Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.

A T-Cell-Derived 3-Gene Signature Distinguishes SARS-CoV-2 from Common Respiratory Viruses.

Research on the host responses to respiratory viruses could help develop effective interventions and therapies against the current and future pandemics from the host perspective. To explore the pathogenesis that distinguishes SARS-CoV-2 infections from other respiratory viruses, we performed a multi-cohort analysis with integrated bioinformatics and machine learning. We collected 3730 blood samples from both asymptomatic and symptomatic individuals infected with SARS-CoV-2, seasonal human coronavirus (sHCoVs), influenza virus (IFV), respiratory syncytial virus (RSV), or human rhinovirus (HRV) across 15 cohorts. First, we identified an enhanced cellular immune response but limited interferon activities in SARS-CoV-2 infection, especially in asymptomatic cases. Second, we identified a SARS-CoV-2-specific 3-gene signature (CLSPN, RBBP6, CCDC91) that was predominantly expressed by T cells, could distinguish SARS-CoV-2 infection, including Omicron, from other common respiratory viruses regardless of symptoms, and was predictive of SARS-CoV-2 infection before detectable viral RNA on RT-PCR testing in a longitude follow-up study. Thereafter, a user-friendly online tool, based on datasets collected here, was developed for querying a gene of interest across multiple viral infections. Our results not only identify a unique host response to the viral pathogenesis in SARS-CoV-2 but also provide insights into developing effective tools against viral pandemics from the host perspective.

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication.

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2α, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

SARS-CoV-2-associated lymphopenia: possible mechanisms and the role of CD147.

T lymphocytes play a primary role in the adaptive antiviral immunity. Both lymphocytosis and lymphopenia were found to be associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While lymphocytosis indicates an active anti-viral response, lymphopenia is a sign of poor prognosis. T-cells, in essence, rarely express ACE2 receptors, making the cause of cell depletion enigmatic. Moreover, emerging strains posed an immunological challenge, potentially alarming for the next pandemic. Herein, we review how possible indirect and direct key mechanisms could contribute to SARS-CoV-2-associated-lymphopenia. The fundamental mechanism is the inflammatory cytokine storm elicited by viral infection, which alters the host cell metabolism into a more acidic state. This "hyperlactic acidemia" together with the cytokine storm suppresses T-cell proliferation and triggers intrinsic/extrinsic apoptosis. SARS-CoV-2 infection also results in a shift from steady-state hematopoiesis to stress hematopoiesis. Even with low ACE2 expression, the presence of cholesterol-rich lipid rafts on activated T-cells may enhance viral entry and syncytia formation. Finally, direct viral infection of lymphocytes may indicate the participation of other receptors or auxiliary proteins on T-cells, that can work alone or in concert with other mechanisms. Therefore, we address the role of CD147-a novel route-for SARS-CoV-2 and its new variants. CD147 is not only expressed on T-cells, but it also interacts with other co-partners to orchestrate various biological processes. Given these features, CD147 is an appealing candidate for viral pathogenicity. Understanding the molecular and cellular mechanisms behind SARS-CoV-2-associated-lymphopenia will aid in the discovery of potential therapeutic targets to improve the resilience of our immune system against this rapidly evolving virus.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

Human SARS-CoV-2 challenge uncovers local and systemic response dynamics.

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.

The tyrosine kinase KDR is essential for the survival of HTLV-1-infected T cells by stabilizing the Tax oncoprotein.

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia/lymphoma (ATLL) and the neuroinflammatory disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax oncoprotein regulates viral gene expression and persistently activates NF-κB to maintain the viability of HTLV-1-infected T cells. Here, we utilize a kinome-wide shRNA screen to identify the tyrosine kinase KDR as an essential survival factor of HTLV-1-transformed cells. Inhibition of KDR specifically induces apoptosis of Tax expressing HTLV-1-transformed cell lines and CD4 + T cells from HAM/TSP patients. Furthermore, inhibition of KDR triggers the autophagic degradation of Tax resulting in impaired NF-κB activation and diminished viral transmission in co-culture assays. Tax induces the expression of KDR, forms a complex with KDR, and is phosphorylated by KDR. These findings suggest that Tax stability is dependent on KDR activity which could be exploited as a strategy to target Tax in HTLV-1-associated diseases.

Viral modulation of type II interferon increases T cell adhesion and virus spread.

