Human Immunodeficiency Virus Type 1 Provirus Research Articles

Identification of HIV-1 Tat-Associated Proteins Contributing to HIV-1 Transcription and Latency

Human immunodeficiency virus type 1 (HIV-1) Tat is a virus-encoded trans-activator that plays a central role in viral transcription. We used our recently developed parallel analysis of in vitro translated open reading frames (ORFs) (PLATO) approach to identify host proteins that associate with HIV-1 Tat. From this proteomic assay, we identify 89 Tat-associated proteins (TAPs). We combine our results with other datasets of Tat or long terminal repeat (LTR)-associated proteins. For some of these proteins (NAT10, TINP1, XRCC5, SIN3A), we confirm their strong association with Tat. These TAPs also suppress Tat-mediated HIV-1 transcription. Removing suppression of HIV-1 transcription benefits the reversal of post-integrated, latent HIV-1 proviruses. We demonstrate that these transcriptionally suppressing TAPs contribute to HIV-1 latency in Jurkat latency (J-LAT) cells. Therefore, our proteomic analysis highlights the previously unappreciated TAPs that play a role in maintaining HIV-1 latency and can be further studied as potential pharmacological targets for the “shock and kill” HIV-1 cure strategy.

Therapeutic Immune Recovery and Reduction of CXCR4-Tropic HIV-1.

In the absence of therapy, CXCR4 (X4)-tropic human immunodeficiency virus type 1 (HIV-1) increases over time, associated with accelerated disease progression. In contrast, the majority of patients receiving long-term combination antiretroviral therapy (cART) present with CCR5 (R5)-tropic HIV-1 variants. It is unclear whether cART itself mediates the reduction of X4-tropic HIV-1. The current study aimed at assessing the tropism of viral integrates in patients' blood during fully suppressive cART. The relative frequencies of X4-tropic proviral HIV-1 variants were determined by means of next-generation sequencing (False Positive Rate (FPR), 3.5%; R5- or X4 tropic variants occurring at less than 2% of the total virus population) for 35 treated patients in the Swiss HIV Cohort Study and followed longitudinally over time. Full viral suppression and a continuous CD4 T-cell recovery during cART were documented for all patients. Viral phylogenetic changes and sequence evolution were analyzed. The majority of patients (80%) experienced no frequency increase in X4-tropic proviruses during therapy. Although some proviral sequence evolution was demonstrable in >50% of these patients during therapy, this growing viral diversity was in no case paralleled by the emergence or expansion of X4-tropic provirus variants. In the remaining 20% of patients, the documented expansion of X4-tropic provirus was based on the outgrowth of single viral variants from minority populations already present before therapy initiation. Our study demonstrates that X4-tropic HIV sharply declines in most patients during successful therapy, which indicates a preferential tropism-dependent provirus elimination in the immunocompetent host. The recently implemented World Health Organization strategies of immediate therapy initiation are fully in line with this gradual loss of X4 tropism during therapy. Moreover, the early use of coreceptor antagonists against the remaining CCR5-tropic viruses may be indicated.

Possible hidden cause of human immunodeficiency virus type 1 latency and role of interferon

Latent human immunodeficiency virus type 1 (HIV-1) infected cells under antiretroviral therapy are reported to be resting memory CD4+ T cells; however, the mechanisms of HIV-1 latency is unclear. We demonstrate that long-term culture of interleukin-2-dependent CD4+ T cells with a memory phenotype mimicked latently HIV-1-infected cells in the presence of interferon-?. These cells are mostly resting and contained HIV-1 proviruses that could be re-activated by stimulation. Our findings suggest a potential role of type-1 interferon in HIV-1 latency.Mediscope Vol. 3, No. 2: July 2016, Pages 11-17

Gp120 binding with DC-SIGN induces reactivation of HIV-1 provirus via the NF-κB signaling pathway.

