Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors

ABSTRACTHIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified viain vitromutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here,nefclones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 downregulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 downregulation ability and modestly impaired CXCR4 downregulation activity compared to those from CP and AP. Nef's ability to downregulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linkedin vivo. Moreover, impairments in Nef's receptor downregulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune escape-associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks.IMPORTANCEHIV-1 Nef, a key factor for viral pathogenesis, downregulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and coreceptors CCR5 and CXCR4. This activity enhances viral replication by protecting infected cells from cytotoxicity associated with superinfection and may also serve as an immune evasion strategy. However, how these activities are maintained under selective pressurein vivoremains elusive. We addressed this question by analyzing functions of primary Nef clones isolated from patients at various infection stages and with different disease phenotypes, including elite controllers, who spontaneously control HIV-1 viremia to undetectable levels. The results indicated that downregulation of HIV-1 entry receptors, particularly CCR5, is impaired in Nef clones from elite controllers. These functional impairments were driven by rare Nef polymorphisms and adaptations associated with cellular immune responses, underscoring the complex molecular pathways responsible for maintaining and attenuating viral protein functionin vivo.

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Nef Proteins from HIV-1 Elite Controllers Are Inefficient at Preventing Antibody-Dependent Cellular Cytotoxicity

Journal of Virology ◽

10.1128/jvi.02973-15 ◽

2015 ◽

Vol 90 (6) ◽

pp. 2993-3002 ◽

Cited By ~ 39

Author(s):

Nirmin Alsahafi ◽

Shilei Ding ◽

Jonathan Richard ◽

Tristan Markle ◽

Nathalie Brassard ◽

...

Keyword(s):

Viral Replication ◽

Antiretroviral Treatment ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Elite Controllers ◽

Viremia Level ◽

Infected Cells ◽

Hiv 1 ◽

Durable Suppression ◽

Increased Susceptibility

ABSTRACTImpairment of Nef function, including reduced CD4 downregulation, was described in a subset of HIV-1-infected individuals that control viral replication without antiretroviral treatment (elite controllers [EC]). Elimination of HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) requires the presence of envelope glycoproteins (Env) in the CD4-bound conformation, raising the possibility that accumulating CD4 at the surface of virus-infected cells in EC could interact with Env and thereby sensitize these cells to ADCC. We observed a significant increase in the exposure of Env epitopes targeted by ADCC-mediating antibodies at the surface of cells expressing Nef isolates from EC; this correlated with enhanced susceptibility to ADCC. Altogether, our results suggest that enhanced susceptibility of HIV-1-infected cells to ADCC may contribute to the EC phenotype.IMPORTANCENef clones derived from elite controllers (EC) have been shown to be attenuated for CD4 downregulation; how this contributes to the nonprogressor phenotype of these infected individuals remains uncertain. Increasing evidence supports a role for HIV-specific antibody-dependent cellular cytotoxicity (ADCC) in controlling viral infection and replication. Here, we show that residual CD4 left at the surface of cells expressing Nef proteins isolated from ECs are sufficient to allow Env-CD4 interaction, leading to increased exposure of Env CD4-induced epitopes and increased susceptibility of infected cells to ADCC. Our results suggest that ADCC might be an active immune mechanism in EC that helps to maintain durable suppression of viral replication and low plasma viremia level in this rare subset of infected individuals. Therefore, targeting Nef's ability to downregulate CD4 could render HIV-1-infected cells susceptible to ADCC and thus have therapeutic utility.

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HIV-1 accessory protein Vpr interacts with REAF/RPRD2 to mitigate its antiviral activity

10.1101/408161 ◽

2018 ◽

Cited By ~ 2

Author(s):

Joseph M Gibbons ◽

Kelly M Marno ◽

Rebecca Pike ◽

Wing-yiu Jason Lee ◽

Christopher E Jones ◽

...

Keyword(s):

T Cells ◽

Viral Replication ◽

Viral Entry ◽

Reverse Transcription ◽

Early Phase ◽

Early Stage ◽

Target Cells ◽

Accessory Protein ◽

Hiv 1

AbstractThe Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement in the early phase of infection. Previously, we described REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without intactvpris more highly restricted by REAF and, using delivery by VLPs, that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells and we detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, which impedes HIV infection in macrophages.ImportanceFor at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages, or to explain why Vpr is carried in the virus particle. Here we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

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Faculty Opinions recommendation of Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726369545.793518839 ◽

2016 ◽

Author(s):

Stephen J Kent

Keyword(s):

Neutralizing Antibodies ◽

Broadly Neutralizing Antibodies ◽

Infected Cells ◽

Hiv 1

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Synergistic In Vitro Antiretroviral Activity of a Humanized Monoclonal Anti-CD4 Antibody (TNX-355) and Enfuvirtide (T-20)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00761-05 ◽

