Suppression of HIV-1 Infection by APOBEC3 Proteins in Primary Human CD4+T Cells Is Associated with Inhibition of Processive Reverse Transcription as Well as Excessive Cytidine Deamination

ABSTRACTThe Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4+T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4+T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3′ termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4+T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself.

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Kinetics of Human Immunodeficiency Virus Type 1 Decay following Entry into Resting CD4+ T Cells

Journal of Virology ◽

10.1128/jvi.79.4.2199-2210.2005 ◽

2005 ◽

Vol 79 (4) ◽

pp. 2199-2210 ◽

Cited By ~ 137

Author(s):

Yan Zhou ◽

Haili Zhang ◽

Janet D. Siliciano ◽

Robert F. Siliciano

Keyword(s):

Gene Expression ◽

Human Immunodeficiency Virus ◽

T Cells ◽

Human Immunodeficiency Virus Type ◽

Reverse Transcription ◽

Half Life ◽

Immunodeficiency Virus ◽

Kinetics Of ◽

Hiv 1

ABSTRACT In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4+ T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4+ cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4+ T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4+ T cells. Decay processes dominate in resting CD4+ T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4+ T cells is highly labile.

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Investigating HIV-Human Interaction Networks to Unravel Pathogenic Mechanism for Drug Discovery: A Systems Biology Approach

Current HIV Research ◽

10.2174/1570162x16666180219155324 ◽

2018 ◽

Vol 16 (1) ◽

pp. 77-95 ◽

Cited By ~ 1

Author(s):

Cheng-Wei Li ◽

Bor-Sen Chen

Keyword(s):

T Cells ◽

Reverse Transcription ◽

Late Stage ◽

Time Course ◽

System Modeling ◽

Human Interaction ◽

Flufenamic Acid ◽

Replication Stage ◽

Core Proteins ◽

Hiv 1

Background: Two big issues in the study of pathogens are determining how pathogens infect hosts and how the host defends itself against infection. Therefore, investigating host-pathogen interactions is important for understanding pathogenicity and host defensive mechanisms and treating infections.Methods: In this study, we used omics data, including time-course data from high-throughput sequencing, real-time polymerase chain reaction, and human microRNA (miRNA) and protein-protein interaction to construct an interspecies protein-protein and miRNA interaction (PPMI) network of human CD4+ T cells during HIV-1 infection through system modeling and identification.Results: By applying a functional annotation tool to the identified PPMI network at each stage of HIV infection, we found that repressions of three miRNAs, miR-140-5p, miR-320a, and miR-941, are involved in the development of autoimmune disorders, tumor proliferation, and the pathogenesis of T cells at the reverse transcription stage. Repressions of miR-331-3p and miR-320a are involved in HIV-1 replication, replicative spread, anti-apoptosis, cell proliferation, and dysregulation of cell cycle control at the integration/replication stage. Repression of miR-341-5p is involved in carcinogenesis at the late stage of HIV-1 infection.Conclusion: By investigating the common core proteins and changes in specific proteins in the PPMI network between the stages of HIV-1 infection, we obtained pathogenic insights into the functional core modules and identified potential drug combinations for treating patients with HIV-1 infection, including thalidomide, oxaprozin, and metformin, at the reverse transcription stage; quercetin, nifedipine, and fenbendazole, at the integration/replication stage; and staurosporine, quercetin, prednisolone, and flufenamic acid, at the late stage.

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Inhibition of Xenotropic Murine Leukemia Virus-Related Virus by APOBEC3 Proteins and Antiviral Drugs

Journal of Virology ◽

10.1128/jvi.00134-10 ◽

2010 ◽

Vol 84 (11) ◽

pp. 5719-5729 ◽

Cited By ~ 63

Author(s):

Tobias Paprotka ◽

Narasimhan J. Venkatachari ◽

Chawaree Chaipan ◽

Ryan Burdick ◽

Krista A. Delviks-Frankenberry ◽

...

Keyword(s):