The reactivation mechanism of latent human immunodeficiency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC). DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 and other pathogens to activate the extracellular regulated protein kinase (ERK) and nuclear factor-kappa B (NF-κB) pathways and regulate cytokine expression. We hypothesized that DC-SIGN-induced signaling pathways may activate HIV-1 provirus. To investigate this hypothesis, we generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and an HIV-1 5' long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 or VSV-G-pNL4.3 pseudotype virus (without gp120 protein). It was found that the HIV-1 5'LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells. Then the HIV-1 chronically infected CEM-Bru cells were transfected with DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein. It was found that early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation. We then investigated the involvement of the ERK, p38 mitogen-activated protein kinases and NF-κB signaling pathways in HIV-1 gp120/DC-SIGN-induced activation of HIV-1 provirus by inhibiting the pathways specifically. Our results indicated that HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.

Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals.

BackgroundHuman immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5’ LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5’ LTR.ResultsIn the Jurkat CD4+ T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5’ LTR sequences. Consecutive stimulations of model CD4+ T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5’ LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5’ LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5’ LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5’ LTR DNA methylation in the resting CD4+ T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5’ LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5’ LTR CpG methylation.ConclusionsOur data showed the presence of 5’ LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0185-6) contains supplementary material, which is available to authorized users.

Short-term Treatment With Interferon Alfa Diminishes Expression of HIV-1 and Reduces CD4+ T-Cell Activation in Patients Coinfected With HIV and Hepatitis C Virus and Receiving Antiretroviral Therapy.

Long-term treatment with interferon (IFN) alfa plus ribavirin decreases the proviral human immunodeficiency virus type 1 (HIV) DNA level. However, the short-term impact of IFN alfa on persistent HIV and its effects on immune activation after antiretroviral therapy remain unknown. Our study showed that the cell-associated HIV RNA level and CD4(+) T-cell activation decreased in the IFN group (n = 10). No changes were detected in levels of residual plasma viremia, replication-competent reservoirs, proviral DNA, or 2-long-terminal repeat circles, although APOBEC3G, TRIM5α, BST2, and TRIM22 were upregulated in the IFN group. These data suggest that short-term treatment with IFN alfa combined with RBV decreases HIV expression, in part through inhibition of HIV transcription by TRIM22 and decrease in T-cell activation.

Procyanidin trimer C1 derived from Theobroma cacao reactivates latent human immunodeficiency virus type 1 provirus

Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replication-competent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation largely depends on the NF-κB and MAPK signaling pathways because either overexpression of a super-repressor form of IκBα or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1 reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation, NF-κB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a potential compound for reactivation of latent HIV-1 reservoirs.

Stochastic Control of Intracellular Latency and Transactivation in Human Immunodeficiency Virus Type 1 (HIV-1)

Efforts for curing HIV infection have been largely unsuccessful. A major obstacle is that the virus forms latent reservoirs in resting CD4+ T cells, which cannot be eradicated by current antiretroviral therapies. However, latently infected cells can be activated at later times to enter the transactivation state of viral production. The selection between latency and transactivation are likely stochastically controlled. However, detailed control mechanisms are unknown. To address this issue, we have developed a stochastic model for the intracellular genetic circuit controlling HIV latency and transactivation, and computed directly the steady state and time evolving probability landscape using the multi-finite buffer discrete Chemical Master Equation (mb-dCME) method. The computed stochastic steady state landscape of the HIV Tat circuit exhibits bistable behavior, with a highly stable latent state with zero Tat protein and a much wider but less stable transactivation state of nonzero Tat protein. Our calculation results agree with experimental observations of latency and transactivation dynamics. To further examine how the rate of intracellular HIV-1 transactivation is affected by the positive feedback of Tat circuit, we construct a mono-stable circuit by removing this feedback. We show that the transactivation rate in the mono-stable circuit is significantly lower than the wild-type bistable circuit. To study how the HIV-1 provirus can be transactivated more effectively, we systematically explored different biochemical properties of Tat binding, reaction rates, and transportation rates between nucleus and cytoplasm. Our results suggest novel approaches for eradicating HIV-1. Our results show that the simple positive feedback loop of Tat self-activation is sufficient to generate bistability, without the need for cooperativity and negative feedbacks. Our approach is general and can be applied to study broad issues in genetic circuits modeling in systems biology.