2006 ◽

Vol 50 (6) ◽

pp. 2231-2233 ◽

Cited By ~ 39

Author(s):

Xing-Quan Zhang ◽

Meredith Sorensen ◽

Michael Fung ◽

Robert T. Schooley

Keyword(s):

Viral Replication ◽

Viral Entry ◽

Antiretroviral Agents ◽

Entry Inhibitors ◽

High Level ◽

Antiretroviral Activity

ABSTRACT Recently, antiretroviral agents directed at several steps involved in viral entry have been shown to reduce viral replication in vitro and in vivo. We have demonstrated a high level of in vitro synergistic antiretroviral activity for two entry inhibitors that are directed at sequential steps in the entry process.

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Notwithstanding Circumstantial Alibis, Cytotoxic T Cells Can Be Major Killers of HIV-1-Infected Cells

Journal of Virology ◽

10.1128/jvi.00306-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7066-7083 ◽

Cited By ~ 14

Author(s):

Saikrishna Gadhamsetty ◽

Tim Coorens ◽

Rob J. de Boer

Keyword(s):

T Cells ◽

Viral Replication ◽

Cytotoxic T Cells ◽

Chronic Phase ◽

Replication Rate ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Eclipse Phase ◽

Hiv 1

ABSTRACTSeveral experiments suggest that in the chronic phase of human immunodeficiency virus type 1 (HIV-1) infection, CD8+cytotoxic T lymphocytes (CTL) contribute very little to the death of productively infected cells. First, the expected life span of productively infected cells is fairly long, i.e., about 1 day. Second, this life span is hardly affected by the depletion of CD8+T cells. Third, the rate at which mutants escaping a CTL response take over the viral population tends to be slow. Our main result is that all these observations are perfectly compatible with killing rates that are much faster than one per day once we invoke the fact that infected cells proceed through an eclipse phase of about 1 day before they start producing virus. Assuming that the major protective effect of CTL is cytolytic, we demonstrate that mathematical models with an eclipse phase account for the data when the killing is fast and when it varies over the life cycle of infected cells. Considering the steady state corresponding to the chronic phase of the infection, we find that the rate of immune escape and the rate at which the viral load increases following CD8+T cell depletion should reflect the viral replication rate, ρ. A meta-analysis of previous data shows that viral replication rates during chronic infection vary between 0.5 ≤ ρ ≤ 1 day−1. Balancing such fast viral replication requires killing rates that are several times larger than ρ, implying that most productively infected cells would die by cytolytic effects.IMPORTANCEMost current data suggest that cytotoxic T cells (CTL) mediate their control of human immunodeficiency virus type 1 (HIV-1) infection by nonlytic mechanisms; i.e., the data suggest that CTL hardly kill. This interpretation of these data has been based upon the general mathematical model for HIV infection. Because this model ignores the eclipse phase between the infection of a target cell and the start of viral production by that cell, we reanalyze the same data sets with novel models that do account for the eclipse phase. We find that the data are perfectly consistent with lytic control by CTL and predict that most productively infected cells are killed by CTL. Because the killing rate should balance the viral replication rate, we estimate both parameters from a large set of published experiments in which CD8+T cells were depleted in simian immunodeficiency virus (SIV)-infected monkeys. This confirms that the killing rate can be much faster than is currently appreciated.

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Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

Journal of Virology ◽

10.1128/jvi.00915-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9864-9878 ◽

Cited By ~ 148

Author(s):

Michael E. Abram ◽

Andrea L. Ferris ◽

Wei Shao ◽

W. Gregory Alvord ◽

Stephen H. Hughes

Keyword(s):

Viral Replication ◽

Mutation Rate ◽

Hot Spots ◽

High Rate ◽

Published Data ◽

Single Cycle ◽

Efficient System ◽

Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

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Excision of Latent HIV-1 from Infected Cells In Vivo: An Important Step Forward

Molecular Therapy ◽

10.1016/j.ymthe.2017.04.009 ◽

2017 ◽

Vol 25 (5) ◽

pp. 1062-1064 ◽

Cited By ~ 1

Author(s):

Harshana S. De Silva Feelixge ◽

Keith R. Jerome

Keyword(s):

Infected Cells ◽

Latent Hiv ◽

Hiv 1

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The characteristics of the HIV-1 Env glycoprotein contribute to viral pathogenesis

10.1101/2021.07.07.451566 ◽

2021 ◽

Author(s):

Silvia Perez-Yanes ◽

Maria Pernas ◽

Silvia Marfil ◽

Romina Cabrera-Rodríguez ◽

Raquel Ortiz ◽

...

