Vpu-Mediated Counteraction of Tetherin Is a Major Determinant of HIV-1 Interferon Resistance

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) groups M, N, O, and P are the result of independent zoonotic transmissions of simian immunodeficiency viruses (SIVs) infecting great apes in Africa. Among these, only Vpu proteins of pandemic HIV-1 group M strains evolved potent activity against the restriction factor tetherin, which inhibits virus release from infected cells. Thus, effective Vpu-mediated tetherin antagonism may have been a prerequisite for the global spread of HIV-1. To determine whether this particular function enhances primary HIV-1 replication and interferon resistance, we introduced mutations into thevpugenes of HIV-1 group M and N strains to specifically disrupt their ability to antagonize tetherin, but not other Vpu functions, such as degradation of CD4, down-modulation of CD1d and NTB-A, and suppression of NF-κB activity. Lack of particular human-specific adaptations reduced the ability of HIV-1 group M Vpu proteins to enhance virus production and release from primary CD4+T cells at high levels of type I interferon (IFN) from about 5-fold to 2-fold. Interestingly, transmitted founder HIV-1 strains exhibited higher virion release capacity than chronic control HIV-1 strains irrespective of Vpu function, and group M viruses produced higher levels of cell-free virions than an N group HIV-1 strain. Thus, efficient virus release from infected cells seems to play an important role in the spread of HIV-1 in the human population and requires a fully functional Vpu protein that counteracts human tetherin.IMPORTANCEUnderstanding which human-specific adaptations allowed HIV-1 to cause the AIDS pandemic is of great importance. One feature that distinguishes pandemic HIV-1 group M strains from nonpandemic or rare group O, N, and P viruses is the acquisition of mutations in the accessory Vpu protein that confer potent activity against human tetherin. Adaptation was required because human tetherin has a deletion that renders it resistant to the Nef protein used by the SIV precursor of HIV-1 to antagonize this antiviral factor. It has been suggested that these adaptations in Vpu were critical for the effective spread of HIV-1 M strains, but direct evidence has been lacking. Here, we show that these changes in Vpu significantly enhance virus replication and release in human CD4+T cells, particularly in the presence of IFN, thus supporting an important role in the spread of pandemic HIV-1.

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Reactivation Kinetics of HIV-1 and Susceptibility of Reactivated Latently Infected CD4+T Cells to HIV-1-Specific CD8+T Cells

Journal of Virology ◽

10.1128/jvi.01454-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9631-9638 ◽

Cited By ~ 10

Author(s):

Victoria E. K. Walker-Sperling ◽

Valerie J. Cohen ◽

Patrick M. Tarwater ◽

Joel N. Blankson

Keyword(s):

T Cells ◽

T Cell ◽

Antiretroviral Therapy ◽

Time Frame ◽

Virus Release ◽

Latently Infected ◽

Shock And Kill ◽

Infected Cells ◽

Kinetics Of ◽

Hiv 1

ABSTRACTThe “shock and kill” model of human immunodeficiency virus type 1 (HIV-1) eradication involves the induction of transcription of HIV-1 genes in latently infected CD4+T cells, followed by the elimination of these infected CD4+T cells by CD8+T cells or other effector cells. CD8+T cells may also be needed to control the spread of new infection if residual infected cells are present at the time combination antiretroviral therapy (cART) is discontinued. In order to determine the time frame needed for CD8+T cells to effectively prevent the spread of HIV-1 infection, we examined the kinetics of HIV transcription and virus release in latently infected cells reactivatedex vivo. Isolated resting, primary CD4+T cells from HIV-positive (HIV+) subjects on suppressive regimens were found to upregulate cell-associated HIV-1 mRNA within 1 h of stimulation and produce extracellular virus as early as 6 h poststimulation. In spite of the rapid kinetics of virus production, we show that CD8+T cells from 2 out of 4 viremic controllers were capable of effectively eliminating reactivated autologous CD4+cells that upregulate cell-associated HIV-1 mRNA. The results have implications for devising strategies to prevent rebound viremia due to reactivation of rare latently infected cells that persist after potentially curative therapy.IMPORTANCEA prominent HIV-1 cure strategy termed “shock and kill” involves the induction of HIV-1 transcription in latently infected CD4+T cells with the goal of elimination of these cells by either the cytotoxic T lymphocyte response or other immune cell subsets. However, the cytotoxic T cell response may also be required after curative treatment if residual latently infected cells remain. The kinetics of HIV-1 reactivation indicate rapid upregulation of cell-associated HIV-1 mRNA and a 5-h window between transcription and virus release. Thus, HIV-specific CD8+T cell responses likely have a very short time frame to eliminate residual latently infected CD4+T cells that become reactivated after discontinuation of antiretroviral therapy following potentially curative treatment strategies.