Prostate Cancer ◽

T Cells ◽

B Cells ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Du145 Cells ◽

Apobec3 Proteins ◽

Hiv 1 ◽

Xmrv Infection ◽

Murine Leukemia

ABSTRACT Xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus, has been isolated from human prostate cancer tissue and from activated CD4+ T cells and B cells of patients with chronic fatigue syndrome, suggesting an association between XMRV infection and these two diseases. Since APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of murine leukemia virus and Vif-deficient human immunodeficiency virus type 1 (HIV-1), are expressed in human CD4+ T cells and B cells, we sought to determine how XMRV evades suppression of replication by APOBEC3 proteins. We found that expression of A3G, A3F, or murine A3 in virus-producing cells resulted in their virion incorporation, inhibition of XMRV replication, and G-to-A hypermutation of the viral DNA with all three APOBEC3 proteins. Quantitation of A3G and A3F mRNAs indicated that, compared to the human T-cell lines CEM and H9, prostate cell lines LNCaP and DU145 exhibited 50% lower A3F mRNA levels, whereas A3G expression in 22Rv1, LNCaP, and DU145 cells was nearly undetectable. XMRV proviral genomes in LNCaP and DU145 cells were hypermutated at low frequency with mutation patterns consistent with A3F activity. XMRV proviral genomes were extensively hypermutated upon replication in A3G/A3F-positive T cells (CEM and H9), but not in A3G/A3F-negative cells (CEM-SS). We also observed that XMRV replication was susceptible to the nucleoside reverse transcriptase (RT) inhibitors zidovudine (AZT) and tenofovir and the integrase inhibitor raltegravir. In summary, the establishment of XMRV infection in patients may be dependent on infection of A3G/A3F-deficient cells, and cells expressing low levels of A3G/A3F, such as prostate cancer cells, may be ideal producers of infectious XMRV. Furthermore, the anti-HIV-1 drugs AZT, tenofovir, and raltegravir may be useful for treatment of XMRV infection.

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Immature Dendritic Cells Selectively Replicate Macrophagetropic (M-Tropic) Human Immunodeficiency Virus Type 1, while Mature Cells Efficiently Transmit both M- and T-Tropic Virus to T Cells

Journal of Virology ◽

10.1128/jvi.72.4.2733-2737.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 2733-2737 ◽

Cited By ~ 221

Author(s):

Angela Granelli-Piperno ◽

Elena Delgado ◽

Victoria Finkel ◽

William Paxton ◽

Ralph M. Steinman

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Dendritic Cells ◽

Human Immunodeficiency Virus Type ◽

Reverse Transcription ◽

Blood Monocytes ◽

Gm Csf ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Dendritic cells (DCs) can develop from CD14+ peripheral blood monocytes cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). By 6 days in culture, the cells have the characteristics of immature DCs and can be further induced to mature by inflammatory stimuli or by monocyte-conditioned medium. After infection with macrophagetropic (M-tropic) human immunodeficiency virus type 1 (HIV-1), monocytes and mature DCs show a block in reverse transcription and only form early transcripts that can be amplified with primers for the R/U5 region. In contrast, immature DCs cultured for 6 or 11 days in GM-CSF and IL-4 complete reverse transcription and show a strong signal when LTR/gag primers are used. Blood monocytes and mature DCs do not replicate HIV-1, whereas immature DCs can be productively infected, but only with M-tropic HIV-1. The virus produced by immature DCs readily infects activated T cells. Although mature DCs do not produce virus, these cells transmit both M- and T-tropic virus to T cells. In the cocultures, both DCs and T cells must express functional chemokine coreceptors for viral replication to occur. Therefore, the developmental stage of DCs can influence the interaction of these cells with HIV-1 and influence the extent to which M-tropic and T-tropic virus can replicate.

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Contribution of MxB Oligomerization to HIV-1 Capsid Binding and Restriction

Journal of Virology ◽

10.1128/jvi.03730-14 ◽

2015 ◽

Vol 89 (6) ◽

pp. 3285-3294 ◽

Cited By ~ 33

Author(s):

Cindy Buffone ◽

Bianca Schulte ◽

Silvana Opp ◽

Felipe Diaz-Griffero

Keyword(s):

T Cells ◽

Structure Function ◽

Capsid Protein ◽

Reverse Transcription ◽

Tertiary Structure ◽

Alpha Interferon ◽

Restriction Factor ◽

Current Structure ◽

Modeled Structure ◽

Hiv 1

ABSTRACTThe alpha interferon (IFN-α)-inducible restriction factor myxovirus B (MxB) blocks HIV-1 infection after reverse transcription but prior to integration. MxB binds to the HIV-1 core, which is composed of capsid protein, and this interaction leads to inhibition of the uncoating process of HIV-1. Previous studies suggested that HIV-1 restriction by MxB requires binding to capsid. This work tests the hypothesis that MxB oligomerization is important for the ability of MxB to bind to the HIV-1 core. For this purpose, we modeled the structure of MxB using the published tertiary structure of MxA. The modeled structure of MxB guided our mutagenic studies and led to the discovery of several MxB variants that lose the capacity to oligomerize. In agreement with our hypothesis, MxB variants that lost the oligomerization capacity also lost the ability to bind to the HIV-1 core. MxB variants deficient for oligomerization were not able to block HIV-1 infection. Overall, our work showed that oligomerization is required for the ability of MxB to bind to the HIV-1 core and block HIV-1 infection.IMPORTANCEMxB is a novel restriction factor that blocks infection of HIV-1. MxB is inducible by IFN-α, particularly in T cells. The current work studies the oligomerization determinants of MxB and carefully explores the contribution of oligomerization to capsid binding and restriction. This work takes advantage of the current structure of MxA and models the structure of MxB, which is used to guide structure-function studies. This work leads to the conclusion that MxB oligomerization is important for HIV-1 capsid binding and restriction.