Keyword(s):

Functional Characteristic ◽

Elite Controllers ◽

Clinical Progression ◽

Viral Control ◽

Clinical Phenotypes ◽

Viral Loads ◽

Variable Contribution ◽

Gp120 Subunit ◽

Hiv 1

The understanding of HIV-1 pathogenesis and clinical progression is incomplete because of the variable contribution of host, immune and viral factors. The involvement of viral factors has been investigated in extreme clinical phenotypes from rapid progressors to long-term non-progressors (LTNPs). Among HIV-1 proteins, the envelope glycoprotein complex (Env) has concentrated many studies for its important role in the immune response and in the first steps of viral replication. In this study, we analyzed the contribution of 41 Envs from 24 patients with different clinical progression rates and viral loads (VLs), LTNP-Elite Controllers (LTNP-ECs); Viremic LTNPs (vLTNPs), and non-controller’s individuals contemporary to LTNPs or recent, named Old and Modern progressors. We analyzed the Env expression, the fusion and cell-to-cell transfer capacities as well as viral infectivity. The sequence and phylogenetic analysis of Envs were also performed. In every functional characteristic, the Envs from subjects with viral control (LTNP-ECs and vLTNPs) showed significant lower performance compared to those from the progressor individuals (Old and Modern). Regarding sequence analysis, the variable loops of the gp120 subunit of the Env (i.e., V2, V4 and mainly V5) of the progressor individuals showed longer and more glycosylated sequences than controller subjects. Therefore, HIV-1 Envs presenting poor viral functions and shorter sequences were associated with viremic control and the non-progressor clinical phenotype, whereas functional Envs were associated with the lack of virological control and progressor clinical phenotypes. These correlations support the central role of Env genotypic and phenotypic characteristics in the in vivo HIV-1 infection and pathogenesis.

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HIV-Infected Macrophages Are Infected and Killed by the Interferon-Sensitive Rhabdovirus MG1

Journal of Virology ◽

10.1128/jvi.01953-20 ◽

2021 ◽

Vol 95 (9) ◽

Author(s):

Teslin S. Sandstrom ◽

Nischal Ranganath ◽

Stephanie C. Burke Schinkel ◽

Syim Salahuddin ◽

Oussama Meziane ◽

...

Keyword(s):

Cancer Cells ◽

Oncolytic Viruses ◽

Type I Interferons ◽

Viral Reservoir ◽

Type I ◽

Antiviral Signaling ◽

Infected Cells ◽

Hiv 1

ABSTRACT The use of unique cell surface markers to target and eradicate HIV-infected cells has been a longstanding objective of HIV-1 cure research. This approach, however, overlooks the possibility that intracellular changes present within HIV-infected cells may serve as valuable therapeutic targets. For example, the identification of dysregulated antiviral signaling in cancer has led to the characterization of oncolytic viruses capable of preferentially killing cancer cells. Since impairment of cellular antiviral machinery has been proposed as a mechanism by which HIV-1 evades immune clearance, we hypothesized that HIV-infected macrophages (an important viral reservoir in vivo) would be preferentially killed by the interferon-sensitive oncolytic Maraba virus MG1. We first showed that HIV-infected monocyte-derived macrophages (MDM) were more susceptible to MG1 infection and killing than HIV-uninfected cells. As MG1 is highly sensitive to type I interferons (IFN-I), we then investigated whether we could identify IFN-I signaling differences between HIV-infected and uninfected MDM and found evidence of impaired IFN-α responsiveness within HIV-infected cells. Finally, to assess whether MG1 could target a relevant, primary cell reservoir of HIV-1, we investigated its effects in alveolar macrophages (AM) obtained from effectively treated individuals living with HIV-1. As observed with in vitro-infected MDM, we found that HIV-infected AM were preferentially eliminated by MG1. In summary, the oncolytic rhabdovirus MG1 appears to preferentially target and kill HIV-infected cells via impairment of antiviral signaling pathways and may therefore provide a novel approach to an HIV-1 cure. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) remains a treatable, but incurable, viral infection. The establishment of viral reservoirs containing latently infected cells remains the main obstacle in the search for a cure. Cure research has also focused on only one cellular target of HIV-1 (the CD4+ T cell) while largely overlooking others (such as macrophages) that contribute to HIV-1 persistence. In this study, we address these challenges by describing a potential strategy for the eradication of HIV-infected macrophages. Specifically, we show that an engineered rhabdovirus—initially developed as a cancer therapy—is capable of preferential infection and killing of HIV-infected macrophages, possibly via the same altered antiviral signaling seen in cancer cells. As this rhabdovirus is currently being explored in phase I/II clinical trials, there is potential for this approach to be readily adapted for use within the HIV-1 cure field.

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