Spatiotemporal binding of cyclophilin A and CPSF6 to capsid regulates HIV-1 nuclear entry and integration.

Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration. However, disruption of CypA binding to the mutant capsid restored nuclear entry, integration, and infection in a CPSF6-dependent manner. Furthermore, relocalization of CypA expression from the cell cytoplasm to the nucleus failed to restore mutant HIV-1 infection. Our results clarify that sequential binding of CypA and CPSF6 to HIV-1 capsid is required for optimal nuclear entry and integration targeting, informing antiretroviral therapies that contain lenacapavir.

Deep analysis of CD4 T cells in the rhesus CNS during SIV infection.

Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS)-known as Neuro-HIV- persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Acute infection led to marked imbalance in the CNS CD4/CD8 ratio which persisted into the chronic phase. These observations underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our findings offer insights for potential T cell-focused interventions while underscoring the challenges in eradicating HIV from the CNS, particularly in the context of sub-optimal ART.

Use of pediatric thymus to humanize mice for HIV-1 mucosal transmission.

Humanized mice have been used to study human immunodeficiency virus type 1 (HIV-1) transmission, pathogenesis, and treatment. The ability of pediatric thymus tissue implanted either in the leg (Leg PedThy) or under the renal capsule (Renal PedThy) with allogeneic CD34+ hematopoietic cells (HSCs) in NSG mice was evaluated for reconstitution of human immune cells and for rectal transmission of HIV-1. These mice were compared to traditional BLT mice implanted with fetal liver and thymus under the renal capsule and mice injected only with HSCs. Renal PedThy mice had similar immune reconstitution in the blood, spleen and intestine as BLT mice, while Leg PedThy mice had transient detection of immune cells, particularly CD4+ T cells and macrophages, the target cells for HIV-1 infection. Rectal transmission and replication of HIV-1 was efficient in BLT mice but lower and more variable in Renal PedThy mice. HIV-1 was poorly transmitted in HSC mice and not transmitted in Leg PedThy mice, which correlated with the frequencies of target cells in the spleen and intestine. Humanization of NSG mice with pediatric thymus was successful when implanted under the kidney capsule, but led to less efficient HIV-1 rectal transmission and replication compared to BLT mice.

CD4 T cell Responses in the CNS during SIV infection.

Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS) - known as Neuro-HIV- persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Despite viral suppression with ART, acute infection led to significant depletion of CNS CD4 T cells, persisting into the chronic phase. These findings underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our results offer insights for potential T cell-focused interventions while also underscoring the challenges in eradicating HIV from the CNS, even with effective ART.

Targeting spike glycans to inhibit SARS-CoV2 viral entry.

SARS-CoV-2 spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical approaches, we demonstrate that the interaction is avidity driven and that BOA cross-links the spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that multivalent glycan-targeting molecules have the potential to act as pan-coronavirus inhibitors.

APOBEC3 selects V179I in HIV-1 reverse transcriptase to provide selective advantage for non-nucleoside reverse transcriptase inhibitor-resistant mutants.

The V179I substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in humans or mouse models treated with certain nonnucleoside reverse transcriptase inhibitors (NNRTIs). While it is often observed together with other NNRTI resistance mutations, V179I does not confer drug resistance. To understand how V179I arises during NNRTI treatment, we characterized it in HIV-1 molecular clones with or without the NNRTI resistance mutations Y181C or Y181V. While V179I alone did not confer resistance to any NNRTIs tested, when present with Y181C/V it enhanced drug resistance to some NNRTIs by 3- to 8-fold. In replication competition experiments in the presence of the NNRTI rilpivirine (RPV), V179I modestly enhanced Y181C HIV-1 or Y181V HIV-1 replication compared to viruses without V179I. As V179I arises from a G to A mutation, we evaluated whether it could arise due to host APOBEC3 deaminase activity and be maintained in the presence of a NNRTI to provide a selective advantage for the virus. V179I was detected in some humanized mice treated with RPV and was associated with G to A mutations characteristic of APOBEC3 activity. In RPV selection experiments, the frequency of V179I in HIV-1 was accelerated in CD4+ T cells expressing higher APOBEC3F and APOBEC3G levels. Our results provide evidence that V179I in HIV-1 RT can arise due to APOBEC-mediated G to A hypermutation and can confer a selective advantage to drug-resistant HIV-1 isolates in the presence of some NNRTIs.

