. To our knowledge, GSK232 has not been tested against HIV-2, and there are limited data regarding the susceptibility of HIV-2 to other HIV-1 maturation inhibitors. To assess the potential utility of GSK232 as an option for HIV-2 treatment, we determined the activity of the compound against a panel of HIV-1, HIV-2, and SIV isolates in culture. GSK232 was highly active against HIV-1 isolates from group M subtypes A, B, C, D, F, and group O, with IC50 values ranging from 0.25-0.92 nM in spreading (multi-cycle) assays and 1.5-2.8 nM in a single cycle of infection. In contrast, HIV-2 isolates from groups A, B, and CRF01_AB, and SIV isolates SIVmac239, SIVmac251, and SIVagm.sab-2, were highly resistant to GSK232. To determine the role of CA/SP1 in the observed phenotypes, we constructed a mutant of HIV-2ROD9 in which the sequence of CA/SP1 was modified to match the corresponding sequence found in HIV-1. The resulting variant was fully susceptible to GSK232 in the single-cycle assay (IC50 = 1.8 nM). Collectively, our data indicate that the HIV-2 and SIV isolates tested in our study are intrinsically resistant to GSK232, and that the determinants of resistance map to CA/SP1. The molecular mechanism(s) responsible for the differential susceptibility of HIV-1 and HIV-2/SIV to GSK232 require further investigation.

High frequency of CD8 escape mutations in elite controllers as new obstacle for HIV cure.

Accumulation of mutations in epitopes of cytolytic-T-lymphocytes immune response (CTL) in HIV-reservoir seems to be one of the reasons for shock-and-kill strategy failure. Ten non-controller patients on successful cART (TX) and seven elite controllers (EC) were included. HIV-Gag gene from purified resting memory CD4+ T-cells was sequenced by Next-Generation-Sequencing. HLA class-I alleles were typed to predict optimal HIV-Gag CTL epitopes. For each subject, the frequency of mutated epitopes in the HIV-Gag gene, the proportion of them considered as CTL-escape variants as well as their effect on antigen recognition by HLA were assessed. The proportion (%) of mutated HIV-Gag CTL epitopes in the reservoir was high and similar in EC and TX (86%[50-100] and 57%[48-82] respectively, p=0.315). Many of them were predicted to negatively impact antigen recognition. Moreover, the proportion of mutated epitopes considered to be CTL-escape variants was also similar in TX and EC (77%[49-92] vs. 50%[33-75] respectively, p=0.117). Thus, the most relevant finding of our study was the high and similar proportions of HIV-Gag CTL-escape mutations in the reservoir of both HIV-noncontroller patients with cART (TX) and patients with spontaneous HIV-control (EC). Our findings suggest that escape mutations of CTL-response may be another obstacle to eliminate the HIV reservoir and constitute a proof of concept that challenges HIV cure strategies focused on the reactivation of reservoirs. Due to the small sample size that could impact the robustness of the study, further studies with larger cohorts of elite controller patients are needed to confirm these results.

Upstream of N-Ras (Unr/CSDE1) Interacts with NCp7 and Gag, Modulating HIV-1 IRES-Mediated Translation Initiation.

The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC) as a mature protein or as a domain of the Gag precursor plays important roles in the early and late phases of the infection. To better understand its roles, we searched for new cellular partners and identified the RNA-binding protein Unr/CSDE1, Upstream of N-ras, whose interaction with Gag and NCp7 was confirmed by co-immunoprecipitation and FRET-FLIM. Unr interaction with Gag was found to be RNA-dependent and mediated by its NC domain. Using a dual luciferase assay, Unr was shown to act as an ITAF (IRES trans-acting factor), increasing the HIV-1 IRES-dependent translation. Point mutations of the HIV-1 IRES in a consensus Unr binding motif were found to alter both the IRES activity and its activation by Unr, suggesting a strong dependence of the IRES on Unr. Interestingly, Unr stimulatory effect is counteracted by NCp7, while Gag increases the Unr-promoted IRES activity, suggesting a differential Unr effect on the early and late phases of viral infection. Finally, knockdown of Unr in HeLa cells leads to a decrease in infection by a non-replicative lentivector, proving its functional implication in the early phase of viral infection.