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Flexibility in Nucleic Acid Binding Is Central to APOBEC3H Antiviral Activity

Journal of Virology ◽

10.1128/jvi.01275-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 4

Author(s):

Jennifer A. Bohn ◽

Justin DaSilva ◽

Siarhei Kharytonchyk ◽

Maria Mercedes ◽

Jennifer Vosters ◽

...

Keyword(s):

Amino Acids ◽

Nucleic Acid ◽

Antiviral Activity ◽

Reverse Transcription ◽

Rna Binding ◽

Viral Dna ◽

Dna Substrates ◽

Apobec3 Proteins ◽

Rna Interaction ◽

Hiv 1

ABSTRACT APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination. IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

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APOBEC3F and APOBEC3G Inhibit HIV-1 DNA Integration by Different Mechanisms

Journal of Virology ◽

10.1128/jvi.02358-09 ◽

2010 ◽

Vol 84 (10) ◽

pp. 5250-5259 ◽

Cited By ~ 83

Author(s):

Jean L. Mbisa ◽

Wei Bu ◽

Vinay K. Pathak

Keyword(s):

Catalytic Domain ◽

Restriction Factors ◽

Viral Dna ◽

Dna Integration ◽

Host Restriction ◽

Antiviral Activities ◽

Viral Cdna ◽

Viral Dna Integration ◽

Cytidine Deamination ◽

Hiv 1

ABSTRACT APOBEC3F (A3F) and APBOBEC3G (A3G) both are host restriction factors that can potently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Their antiviral activities are at least partially mediated by cytidine deamination, which causes lethal mutations of the viral genome. We recently showed that A3G blocks viral plus-strand DNA transfer and inhibits provirus establishment in the host genome (J. L. Mbisa, R. Barr, J. A. Thomas, N. Vandegraaff, I. J. Dorweiler, E. S. Svarovskaia, W. L. Brown, L. M. Mansky, R. J. Gorelick, R. S. Harris, A. Engelman, and V. K. Pathak, J. Virol. 81:7099-7110, 2007). Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blot analysis. A3G generates a 6-bp extension at the viral U5 end of the 3′ long terminal repeat (3′-LTR), which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing the 3′ processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3′-LTR double-stranded DNA (dsDNA) oligonucleotide template than A3G. Taking these results together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.

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Foamy Virus Bet Proteins Function as Novel Inhibitors of the APOBEC3 Family of Innate Antiretroviral Defense Factors

Journal of Virology ◽

10.1128/jvi.79.14.8724-8731.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 8724-8731 ◽

Cited By ~ 130

Author(s):

Rebecca A. Russell ◽

Heather L. Wiegand ◽

Michael D. Moore ◽

Alexandra Schäfer ◽

Myra O. McClure ◽

...

Keyword(s):

Foamy Virus ◽

African Green Monkey ◽

Apobec3 Protein ◽

Zoonotic Infections ◽

Wide Range ◽

Green Monkey ◽

Auxiliary Protein ◽

Apobec3 Proteins ◽

Bet Protein ◽

Hiv 1

ABSTRACT Foamy viruses are a family of complex retroviruses that establish common, productive infections in a wide range of nonhuman primates. In contrast, humans appear nonpermissive for foamy virus replication, although zoonotic infections do occur. Here we have analyzed the ability of primate and mouse APOBEC3G proteins to inhibit the infectivity of primate foamy virus (PFV) virions produced in their presence. We demonstrate that several APOBEC3 proteins can potently inhibit the infectivity of a PFV-based viral vector. This inhibition correlated with the packaging of inhibitory APOBEC3 proteins into PFV virions, due to a specific PFV Gag/APOBEC3 interaction, and resulted in the G to A hypermutation of PFV reverse transcripts. While inhibition of PFV virion infectivity by primate APOBEC3 proteins was largely relieved by coexpression of the PFV Bet protein, a cytoplasmic auxiliary protein of previously uncertain function, Bet failed to relieve inhibition caused by murine APOBEC3. PFV Bet bound to human, but not mouse, APOBEC3 proteins in coexpressing cells, and this binding correlated with the specific inhibition of their incorporation into PFV virions. Of note, both PFV Bet and a second Bet protein, derived from an African green monkey foamy virus, rescued the infectivity of Vif-deficient human immunodeficiency virus type 1 (HIV-1) virions produced in the presence of African green monkey APOBEC3G and blocked the incorporation of this host factor into HIV-1 virion particles. However, neither foamy virus Bet protein reduced APOBEC3 protein expression levels in virion producer cells. While these data identify the foamy virus Bet protein as a functional ortholog of the HIV-1 Vif auxiliary protein, they also indicate that Vif and Bet block APOBEC3 protein function by distinct mechanisms.

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