Construction and characterization of two SARS-CoV-2 minigenome replicon systems.

The ongoing COVID-19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virology and antiviral discovery, a noninfectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS-CoV-2 minigenome replicon systems. First, we constructed the IVT-CoV2-Rep complementary DNA template to generate a replicon messenger RNA (mRNA) with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT-CoV2-Rep in a variety of cell lines, which could be completely abolished by known SARS-CoV-2 replication inhibitors. Our data also suggest that the transient phenotype of IVT-CoV2-Rep is not due to host innate antiviral responses. In addition, we have developed a DNA-launched replicon BAC-CoV2-Rep, which supports the in-cell transcription of a replicon mRNA as initial replication template. The BAC-CoV2-Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT-CoV2-Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC-CoV2-Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS-CoV-2 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization.

Targeting Spike Glycans to Inhibit SARS-CoV2 Viral Entry.

SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.

Disassembling the Nature of Capsid: Biochemical, Genetic, and Imaging Approaches to Assess HIV-1 Capsid Functions.

The human immunodeficiency virus type 1 (HIV-1) capsid and its disassembly, or capsid uncoating, has remained an active area of study over the past several decades. Our understanding of the HIV-1 capsid as solely a protective shell has since shifted with discoveries linking it to other complex replication events. The interplay of the HIV-1 capsid with reverse transcription, nuclear import, and integration has led to an expansion of knowledge of capsid functionality. Coincident with advances in microscopy, cell, and biochemistry assays, several models of capsid disassembly have been proposed, in which it occurs in either the cytoplasmic, nuclear envelope, or nuclear regions of the cell. Here, we discuss how the understanding of the HIV-1 capsid has evolved and the key methods that made these discoveries possible.

CG Dinucleotide Removal in Bioluminescent and Fluorescent Reporters Improves HIV-1 Replication and Reporter Gene Expression for Dual Imaging in Humanized Mice.

Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging in vivo. Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a near-infrared fluorescent protein (iRFP) upstream of nef. HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression in vivo correlated initially with plasma viremia, expression decreased after 4 to 5 weeks despite high plasma viremia. The reporter genes were codon optimized to remove cytosine/guanine (CG) dinucleotides, and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication in vitro and reporter expression in vivo and ex vivo. Both codon-optimized reporter viruses could be visualized during coinfection and in vivo reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. IMPORTANCE Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole-body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.

Cytoplasmic CPSF6 Regulates HIV-1 Capsid Trafficking and Infection in a Cyclophilin A-Dependent Manner.

Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain variations in HIV-1 capsid trafficking and uncoating in CD4+ T cells and macrophages.IMPORTANCE HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps. HIV capsid has been shown to interact with multiple cell proteins during movement to the cell nucleus in a poorly understood process that may differ during infection of different cell types. In this study, we show that premature or too much binding of one human protein, cleavage and polyadenylation specificity factor 6 (CPSF6), disrupts the ability of the capsid to deliver the viral genome to the cell nucleus. Another human protein, cyclophilin A (CypA), can shield HIV capsid from premature binding to CPSF6, which can differ in CD4+ T cells and macrophages. Better understanding of how HIV infects cells will allow better drugs to prevent or inhibit infection and pathogenesis.

SIV Evolutionary Dynamics in Cynomolgus Macaques during SIV-Mycobacterium tuberculosis Co-Infection.

Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a worldwide public health concern, leading to worse clinical outcomes caused by both pathogens. We used a non-human primate model of simian immunodeficiency virus (SIV)-Mtb co-infection, in which latent Mtb infection was established prior to SIVmac251 infection. The evolutionary dynamics of SIV env was evaluated from samples in plasma, lymph nodes, and lungs (including granulomas) of SIV-Mtb co-infected and SIV only control animals. While the diversity of the challenge virus was low and overall viral diversity remained relatively low over 6-9 weeks, changes in viral diversity and divergence were observed, including evidence for tissue compartmentalization. Overall, viral diversity was highest in SIV-Mtb animals that did not develop clinical Mtb reactivation compared to animals with Mtb reactivation. Among lung granulomas, viral diversity was positively correlated with the frequency of CD4+ T cells and negatively correlated with the frequency of CD8+ T cells. SIV diversity was highest in the thoracic lymph nodes compared to other sites, suggesting that lymphatic drainage from the lungs in co-infected animals provides an advantageous environment for SIV replication. This is the first assessment of SIV diversity across tissue compartments during SIV-Mtb co-infection after established Mtb latency.