During primary varicella zoster virus (VZV) infection, infected lymphocytes drive primary viremia, causing systemic dissemination throughout the host, including the skin. This results in cytokine expression, including interferons (IFNs), which partly limit infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. It is not clear how VZV achieves this while evading the cytokine response. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity, increasing the expression of a subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of keratinocytes facilitates lymphocyte function-associated antigen 1-dependent T cell adhesion and expression of gC during infection increases VZV spread to peripheral blood mononuclear cells. This constitutes the discovery of a strategy to modulate IFN-γ activity, upregulating a subset of ISGs, promoting enhanced lymphocyte adhesion and virus spread.

Human cytomegalovirus UL23 exploits PD-L1 inhibitory signaling pathway to evade T cell-mediated cytotoxicity.

Human cytomegalovirus (HCMV), a widely prevalent human beta-herpesvirus, establishes lifelong persistence in the host following primary infection. In healthy individuals, the virus is effectively controlled by HCMV-specific T cells and typically exhibits asymptomatic. The T cell immune response plays a pivotal role in combating HCMV infection, while HCMV employs various strategies to counteract it within the host. Previously, we reported that UL23, a tegument protein of HCMV, facilitates viral immune evasion from interferon-gamma (IFN-γ) responses, and it is well known that IFN-γ is mainly derived from T cells. However, the involvement of UL23 in viral immune evasion from T cell-mediated immunity remains unclear. Herein, we present compelling evidence that UL23 significantly enhances viral resistance against T cell-mediated cytotoxicity during HCMV infection from the co-culture assays of HCMV-infected cells with T cells. We found that IFN-γ plays a major role in regulating T cell cytotoxicity mediated by UL23. More interestingly, we demonstrated that UL23 not only regulates the IFN-γ downstream responses but also modulates the IFN-γ secretion by regulating T cell activities. Further experiments indicate that UL23 upregulates the expression and signaling of programmed death ligand 1 (PD-L1), which is responsible for inhibiting multiple aspects of T cell activities, including activation, apoptosis, and IFN-γ secretion, as determined through RNA-seq analysis and inhibitor-blocking experiments, ultimately facilitating viral replication and spread. Our findings highlight the potential role of UL23 as an alternative antagonist in suppressing T cell cytotoxicity and unveil a novel strategy for HCMV to evade T cell immunity. IMPORTANCE: T cell immunity is pivotal in controlling primary human cytomegalovirus (HCMV) infection, restricting periodic reactivation, and preventing HCMV-associated diseases. Despite inducing a robust T cell immune response, HCMV has developed sophisticated immune evasion mechanisms that specifically target T cell responses. Although numerous studies have been conducted on HCMV-specific T cells, the primary focus has been on the impact of HCMV on T cell recognition via major histocompatibility complex molecules. Our studies show for the first time that HCMV exploits the programmed death ligand 1 (PD-L1) inhibitory signaling pathway to evade T cell immunity by modulating the activities of T cells and thereby blocking the secretion of IFN-γ, which is directly mediated by HCMV-encoded tegument protein UL23. While PD-L1 has been extensively studied in the context of tumors and viruses, its involvement in HCMV infection and viral immune evasion is rarely reported. We observed an upregulation of PD-L1 in normal cells during HCMV infection and provided strong evidence supporting its critical role in UL23-induced inhibition of T cell-mediated cytotoxicity. The novel strategy employed by HCMV to manipulate the inhibitory signaling pathway of T cell immune activation for viral evasion through its encoded protein offers valuable insights for the understanding of HCMV-mediated T cell immunomodulation and developing innovative antiviral treatment strategies.

OMIP-103: A 35-marker imaging mass cytometry panel for the co-detection of HIV and immune cell populations in human formalin fixed paraffin embedded intestinal tissue.

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature. The described method for HIV RNA detection can be generalized to other low abundance RNA targets, whether endogenous or pathogen derived. As such, the panel presented here is useful for high parameter spatial mapping of intestinal immune cells and their interactions with pathogens such as HIV.

HIV-1 Capsid Rapidly Induces Long-Lived CPSF6 Puncta in Non-Dividing Cells, but Similar Puncta Already Exist in Uninfected T-Cells.

The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.

The Disassociation of A3G-Related HIV-1 cDNA G-to-A Hypermutation to Viral Infectivity.

APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.

Visualizing HIV-1 Assembly at the T-Cell Plasma Membrane Using Single-Molecule Localization Microscopy.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.

HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

IMPORTANCE: The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.

Virus update for the M2 "mac-in-touch".