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The Nef protein of the macrophage tropic HIV-1 strain AD8 counteracts human Bst-2/tetherin

10.1101/2020.02.07.938464 ◽

2020 ◽

Author(s):

Sebastian Giese ◽

Scott P. Lawrence ◽

Michela Mazzon ◽

Bernadien M. Nijmeijer ◽

Mark Marsh

Keyword(s):

Cell Surface ◽

Virus Release ◽

Viral Budding ◽

Primary Monocyte ◽

Human Tetherin ◽

Human Immunodeficiency Viruses ◽

Infected Cells ◽

Translation Initiation Codon ◽

Hiv 1 ◽

Tetherin Antagonism

AbstractBst-2/tetherin inhibits the release of numerous enveloped viruses by physically attaching nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency viruses (HIV), and pandemic main (M) group HIV-1s are thought to exclusively rely on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 was able to use its Nef protein to compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous tetherin levels from the cell surface, physically separating it from the site of viral budding and thus preventing HIV retention. Mechanistically, AD8 Nef enhances l-tetherin internalisation, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, this is the first report showing that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

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Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1

Journal of Virology ◽

10.1128/jvi.02736-15 ◽

2015 ◽

Vol 90 (5) ◽

pp. 2486-2502 ◽

Cited By ~ 6

Author(s):

Andrey Tokarev ◽

Charlotte Stoneham ◽

Mary K. Lewinski ◽

Amey Mukim ◽

Savitha Deshmukh ◽

...

Keyword(s):

T Cells ◽

Ubiquitin Ligase ◽

Immune Surveillance ◽

Hek293 Cells ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Surface Level ◽

Virion Release ◽

Infected Cells ◽

Hiv 1

ABSTRACTHIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence ofvpuornefand even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC.IMPORTANCEPathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.

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Kinetics of Antiviral Activity by Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes (CTL) and Rapid Selection of CTL Escape Virus In Vitro

Journal of Virology ◽

10.1128/jvi.72.8.6851-6857.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6851-6857 ◽

Cited By ~ 59

Author(s):

C. A. Van Baalen ◽

M. Schutten ◽

R. C. Huisman ◽

P. H. M. Boers ◽

R. A. Gruters ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Activity ◽

Virus Production ◽

Virus Release ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Kinetics Of ◽

Hiv 1 ◽

Selection Of

ABSTRACT The antiviral activity of a CD8+ cytotoxic T-lymphocyte (CTL) clone (TCC108) directed against a newly identified HLA-B14-restricted epitope, human immunodeficiency virus type 1 (HIV-1) Rev(67-75) SAEPVPLQL, was analyzed with respect to its kinetics of target cell lysis and inhibition of HIV-1 production. Addition of TCC108 cells or CD8+ reverse transcriptase-specific CTLs to HLA-matched CD4+ T cells at different times after infection with HIV-1 IIIB showed that infected cells became susceptible to CTL-mediated lysis before peak virus production but after the onset of progeny virus release. When either of these CTLs were added to part of the infected cells immediately after infection, p55 expression and virus production were significantly suppressed. These data support a model in which CTLs, apart from exerting cytolytic activity which may prevent continued virus release, can interfere with viral protein expression during the eclipse phase via noncytolytic mechanisms. TCC108-mediated inhibition of virus replication in peripheral blood mononuclear cells caused rapid selection of a virus with a mutation (69E→K) in the Rev(67-75) CTL epitope which abolished recognition by TCC108 cells. Taken together, these data suggest that both cytolytic and noncytolytic antiviral mechanisms of CTLs can be specifically targeted to HIV-1-infected cells.