Human Immunodeficiency Virus Type 2 Capsid Protein Mutagenesis Reveals Amino Acid Residues Important for Virus Particle Assembly.

Human immunodeficiency virus (HIV) Gag drives virus particle assembly. The capsid (CA) domain is critical for Gag multimerization mediated by protein-protein interactions. The Gag protein interaction network defines critical aspects of the retroviral lifecycle at steps such as particle assembly and maturation. Previous studies have demonstrated that the immature particle morphology of HIV-2 is intriguingly distinct relative to that of HIV-1. Based upon this observation, we sought to determine the amino acid residues important for virus assembly that might help explain the differences between HIV-1 and HIV-2. To do this, we conducted site-directed mutagenesis of targeted locations in the HIV-2 CA domain of Gag and analyzed various aspects of virus particle assembly. A panel of 31 site-directed mutants of residues that reside at the HIV-2 CA inter-hexamer interface, intra-hexamer interface and CA inter-domain linker were created and analyzed for their effects on the efficiency of particle production, particle morphology, particle infectivity, Gag subcellular distribution and in vitro protein assembly. Seven conserved residues between HIV-1 and HIV-2 (L19, A41, I152, K153, K157, N194, D196) and two non-conserved residues (G38, N127) were found to significantly impact Gag multimerization and particle assembly. Taken together, these observations complement structural analyses of immature HIV-2 particle morphology and Gag lattice organization as well as provide important comparative insights into the key amino acid residues that can help explain the observed differences between HIV immature particle morphology and its association with virus replication and particle infectivity.

HIV-1 mutants expressing B cell clonogenic matrix protein p17 variants are increasing their prevalence worldwide.

AIDS-defining cancers declined after combined antiretroviral therapy (cART) introduction, but lymphomas are still elevated in HIV type 1 (HIV-1)-infected patients. In particular, non-Hodgkin's lymphomas (NHLs) represent the majority of all AIDS-defining cancers and are the most frequent cause of death in these patients. We have recently demonstrated that amino acid (aa) insertions at the HIV-1 matrix protein p17 COOH-terminal region cause protein destabilization, leading to conformational changes. Misfolded p17 variants (vp17s) strongly impact clonogenic B cell growth properties that may contribute to B cell lymphomagenesis as suggested by the significantly higher frequency of detection of vp17s with COOH-terminal aa insertions in plasma of HIV-1-infected patients with NHL. Here, we expand our previous observations by assessing the prevalence of vp17s in large retrospective cohorts of patients with and without lymphoma. We confirm the significantly higher prevalence of vp17s in lymphoma patients than in HIV-1-infected individuals without lymphoma. Analysis of 3,990 sequences deposited between 1985 and 2017 allowed us to highlight a worldwide increasing prevalence of HIV-1 mutants expressing vp17s over time. Since genomic surveillance uncovered a cluster of HIV-1 expressing a B cell clonogenic vp17 dated from 2011 to 2019, we conclude that aa insertions can be fixed in HIV-1 and that mutant viruses displaying B cell clonogenic vp17s are actively spreading.

Specific Interaction of DARPin with HIV-1 CANTD Disturbs the Distribution of Gag, RNA Packaging, and Tetraspanin Remodelling in the Membrane.

A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule.

Comparison of the Immunogenicity of HIV-1 CRF07_BC Gag Antigen With or Without a Seven Amino Acid Deletion in p6 Region.

HIV-1 CRF07_BC-p6Δ7, a strain with a seven amino acid deletion in the p6 region of the Gag protein, is becoming the dominant strain of HIV transmission among men who have sex with men (MSM) in China. Previous studies demonstrated that HIV-1 patients infected by CRF07_BC-p6Δ7 strain had lower viral load and slower disease progression than those patients infected with CRF07_BC wild-type strain. However, the underlying mechanism for this observation is not fully clarified yet. In this study, we constructed the recombinant DNA plasmid and adenovirus type 2 (Ad2) vector-based constructs to express the HIV-1 CRF07_BC Gag antigen with or without p6Δ7 mutation and then investigated their immunogenicity in mice. Our results showed that HIV-1 CRF07_BC Gag antigen with p6Δ7 mutation induced a comparable level of Gag-specific antibodies but stronger CD4+ and CD8+ T-cell immune responses than that of CRF07_BC Gag (07_BC-wt). Furthermore, we identified a series of T-cell epitopes, which induced strong T-cell immune response and cross-immunity with CRF01_AE Gag. These findings implied that the p6Gag protein with a seven amino acid deletion might enhance the Gag immunogenicity in particular cellular immunity, which provides valuable information to clarify the pathogenic mechanism of HIV-1 CRF07_BC-p6Δ7 and to develop precise vaccine strategies against HIV-1 infection.