While HIV-1 infection of macrophages plays a major role in viral persistence and pathogenesis, how HIV-1 transfers from infected T cells to macrophages remains elusive. In this issue, Mascarau et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202205103) demonstrate how macrophage polarization drives their ability to fuse with HIV-1 infected T cells via the CD81/RhoA-ROCK/Myosin axis.

FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

A Morbillivirus Infection Shifts DC Maturation Toward a Tolerogenic Phenotype to Suppress T Cell Activation.

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.

Avatar Mice Underscore the Role of the T Cell-Dendritic Cell Crosstalk in Ebola Virus Disease and Reveal Mechanisms of Protection in Survivors.

Ebola virus disease (EVD) is a complex infectious disease characterized by high inflammation, multiorgan failure, the dysregulation of innate and adaptive immune responses, and coagulation abnormalities. Evidence accumulated over the last 2 decades indicates that, during fatal EVD, the infection of antigen-presenting cells (APC) and the dysregulation of T cell immunity preclude a successful transition between innate and adaptive immunity, which constitutes a key disease checkpoint. In order to better understand the contribution of the APC-T cell crosstalk to EVD pathophysiology, we have developed avatar mice transplanted with human, donor-specific APCs and T cells. Here, we show that the transplantation of T cells and APCs from Ebola virus (EBOV)-naive individuals into avatar mice results in severe disease and death and that this phenotype is dependent on T cell receptor (TCR)-major histocompatibility complex (MCH) recognition. Conversely, avatar mice were rescued from death induced by EBOV infection after the transplantation of both T cells and plasma from EVD survivors. These results strongly suggest that protection from EBOV reinfection requires both cellular and humoral immune memory responses. IMPORTANCE The crosstalk between dendritic cells and T cells marks the transition between innate and adaptive immune responses, and it constitutes an important checkpoint in EVD. In this study, we present a mouse avatar model in which T cell and dendritic cell interactions from a specific donor can be studied during EVD. Our findings indicate that T cell receptor-major histocompatibility complex-mediated T cell-dendritic cell interactions are associated with disease severity, which mimics the main features of severe EVD in these mice. Resistance to an EBOV challenge in the model was achieved via the transplantation of both survivor T cells and plasma.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.

Endocytosed HIV-1 Envelope Glycoprotein Traffics to Rab14+ Late Endosomes and Lysosomes to Regulate Surface Levels in T-Cell Lines.

Production of infectious HIV-1 particles requires incorporation of the viral envelope glycoprotein (Env) at the plasma membrane (PM) of infected CD4+ T cells. Env trafficking to the PM exposes viral epitopes that can be exploited by the host immune system; however, HIV-1 can evade this response by endocytosis of excess Env from the PM. The fate of Env after internalization remains unclear, with evidence suggesting several different vesicular trafficking steps may be involved, including recycling pathways. To date, there have been very few studies documenting the trafficking pathways of native Env in infected T cells. Furthermore, it remains unclear whether there are T-cell-specific endosomal pathways regulating the fate of endocytic Env. Here, we use a pulse-labeling approach with a monovalent anti-Env Fab probe to characterize the trafficking of internalized Env within infected CD4+ T-cell lines, together with CRISPR/Cas9-mediated endogenous protein tagging, to assess the role of host cell Rab GTPases in Env trafficking. We show that endocytosed Env traffics to Rab14+ compartments that possess hallmarks of late endosomes and lysosomes. We also demonstrate that Env can recycle back to the PM, although we find that recycling does not occur at high rates when compared to the model recycling protein transferrin. These results help to resolve open questions about the fate and relevance of endocytosed Env in HIV-infected cells and suggest a novel role for Rab14 in a cell-type-specific late-endosomal/lysosomal trafficking pathway in T cells. IMPORTANCE HIV-1 envelope glycoprotein (Env) evades immune neutralization through many mechanisms. One immune evasion strategy may result from the internalization of excess surface-exposed Env to prevent antibody-dependent cellular cytotoxicity or neutralization. Characterization of the fate of endocytosed Env is critical to understand which vesicular pathways could be targeted to promote display of Env epitopes to the immune system. In this study, we characterize the endocytic fate of native Env, expressed from infected human T-cell lines. We demonstrate that Env is rapidly trafficked to a late-endosome/lysosome-like compartment and can be recycled to the cell surface for incorporation into virus assembly sites. This study implicates a novel intracellular compartment, marked by host-cell Rab14 GTPases, for the sequestration of Env. Therapeutic approaches aimed at mobilizing this intracellular pool of Env could lead to stronger immune control of HIV-1 infection via antibody-dependent cell-mediated cytotoxicity.