Graft Versus HIV-1 Reservoir Effect after Allogeneic Stem Cell Transplantation

Background: The use of a stem cell source homozygous for the CCR5 delta32 mutation is of particular interest in HIV+ patients in need of an allogeneic hematopoietic stem cell transplantation (SCT) given the high natural resistance against HIV-1 acquisition. In the absence of such cell source, the impact of allogeneic SCT on human immunodeficiency virus type 1 (HIV-1) reservoirs in patients receiving combination antiretroviral therapy (cART) is unknown.Methods: Size of HIV-1 reservoirs was investigated in 3 patients with hematological malignancies receiving cART, who underwent allogeneic SCT (Table 1). Patient 1 received a myeloablative single cord blood (wild type (WT) CCR5), supported with third party HLA-mismatched CD34+ cells (haplo-cord SCT). Patients 2 and 3 underwent reduced intensity conditioning SCT of an HLA-matched sibling (heterozygous CCR5 delta32 donor and WT CCR5 donor, respectively). In-depth analysis of the HIV-1 reservoir was performed in the post-transplant period using droplet digital PCR and hemi-nested qPCR (proviral DNA copies/million PBMC). Chimerism analysis was performed by short tandem repeat PCR (STR-PCR).Results: Proviral DNA showed a decreasing dynamics after allogeneic SCT and became undetectable in all 3 patients at 9, 18 and 1 months, respectively (Table 1). In patient 1, negativity of proviral HIV-1 DNA coincided with achievement of complete chimerism while being free of immunosuppression. Patient 2 showed undetectable proviral DNA together with the development of skin chronic GvHD. Finally, undetectable proviral DNA and complete chimerism was achieved in patient 3 early after transplantation. All patients remained in complete hematological remission.Conclusion: All our patients showed a small HIV-1 reservoir after allogeneic SCT. The coincidence of achievement of negative proviral HIV-1 DNA and complete chimerism and/or development of GvHD suggests a role of Ògraft versus HIV-1 reservoir effectÓ in the control of HIV infection. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genital Herpes Simplex Virus Type 2 Infection in Humanized HIV-Transgenic Mice Triggers HIV Shedding and Is Associated With Greater Neurological Disease

Epidemiological studies consistently demonstrate synergy between herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus type 1 (HIV-1). Higher HIV-1 loads are observed in coinfected individuals, and conversely, HIV-1 is associated with more-severe herpetic disease. A small animal model of coinfection would facilitate identification of the biological mechanisms underlying this synergy and provide the opportunity to evaluate interventions. Mice transgenic for HIV-1 provirus and human cyclin T1 under the control of a CD4 promoter (JR-CSF/hu-cycT1) were intravaginally infected with HSV-2 and evaluated for disease progression, HIV shedding, and mucosal immune responses. HSV-2 infection resulted in higher vaginal HIV loads and genital tissue expression of HIV RNA, compared with HSV-uninfected JR-CSF/hu-cycT1 mice. There was an increase in genital tract inflammatory cells, cytokines, chemokines, and interferons in response to HSV-2, although the kinetics of the response were delayed in HIV-transgenic, compared with control mice. Moreover, the JR-CSF/hu-cycT1 mice exhibited earlier and more-severe neurological disease. The latter was associated with downregulation of secretory leukocyte protease inhibitor expression in neuronal tissue, a molecule with antiinflammatory, antiviral, and neuroprotective properties. JR-CSF/hu-cycT1 mice provide a valuable model to study HIV/HSV-2 coinfection and identify potential mechanisms by which HSV-2 facilitates HIV-1 transmission and HIV modulates HSV-2-mediated disease.