Structural Basis for Unusual TCR CDR3ß Usage Against an Immunodominant HIV-1 Gag Protein Peptide Restricted to an HLA-B*81:01 Molecule.

In HIV infection, some closely associated human leukocyte antigen (HLA) alleles are correlated with distinct clinical outcomes although presenting the same HIV epitopes. The mechanism that underpins this observation is still unknown, but may be due to the essential features of HLA alleles or T cell receptors (TCR). In this study, we investigate how T18A TCR, which is beneficial for a long-term control of HIV in clinic, recognizes immunodominant Gag epitope TL9 (TPQDLTML180-188) from HIV in the context of the antigen presenting molecule HLA-B*81:01. We found that T18A TCR exhibits differential recognition for TL9 restricted by HLA-B*81:01. Furthermore, via structural and biophysical approaches, we observed that TL9 complexes with HLA-B*81:01 undergoes no conformational change after TCR engagement. Remarkably, the CDR3ß in T18A complexes does not contact with TL9 at all but with intensive contacts to HLA-B*81:01. The binding kinetic data of T18A TCR revealed that this TCR can recognize TL9 epitope and several mutant versions, which might explain the correlation of T18A TCR with better clinic outcomes despite the relative high mutation rate of HIV. Collectively, we provided a portrait of how CD8+ T cells engage in HIV-mediated T cell response.

Dynamics of HIV-1 Gag Processing as Revealed by Fluorescence Lifetime Imaging Microscopy and Single Virus Tracking.

The viral polyprotein Gag plays a central role for HIV-1 assembly, release and maturation. Proteolytic processing of Gag by the viral protease is essential for the structural rearrangements that mark the transition from immature to mature, infectious viruses. The timing and kinetics of Gag processing are not fully understood. Here, fluorescence lifetime imaging microscopy and single virus tracking are used to follow Gag processing in nascent HIV-1 particles in situ. Using a Gag polyprotein labelled internally with eCFP, we show that proteolytic release of the fluorophore from Gag is accompanied by an increase in its fluorescence lifetime. By tracking nascent virus particles in situ and analyzing the intensity and fluorescence lifetime of individual traces, we detect proteolytic cleavage of eCFP from Gag in a subset (6.5%) of viral particles. This suggests that for the majority of VLPs, Gag processing occurs with a delay after particle assembly.

Detection of Gag C-terminal mutations among HIV-1 non-B subtypes in a subset of Cameroonian patients.

Response to ritonavir-boosted-protease inhibitors (PI/r)-based regimen is associated with some Gag mutations among HIV-1 B-clade. There is limited data on Gag mutations and their covariation with mutations in protease among HIV-1 non-B-clades at PI/r-based treatment failure. Thus, we characterized Gag mutations present in isolates from HIV-1 infected individuals treated with a PI/r-regimen (n = 143) and compared them with those obtained from individuals not treated with PI/r (ART-naïve [n = 101] or reverse transcriptase inhibitors (RTI) treated [n = 118]). The most frequent HIV-1 subtypes were CRF02_AG (54.69%), A (13.53%), D (6.35%) and G (4.69%). Eighteen Gag mutations showed a significantly higher prevalence in PI/r-treated isolates compared to ART-naïve (p < 0.05): Group 1 (prevalence < 1% in drug-naïve): L449F, D480N, L483Q, Y484P, T487V; group 2 (prevalence 1-5% in drug-naïve): S462L, I479G, I479K, D480E; group 3 (prevalence ≥ 5% in drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Five Gag mutations (L449F, P453L, D480E, S465F, Y484P) positively correlated (Phi ≥ 0.2, p < 0.05) with protease-resistance mutations. At PI/r-failure, no significant difference was observed between patients with and without these associated Gag mutations in term of viremia or CD4 count. This analysis suggests that some Gag mutations show an increased frequency in patients failing PIs among HIV-1 non-B clades.