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis.

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.

SIV and Mycobacterium tuberculosis synergy within the granuloma accelerates the reactivation pattern of latent tuberculosis.

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to CD4 depleted animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and reduced Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.

Long-Acting Rilpivirine (RPV) Preexposure Prophylaxis Does Not Inhibit Vaginal Transmission of RPV-Resistant HIV-1 or Select for High-Frequency Drug Resistance in Humanized Mice.

As a long-acting formulation of the nonnucleoside reverse transcriptase inhibitor rilpivirine (RPV LA) has been proposed for use as preexposure prophylaxis (PrEP) and the prevalence of transmitted RPV-resistant viruses can be relatively high, we evaluated the efficacy of RPV LA to inhibit vaginal transmission of RPV-resistant HIV-1 in humanized mice. Vaginal challenges of wild-type (WT), Y181C, and Y181V HIV-1 were performed in mice left untreated or after RPV PrEP. Plasma viremia was measured for 7 to 10 weeks, and single-genome sequencing was performed on plasma HIV-1 RNA in mice infected during PrEP. RPV LA significantly prevented vaginal transmission of WT HIV-1 and Y181C HIV-1, which is 3-fold resistant to RPV. However, it did not prevent transmission of Y181V HIV-1, which has 30-fold RPV resistance in the viruses used for this study. RPV LA did delay WT HIV-1 dissemination in infected animals until genital and plasma RPV concentrations waned. Animals that became infected despite RPV LA PrEP did not acquire new RPV-resistant mutations above frequencies in untreated mice or untreated people living with HIV-1, and the mutations detected conferred low-level resistance. These data suggest that high, sustained concentrations of RPV were required to inhibit vaginal transmission of HIV-1 with little or no resistance to RPV but could not inhibit virus with high resistance. HIV-1 did not develop high-level or high-frequency RPV resistance in the majority of mice infected after RPV LA treatment. However, the impact of low-frequency RPV resistance on virologic outcome during subsequent antiretroviral therapy still is unclear.IMPORTANCE The antiretroviral drug rilpivirine was developed into a long-acting formulation (RPV LA) to improve adherence for preexposure prophylaxis (PrEP) to prevent HIV-1 transmission. A concern is that RPV LA will not inhibit transmission of drug-resistant HIV-1 and may select for drug-resistant virus. In female humanized mice, we found that RPV LA inhibited vaginal transmission of WT or 3-fold RPV-resistant HIV-1 but not virus with 30-fold RPV resistance. In animals that became infected despite RPV LA PrEP, WT HIV-1 dissemination was delayed until genital and plasma RPV concentrations waned. RPV resistance was detected at similar low frequencies in untreated and PrEP-treated mice that became infected. These results indicate the importance of maintaining RPV at a sustained threshold after virus exposure to prevent dissemination of HIV-1 after vaginal infection and low-frequency resistance mutations conferred low-level resistance, suggesting that RPV resistance is difficult to develop after HIV-1 infection during RPV LA PrEP.

AutoCLEM: An Automated Workflow for Correlative Live-Cell Fluorescence Microscopy and Cryo-Electron Tomography.

Correlative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

A Novel Phenotype Links HIV-1 Capsid Stability to cGAS-Mediated DNA Sensing.