Histone Deactylase Inhibitor SAHA Induces a Synergistic HIV-1 Reactivation by 12-O-Tetradecanoylphorbol-13-Acetate in Latently Infected Cells

Objectives: Recent studies have reported that human immunodeficiency virus type 1 (HIV-1) proviruses are strongly suppressed in the unique epigenetic environments caused by chromatin modifications such as acetylation and methylation. Therefore, optimized therapeutic strategies directed against the virus reservoir using these epigenetic modifying agents (EMAs) should cure HIV infection. Methods: Cytotoxicity and HIV-1 reactivation were determined using the PrestoBlue™ Cell Viability Reagent and p24 HIV ELISA, respectively. Results: EMAs, including histone deacetylase inhibitors (VPA and SAHA), DNA methyltransferase inhibitor (5′-Aza-CdR), histone methyltransferase inhibitor (ADOX) and 12-O-tetradecanoylphorbol-13-acetate (TPA), were used to reactivate proviruses in HIV-1 latently infected cells. The effect of monotreatment with these EMAs on HIV-1 reactivation was VPA or SAHA > 5′-Aza-CdR > ADOX. Even though cotreatment with these potential HIV-1 reactivating agents did not show any significant reactivation effects in HIV-1 latently infected cells, employing SAHA under TPA treatment demonstrated a dramatic synergistic effect on purging HIV-1 proviruses in all HIV-1 latently infected cells via the ERK and AP-1 pathways. Conclusions: These results suggest that the combined approaches of EMAs, cotreatment of SAHA and TPA, could provide an effective way to lead a decline of HIV-1 reservoirs in patients.

APOBEC3G expression and hypermutation are inversely associated with human immunodeficiency virus type 1 (HIV-1) burden in vivo

APOBEC3G (A3G) and APOBEC3F (A3F) reduce Vif-negative HIV-1 provirus formation and cause disabling provirus G-to-A hypermutation in vitro. However, evidence conflicts about whether they negatively impact Vif-positive HIV-1, or only enhance virus genetic diversity, in vivo. We studied peripheral blood mononuclear cells (PBMC) from 19 antiretroviral-naïve, HIV-infected adults: 12 long-term non-progressors (LTNP) and 7 non-controllers (NC). Cells from LTNP had higher A3G and A3F mRNA levels, lower provirus burden, and more A3G-hypermutated positions in provirus sequence than cells from NC. A3G mRNA level was directly associated with its Hypermutation Index (HI) and inversely associated with provirus burden. Plasma HIV-1 RNA levels were inversely associated with A3G expression levels and with HI only among subjects who had HI>1. A3G HI was not associated with provirus burden. These results indicate that A3G deaminase-dependent activity above a threshold level, and its deaminase-independent functions, contribute to decreasing Vif-positive virus replication in vivo.

Changes in the topology of gene expression networks by human immunodeficiency virus type 1 (HIV-1) integration in macrophages

One key step of human immunodeficiency virus type 1 (HIV-1) infection is the integration of its viral cDNA. This process is mediated through complex networks of host–virus interactions that alter several normal cell functions of the host. To study the complexity of disturbances in cell gene expression networks by HIV-1 integration, we constructed a network of human macrophage genes located close to chromatin regions rich in proviruses. To perform the network analysis, we selected 28 genes previously identified as the target of cDNA integration and their transcriptional profiles were obtained from GEO Profiles (NCBI).A total of 2770 interactions among the 28 genes located around the HIV-1 proviruses in human macrophages formed a highly dense main network connected to five sub-networks. The overall network was significantly enriched by genes associated with signal transduction, cellular communication and regulatory processes. To simulate the effects of HIV-1 integration in infected macrophages, five genes with the most number of interaction in the normal network were turned off by putting in zero the correspondent expression values. The HIV-1 infected network showed changes in its topology and alteration in the macrophage functions reflected in a re-programming of biosynthetic and general metabolic process.Understanding the complex virus-host interactions that occur during HIV-1 integration, may provided valuable genomic information to develop new antiviral treatments focusing on the management of some specific gene expression networks associated with viral integration. This is the first gene network which describes the human macrophages genes interactions related with HIV-1 integration.