Rational engineering enables co-crystallization and structural determination of the HIV-1 matrix-tRNA complex.

Host tRNAs specifically interact with the matrix domain (MA) of HIV-1 major structural polyprotein, Gag, to control its membrane localization and virion assembly. In this protocol, we describe the purification and engineering of HIV-1 MA and tRNA, and the co-crystallization and structure determination of the complex using X-ray crystallography. Rational engineering of the tRNA surface created tRNA-tRNA packing contacts that drove the formation of diffraction-quality co-crystals. This protocol can be adapted to solve other ribonucleoprotein complex structures containing structured RNAs. For complete details on the use and execution of this protocol, please refer to Bou-Nader et al. (2021).

GSK3640254 Is a Novel HIV-1 Maturation Inhibitor with an Optimized Virology Profile.

HIV-1 maturation inhibitors (MIs) offer a novel mechanism of action and potential for use in HIV-1 treatment. Prior MIs displayed clinical efficacy but were associated with the emergence of resistance and some gastrointestinal tolerability events. Treatment with the potentially safer next-generation MI GSK3640254 (GSK'254) resulted in up to a 2-log10 viral load reduction in a phase IIa proof-of-concept study. In vitro experiments have defined the antiviral and resistance profiles for GSK'254. The compound displayed strong antiviral activity against a library of subtype B and C chimeric viruses containing Gag polymorphisms and site-directed mutants previously shown to affect potency of earlier-generation MIs, with a mean protein-binding adjusted 90% effective concentration (EC90) of 33 nM. Furthermore, GSK'254 exhibited robust antiviral activity against a panel of HIV-1 clinical isolates, with a mean EC50 of 9 nM. Mechanistic studies established that bound GSK'254 dissociated on average 7.1-fold more slowly from wild-type Gag virus-like particles (VLPs) than a previous-generation MI. In resistance studies, the previously identified A364V Gag region mutation was selected under MI pressure in cell culture and during the phase IIa clinical study. As expected, GSK'254 inhibited cleavage of p25 in a range of polymorphic HIV-1 Gag VLPs. Virus-like particles containing the A364V mutation exhibited a p25 cleavage rate 9.3 times higher than wild-type particles, providing a possible mechanism for MI resistance. The findings demonstrate that GSK'254 potently inhibits a broad range of HIV-1 strains expressing Gag polymorphisms.

Two Distinct Mechanisms Leading to Loss of Virological Control in the Rare Group of Antiretroviral Therapy-Naive, Transiently Aviremic Children Living with HIV.

HIV-specific CD8+ T cells play a central role in immune control of adult HIV, but their contribution in pediatric infection is less well characterized. Previously, we identified a group of ART-naive children with persistently undetectable plasma viremia, termed "elite controllers," and a second group who achieved aviremia only transiently. To investigate the mechanisms of failure to maintain aviremia, we characterized in three transient aviremic individuals (TAs), each of whom expressed the disease-protective HLA-B*81:01, longitudinal HIV-specific T-cell activity, and viral sequences. In two TAs, a CD8+ T-cell response targeting the immunodominant epitope TPQDLNTML (Gag-TL9) was associated with viral control, followed by viral rebound and the emergence of escape variants with lower replicative capacity. Both TAs mounted variant-specific responses, but only at low functional avidity, resulting in immunological progression. In contrast, in TA-3, intermittent viremic episodes followed aviremia without virus escape or a diminished CD4+ T-cell count. High quality and magnitude of the CD8+ T-cell response were associated with aviremia. We therefore identify two distinct mechanisms of loss of viral control. In one scenario, CD8+ T-cell responses initially cornered low-replicative-capacity escape variants, but with insufficient avidity to prevent viremia and disease progression. In the other, loss of viral control was associated with neither virus escape nor progression but with a decrease in the quality of the CD8+ T-cell response, followed by recovery of viral control in association with improved antiviral response. These data suggest the potential for a consistently strong and polyfunctional antiviral response to achieve long-term viral control without escape. IMPORTANCE Very early initiation of antiretroviral therapy (ART) in pediatric HIV infection offers a unique opportunity to limit the size and diversity of the viral reservoir. However, only rarely is ART alone sufficient to achieve remission. Additional interventions that likely include contributions from host immunity are therefore required. The HIV-specific T-cell response plays a central role in immune control of adult HIV, often mediated through protective alleles such as HLA-B*57/58:01/81:01. However, due to the tolerogenic and type 2 biased immune response in early life, HLA-I-mediated immune suppression of viremia is seldom observed in children. We assessed a rare group of HLA-B*81:01-positive, ART-naive children who achieved aviremia, albeit only transiently, and investigated the role of the CD8+ T-cell response in the establishment and loss of viral control. We identified a mechanism by which the HIV-specific response can achieve viremic control without viral escape that can be explored in strategies to achieve remission.