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The Nef Protein of the Macrophage Tropic HIV-1 Strain AD8 Counteracts Human BST-2/Tetherin

Viruses ◽

10.3390/v12040459 ◽

2020 ◽

Vol 12 (4) ◽

pp. 459 ◽

Cited By ~ 1

Author(s):

Sebastian Giese ◽

Scott P. Lawrence ◽

Michela Mazzon ◽

Bernadien M. Nijmeijer ◽

Mark Marsh

Keyword(s):

Cell Surface ◽

Virus Release ◽

Viral Budding ◽

Primary Monocyte ◽

Human Tetherin ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Translation Initiation Codon ◽

Hiv 1 ◽

Tetherin Antagonism

Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

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Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1620144114 ◽

2017 ◽

Vol 114 (4) ◽

pp. E590-E599 ◽

Cited By ~ 77

Author(s):

Shilpa S. Iyer ◽

Frederic Bibollet-Ruche ◽

Scott Sherrill-Mix ◽

Gerald H. Learn ◽

Lindsey Plenderleith ◽

...

Keyword(s):

T Cells ◽

Sexual Transmission ◽

Type I ◽

Type I Ifns ◽

The Face ◽

Infected Cells ◽

Sexual Transmission Of Hiv ◽

Distinguishing Property ◽

Hiv 1

Sexual transmission of HIV-1 is an inefficient process, with only one or few variants of the donor quasispecies establishing the new infection. A critical, and as yet unresolved, question is whether the mucosal bottleneck selects for viruses with increased transmission fitness. Here, we characterized 300 limiting dilution-derived virus isolates from the plasma, and in some instances genital secretions, of eight HIV-1 donor and recipient pairs. Although there were no differences in the amount of virion-associated envelope glycoprotein, recipient isolates were on average threefold more infectious (P= 0.0001), replicated to 1.4-fold higher titers (P= 0.004), were released from infected cells 4.2-fold more efficiently (P< 0.00001), and were significantly more resistant to type I IFNs than the corresponding donor isolates. Remarkably, transmitted viruses exhibited 7.8-fold higher IFNα2 (P< 0.00001) and 39-fold higher IFNβ (P< 0.00001) half-maximal inhibitory concentrations (IC50) than did donor isolates, and their odds of replicating in CD4+T cells at the highest IFNα2 and IFNβ doses were 35-fold (P< 0.00001) and 250-fold (P< 0.00001) greater, respectively. Interestingly, pretreatment of CD4+T cells with IFNβ, but not IFNα2, selected donor plasma isolates that exhibited a transmitted virus-like phenotype, and such viruses were also detected in the donor genital tract. These data indicate that transmitted viruses are phenotypically distinct, and that increased IFN resistance represents their most distinguishing property. Thus, the mucosal bottleneck selects for viruses that are able to replicate and spread efficiently in the face of a potent innate immune response.

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Exosomes from Nef expressing monocytic cells restrict HIV-1 replication in infected cells through the assembly of stress granules

10.1101/148791 ◽

2017 ◽

Author(s):

Mohammad Yunus Ansari ◽

Hasan Imam ◽

Nishant Kumar ◽

Zulfazal Ahmed ◽

Shahid Jameel

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Virus Production ◽

Stress Granules ◽

Monocytic Cells ◽

Activation Induced Cell Death ◽

Arginine Residues ◽

Infected Cells ◽

Almost All ◽

Hiv 1

AbstractExosomes are membranous vesicles secreted from almost all types of cells, carry proteins and nucleic acids and function as vehicles for intercellular communication. Cells infected with HIV-1 or expressing the viral Nef protein secrete more exosomes than uninfected cells or those not expressing this protein. We used stably transfected, Nef-expressing U937 human monocytic cells and exosomes purified from these cells to study their effects on HIV-1 infected and uninfected CD4+ T-cells. The Nef exosomes inhibited virus production from HIV-1 infected CD4+ T-cells, but caused activation induced cell death in uninfected bystander cells. Mutations in its conserved Arginine residues and in the secretion-modification-region failed to secrete Nef into exosomes. Cell lines expressing these mutant Nef proteins did not deliver it to the target CD4+ T-cells, and exosomes prepared from these mutant Nef-expressing cells also did not inhibit virus production. Nef exosomes inhibited virus production by inducing the assembly of stress granules in HIV-1 infected cells, which sequestered increased amounts of gag mRNA. This is a novel mechanism wherein we show the effects of exosomes on the assembly of stress granules and viral translational repression.