Varying Modulation of HIV-1 LTR Activity by BAF Complexes

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat is present on both ends of the integrated viral genome and contains regulatory elements needed for transcriptional initiation and elongation. Post-integration, a highly ordered chromatin structure consisting of at least five nucleosomes, is found at the 5′ long terminal repeat, the location and modification state of which control the state of active viral replication as well as silencing of the latent HIV-1 provirus. In this context, the chromatin remodeling field rapidly emerges as having a critical role in the control of viral gene expression. In the current study, we focused on unique Baf subunits that are common to the most highly recognized of chromatin remodeling proteins, the SWI/SNF (switching-defective–sucrose non-fermenting) complexes. We find that at least two Baf proteins, Baf53 and Baf170, are highly regulated in HIV-1-infected cells. Previously, studies have shown that the depletion of Baf53 in uninfected cells leads to the expansion of chromosomal territories and the decompaction of the chromatin. Baf53, in the presence of HIV-1 infection, co-elutes off of a chromatographic column as a different-sized complex when compared to uninfected cells and appears to be predominantly phosphorylated. The innate function of Baf53-containing complexes appears to be transcriptionally suppressive, in that knocking down Baf53 increases viral gene expression from cells both transiently and chronically infected with HIV-1. Additionally, cdk9/cyclin T in the presence of Tat is able to phosphorylate Baf53 in vitro, implying that this posttranslationally modified form relieves the suppressive effect and allows for viral transcription to proceed.

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

Multiploid Inheritance of HIV-1 during Cell-to-Cell Infection

During cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), many viral particles can be simultaneously transferred from infected to uninfected CD4 T cells through structures called virological synapses (VS). Here we directly examine how cell-free and cell-to-cell infections differ from infections initiated with cell-free virus in the number of genetic copies that are transmitted from one generation to the next, i.e., the genetic inheritance. Following exposure to HIV-1-expressing cells, we show that target cells with high viral uptake are much more likely to become infected. Using T cells that coexpress distinct fluorescent HIV-1 variants, we show that multiple copies of HIV-1 can be cotransmitted across a single VS. In contrast to cell-free HIV-1 infection, which titrates with Poisson statistics, the titration of cell-associated HIV-1 to low rates of overall infection generates a constant fraction of the newly infected cells that are cofluorescent. Triple infection was also readily detected when cells expressing three fluorescent viruses were used as donor cells. A computational model and a statistical model are presented to estimate the degree to which cofluorescence underestimates coinfection frequency. Lastly, direct detection of HIV-1 proviruses using fluorescence in situ hybridization confirmed that significantly more HIV-1 DNA copies are found in primary T cells infected with cell-associated virus than in those infected with cell-free virus. Together, the data suggest that multiploid inheritance is common during cell-to-cell HIV-1 infection. From this study, we suggest that cell-to-cell infection may explain the high copy numbers of proviruses found in infected cells in vivo and may provide a mechanism through which HIV preserves sequence heterogeneity in viral quasispecies through genetic complementation.

Human Immunodeficiency Virus Type 1 Modified To Package Simian Immunodeficiency Virus Vpx Efficiently Infects Macrophages and Dendritic Cells

The lentiviral accessory protein Vpx is thought to facilitate the infection of macrophages and dendritic cells by counteracting an unidentified host restriction factor. Although human immunodeficiency virus type 1 (HIV-1) does not encode Vpx, the accessory protein can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) in virus-like particles, dramatically enhancing their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. We localized the minimal Vpx packaging motif of simian immunodeficiency virus SIVmac(239) p6 to a 10-amino-acid motif and introduced this sequence into an infectious HIV-1 provirus. The chimeric virus packaged Vpx that was provided in trans and was substantially more infectious on MDDC and MDM than the wild-type virus. We further modified the virus by introducing the Vpx coding sequence in place of nef. The resulting virus produced Vpx and replicated efficiently in MDDC and MDM. The virus also induced a potent type I interferon response in MDDC. In a coculture system, the Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo, Vpx may facilitate transmission of the virus from dendritic cells to T cells. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells.