The HIV-1 capsid executes essential functions that are regulated by capsid stability and host factors. In contrast to increasing knowledge on functional roles of capsid-interacting host proteins during postentry steps, less is known about capsid stability and its impact on intracellular events. Here, using the antiviral compound PF-3450074 (PF74) as a probe for capsid function, we uncovered a novel phenotype of capsid stability that has a profound effect on innate sensing of viral DNA by the DNA sensor cGAS. A single mutation, R143A, in the capsid protein conferred resistance to high concentrations of PF74, without affecting capsid binding to PF74. A cell-free assay showed that the R143A mutant partially counteracted the capsid-destabilizing activity of PF74, pointing to capsid stabilization as a resistance mechanism for the R143A mutant. In monocytic THP-1 cells, the R143A virus, but not the wild-type virus, suppressed cGAS-dependent innate immune activation. These results suggest that capsid stabilization improves the shielding of viral DNA from innate sensing. We found that a naturally occurring transmitted founder (T/F) variant shares the same properties as the R143A mutant with respect to PF74 resistance and DNA sensing. Imaging assays revealed delayed uncoating kinetics of this T/F variant and the R143A mutant. All these phenotypes of this T/F variant were controlled by a genetic polymorphism located at the trimeric interface between capsid hexamers, thus linking these capsid-dependent properties. Overall, this work functionally connects capsid stability to innate sensing of viral DNA and reveals naturally occurring phenotypic variation in HIV-1 capsid stability.IMPORTANCE The HIV-1 capsid, which is made from individual viral capsid proteins (CA), is a target for a number of antiviral compounds, including the small-molecule inhibitor PF74. In the present study, we utilized PF74 to identify a transmitted/founder (T/F) strain that shows increased capsid stability. Interestingly, PF74-resistant variants prevented cGAS-dependent innate immune activation under a condition where the other T/F strains induced type I interferon. These observations thus reveal a new CA-specific phenotype that couples capsid stability to viral DNA recognition by cytosolic DNA sensors.

Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs.

Two mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the "primer grip" region and could be influenced by the M230I mutation.IMPORTANCE Although antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

CA Mutation N57A Has Distinct Strain-Specific HIV-1 Capsid Uncoating and Infectivity Phenotypes.

The ability of human immunodeficiency virus type 1 (HIV-1) to transduce nondividing cells is key to infecting terminally differentiated macrophages, which can serve as a long-term reservoir of HIV-1 infection. The mutation N57A in the viral CA protein renders HIV-1 cell cycle dependent, allowing examination of HIV-1 infection of nondividing cells. Here, we show that the N57A mutation confers a postentry infectivity defect that significantly differs in magnitude between the common lab-adapted molecular clones HIV-1NL4-3 (>10-fold) and HIV-1LAI (2- to 5-fold) in multiple human cell lines and primary CD4+ T cells. Capsid permeabilization and reverse transcription are altered when N57A is incorporated into HIV-1NL4-3 but not HIV-1LAI The N57A infectivity defect is significantly exacerbated in both virus strains in the presence of cyclosporine (CsA), indicating that N57A infectivity is dependent upon CA interacting with host factor cyclophilin A (CypA). Adaptation of N57A HIV-1LAI selected for a second CA mutation, G94D, which rescued the N57A infectivity defect in HIV-1LAI but not HIV-1NL4-3 The rescue of N57A by G94D in HIV-1LAI is abrogated by CsA treatment in some cell types, demonstrating that this rescue is CypA dependent. An examination of over 40,000 HIV-1 CA sequences revealed that the four amino acids that differ between HIV-1NL4-3 and HIV-1LAI CA are polymorphic, and the residues at these positions in the two strains are widely prevalent in clinical isolates. Overall, a few polymorphic amino acid differences between two closely related HIV-1 molecular clones affect the phenotype of capsid mutants in different cell types.IMPORTANCE The specific mechanisms by which HIV-1 infects nondividing cells are unclear. A mutation in the HIV-1 capsid protein abolishes the ability of the virus to infect nondividing cells, serving as a tool to examine cell cycle dependence of HIV-1 infection. We have shown that two widely used HIV-1 molecular clones exhibit significantly different N57A infectivity phenotypes due to fewer than a handful of CA amino acid differences and that these clones are both represented in HIV-infected individuals. As such minor differences in closely related HIV-1 strains may impart significant infectivity differences, careful consideration should be given to drawing conclusions from one particular HIV-1 clone. This study highlights the potential for significant variation in results with the use of multiple strains and possible unanticipated effects of natural polymorphisms.

Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro-assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.