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Tetherin Antagonism by HIV-1 Group M Nef Proteins

Journal of Virology ◽

10.1128/jvi.01465-16 ◽

2016 ◽

Vol 90 (23) ◽

pp. 10701-10714 ◽

Cited By ~ 13

Author(s):

Juan F. Arias ◽

Marta Colomer-Lluch ◽

Benjamin von Bredow ◽

Justin M. Greene ◽

Julie MacDonald ◽

...

Keyword(s):

Transmembrane Protein ◽

Viral Gene ◽

Significant Activity ◽

Virus Release ◽

Aids Pandemic ◽

Human Tetherin ◽

Dependent Cell ◽

Infected Cells ◽

Hiv 1 ◽

Tetherin Antagonism

ABSTRACTAlthough Nef is the viral gene product used by most simian immunodeficiency viruses to overcome restriction by tetherin, this activity was acquired by the Vpu protein of HIV-1 group M due to the absence of sequences in human tetherin that confer susceptibility to Nef. Thus, it is widely accepted that HIV-1 group M uses Vpu instead of Nef to counteract tetherin. Challenging this paradigm, we identified Nef alleles of HIV-1 group M isolates with significant activity against human tetherin. These Nef proteins promoted virus release and tetherin downmodulation from the cell surface and, in the context ofvpu-deleted HIV-1 recombinants, enhanced virus replication and resistance to antibody-dependent cell-mediated cytotoxicity (ADCC). Further analysis revealed that the Vpu proteins from several of these viruses lack antitetherin activity, suggesting that under certain circumstances, HIV-1 group M Nef may acquire the ability to counteract tetherin to compensate for the loss of this function by Vpu. These observations illustrate the remarkable plasticity of HIV-1 in overcoming restriction by tetherin and challenge the prevailing view that all HIV-1 group M isolates use Vpu to counteract tetherin.IMPORTANCEMost viruses of HIV-1 group M, the main group of HIV-1 responsible for the global AIDS pandemic, use their Vpu proteins to overcome restriction by tetherin (BST-2 or CD317), which is a transmembrane protein that inhibits virus release from infected cells. Here we show that the Nef proteins of certain HIV-1 group M isolates can acquire the ability to counteract tetherin. These results challenge the current paradigm that HIV-1 group M exclusively uses Vpu to counteract tetherin and underscore the importance of tetherin antagonism for efficient viral replication.

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HIV-1 Infection Transcriptomics: Meta-Analysis of CD4+ T Cells Gene Expression Profiles

Viruses ◽

10.3390/v13020244 ◽

2021 ◽

Vol 13 (2) ◽

pp. 244 ◽

Cited By ~ 1

Author(s):

Antonio Victor Campos Coelho ◽

Rossella Gratton ◽

João Paulo Britto de Melo ◽

José Leandro Andrade-Santos ◽

Rafael Lima Guimarães ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Differentially Expressed Genes ◽

Cd4 T Cells ◽

Expression Profiles ◽

Meta Analysis ◽

Differentially Expressed ◽

Rna Seq ◽

Infected Cells ◽

Hiv 1

HIV-1 infection elicits a complex dynamic of the expression various host genes. High throughput sequencing added an expressive amount of information regarding HIV-1 infections and pathogenesis. RNA sequencing (RNA-Seq) is currently the tool of choice to investigate gene expression in a several range of experimental setting. This study aims at performing a meta-analysis of RNA-Seq expression profiles in samples of HIV-1 infected CD4+ T cells compared to uninfected cells to assess consistently differentially expressed genes in the context of HIV-1 infection. We selected two studies (22 samples: 15 experimentally infected and 7 mock-infected). We found 208 differentially expressed genes in infected cells when compared to uninfected/mock-infected cells. This result had moderate overlap when compared to previous studies of HIV-1 infection transcriptomics, but we identified 64 genes already known to interact with HIV-1 according to the HIV-1 Human Interaction Database. A gene ontology (GO) analysis revealed enrichment of several pathways involved in immune response, cell adhesion, cell migration, inflammation, apoptosis, Wnt, Notch and ERK/MAPK signaling.

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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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