Remarkable Lethal G-to-A Mutations in vif- Proficient HIV-1 Provirus by Individual APOBEC3 Proteins in Humanized Mice

Genomic hypermutation of RNA viruses, including human immunodeficiency virus type 1 (HIV-1), can be provoked by intrinsic and extrinsic pressures, which lead to the inhibition of viral replication and/or the progression of viral diversity. Human APOBEC3G was identified as an HIV-1 restriction factor, which edits nascent HIV-1 DNA by inducing G-to-A hypermutations and debilitates the infectivity of vif-deficient HIV-1. On the other hand, HIV-1 Vif protein has the robust potential to degrade APOBEC3G protein. Although subsequent investigations have revealed that lines of APOBEC3 family proteins have the capacity to mutate HIV-1 DNA, it remains unclear whether these endogenous APOBEC3s, including APOBEC3G, contribute to mutations of vif-proficient HIV-1 provirus in vivo and, if so, what is the significance of these mutations. In this study, we use a human hematopoietic stem cell-transplanted humanized mouse (NOG-hCD34 mouse) model and demonstrate the predominant accumulation of G-to-A mutations in vif-proficient HIV-1 provirus displaying characteristics of APOBEC3-mediated mutagenesis. Notably, the APOBEC3-associated G-to-A mutation of HIV-1 DNA that leads to the termination of translation was significantly observed. We further provide a novel insight suggesting that HIV-1 G-to-A hypermutation is independently induced by individual APOBEC3 proteins. In contrast to the prominent mutation in intracellular proviral DNA, viral RNA in plasma possessed fewer G-to-A mutations. Taken together, these results provide the evidence indicating that endogenous APOBEC3s are associated with G-to-A mutation of HIV-1 provirus in vivo, which can result in the abrogation of HIV-1 infection.

HIV-1 DNA proviral load in treated and untreated HIV-1 sero-positive patients

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon activation, its detection might offer significant therapeutic information, complementing the input provided by plasma RNA determination in the follow-up of infected individuals. A selected group of acutely infected subjects was studied to verify both total and 2-long terminal repeat (2-LTR) DNA proviral load during the acute phase of infection and thereafter. Patients were divided in two sex- and age-matched groups: 19 naive individuals who did not receive antiretroviral therapy during the observation period and 20 subjects treated according to current guidelines. Total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load and CD4 cell count, were determined in peripheral blood mononuclear cells (PBMC) at baseline, 6 and 12 months after the first sampling. Total and 2-LTR HIV-1 DNA proviral load exhibited no significant variation at any time in the naive patients (total HIV-1 DNA ranging from 896 ± 731 to 715 ± 673 copies/105 PBMC and 2-LTR HIV-1 DNA ranging from 94 ± 105 to 65 ± 44 copies/105 PBMC), whereas a significant reduction in both total HIV-1 DNA (ranging from 997 ± 676 to 262 ± 174 copies/105 PBMC) and 2-LTR HIV-1 DNA proviral load (ranging from 116 ± 55 to 26 ± 35 copies/105 PBMC) was detected in highly active antiretroviral therapy (HAART) patients, together with a CD4+ T cell count increase and RNA load decrease. HAART negatively affects both the labile HIV burden and the integrated proviral DNA, at least in the initial period of successful treatment, suggesting that quantification of HIV-1 DNA proviral load may be an important parameter in monitoring HIV infection.

APOBEC3F and APOBEC3G inhibit HIV-1 DNA integration by different mechanisms.

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.