Rational design of self-assembled RNA nanostructures for HIV-1 virus assembly blockade.

Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem-loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions. The resulting STL-decorated nanostructures (double and triple stem-loop structures denoted as Dumbbell and Tribell, respectively) can elicit more pronounced viral blockade than their building blocks, with the inhibition arising as a result of nanostructures interfering with Gag multimerization. These findings could open up new avenues for RNA-based therapy.

Viral and Cellular Factors Leading to the Loss of CD4 Homeostasis in HIV-1 Viremic Nonprogressors.

Human immunodeficiency virus type 1 (HIV-1) viremic nonprogressors (VNPs) represent a very rare HIV-1 extreme phenotype. VNPs are characterized by persistent high plasma viremia and maintenance of CD4+ T-cell counts in the absence of treatment. However, the causes of nonpathogenic HIV-1 infection in VNPs remain elusive. Here, we identified for the first time two VNPs who experienced the loss of CD4+ homeostasis (LoH) after more than 13 years. We characterized in deep detail viral and host factors associated with the LoH and compared with standard VNPs and healthy controls. The viral factors determined included HIV-1 coreceptor usage and replicative capacity. Changes in CD4+ and CD8+ T-cell activation, maturational phenotype, and expression of CCR5 and CXCR6 in CD4+ T-cells were also evaluated as host-related factors. Consistently, we determined a switch in HIV-1 coreceptor use to CXCR4 concomitant with an increase in replicative capacity at the LoH for the two VNPs. Moreover, we delineated an increase in the frequency of HLA-DR+CD38+ CD4+ and CD8+ T cells and traced the augment of naive T-cells upon polyclonal activation with LoH. Remarkably, very low and stable levels of CCR5 and CXCR6 expression in CD4+ T-cells were measured over time. Overall, our results demonstrated HIV-1 evolution toward highly pathogenic CXCR4 strains in the context of very limited and stable expression of CCR5 and CXCR6 in CD4+ T cells as potential drivers of LoH in VNPs. These data bring novel insights into the correlates of nonpathogenic HIV-1 infection. IMPORTANCE The mechanism behind nonpathogenic human immunodeficiency virus type 1 (HIV-1) infection remains poorly understood, mainly because of the very low frequency of viremic nonprogressors (VNPs). Here, we report two cases of VNPs who experienced the loss of CD4+ T-cell homeostasis (LoH) after more than 13 years of HIV-1 infection. The deep characterization of viral and host factors supports the contribution of viral and host factors to the LoH in VNPs. Thus, HIV-1 evolution toward highly replicative CXCR4 strains together with changes in T-cell activation and maturational phenotypes were found. Moreover, we measured very low and stable levels of CCR5 and CXCR6 in CD4+ T-cells over time. These findings support viral evolution toward X4 strains limited by coreceptor expression to control HIV-1 pathogenesis and demonstrate the potential of host-dependent factors, yet to be fully elucidated in VNPs, to control HIV-1 pathogenesis.

Perturbing HIV-1 Ribosomal Frameshifting Frequency Reveals a cis Preference for Gag-Pol Incorporation into Assembling Virions.

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks" scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a "weighted Goldilocks" scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.

HIV-1 Packaging Visualised by In-Gel SHAPE.

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA-protein complex. Here, we apply the in-gel SHAPE (selective 2'OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag-dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.