CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue

A rapid decline in T-cell counts and the progression to AIDS is often associated with a switch from CCR5-tropic (R5) HIV-1 to CXCR4-tropic (X4) HIV-1 or R5/X4 HIV-1 variants. Experimental infection with R5 HIV-1 causes less T-cell depletion than infection with X4 or R5/X4 variants in T-cell cultures, in ex vivo infected human lymphoid tissue and in SCID/hu mice, despite similar replication levels. Experimental genetic changes in those sequences in gp120 that transform R5 HIV-1 variants into otherwise isogenic X4 viruses make them highly cytopathic. Thus, it is now believed that R5 variants are less cytopathic for T cells than are X4 variants. However, it is not known why CCR5-mediated HIV-1 infection does not lead to a massive CD4+ T-cell depletion, as occurs in CXCR4-mediated HIV-1 infection. Here we demonstrate that R5 HIV-1 isolates are indeed highly cytopathic, but only for CCR5+/CD4+ T cells. Because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T-cell count. These results may explain why the clinical stage of HIV disease correlates with viral tropism.     

Variation in the biological properties of HIV-1 R5 envelopes: implications of envelope structure, transmission and pathogenesis

HIV-1 R5 viruses predominantly use CCR5 as a coreceptor to infect CD4(+) T cells and macrophages. While R5 viruses generally infect CD4(+) T cells, research over the past few years has demonstrated that they vary extensively in their capacity to infect macrophages. Thus, R5 variants that are highly macrophage tropic have been detected in late disease and are prominent in brain tissue of subjects with neurological complications. Other R5 variants that are less sensitive to CCR5 antagonists and use CCR5 differently have also been identified in late disease. These latter variants have faster replication kinetics and may contribute to CD4 T-cell depletion. In addition, R5 viruses are highly variable in many other properties, including sensitivity to neutralizing antibodies and inhibitors that block HIV-1 entry into cells. Here, we review what is currently known about how HIV-1 R5 viruses vary in cell tropism and other properties, and discuss the implications of this variation on transmission, pathogenesis, therapy and vaccines.     

Biological characterization of human immunodeficiency virus type 1 clones derived from different organs of an AIDS patient by long-range PCR.

In order to characterize the biological properties of human immunodeficiency virus type 1 (HIV-1) variants from different tissues (peripheral blood mononuclear cells [PBMC], lymph node, spleen, brain, and lung) of one patient, we have chosen long-range PCR to amplify virtually full-length HIV proviruses and to construct replication-competent viruses by adding a patient-specific 5' long terminal repeat. To avoid selection during propagation in CD4+ target cells, we transfected 293 cells and used the supernatants from these cells as challenge viruses for tropism studies after titration on human PBMC. Despite differences in the V3 loop of the major variants found in brain and lung compared to lymphoid tissues all recombinant HIV clones obtained showed identical cell tropism and replicative kinetics. After infection of human PBMC these viruses replicated with similar kinetics, with a slow/low-titer, non-syncytium-inducing phenotype. In contrast to the prediction of macrophage tropism, drawn from the V3 loop sequence, none of these viruses infected monocyte-derived macrophages. The challenge of blood dendritic cells by these recombinant viruses in the presence of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-4 resulted in a productive infection only after adding stimulated CD4+ T lymphocytes. Therefore, the biological properties of the HIV-1 variants derived from nonlymphoid tissue of this patient did not differ from those of HIV-1 variants from lymphoid tissue with respect to tropism for primary cells such as PBMC, macrophages, and blood dendritic cells.     

Differential pathogenesis of primary CCR5-using human immunodeficiency virus type 1 isolates in ex vivo human lymphoid tissue

In the course of human immunodeficiency virus (HIV) disease, CCR5-utilizing HIV type 1 (HIV-1) variants (R5), which typically transmit infection and dominate its early stages, persist in approximately half of the infected individuals (nonswitch virus patients), while in the other half (switch virus patients), viruses using CXCR4 (X4 or R5X4) emerge, leading to rapid disease progression. Here, we used a system of ex vivo tonsillar tissue to compare the pathogeneses of sequential primary R5 HIV-1 isolates from patients in these two categories. The absolute replicative capacities of HIV-1 isolates seemed to be controlled by tissue factors. In contrast, the replication level hierarchy among sequential isolates and the levels of CCR5(+) CD4(+) T-cell depletion caused by the R5 isolates seemed to be controlled by viral factors. R5 viruses isolated from nonswitch virus patients depleted more target cells than R5 viruses isolated from switch virus patients. The high depletion of CCR5(+) cells by HIV-1 isolates from nonswitch virus patients may explain the steady decline of CD4(+) T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value reflecting whether, in an infected individual, X4 HIV-1 will eventually dominate.     

Vaginal Myeloid Dendritic Cells Transmit Founder HIV-1

We report that primary human vaginal dendritic cells (DCs) display a myeloid phenotype and express CD4, CCR5, and CXCR4. Vaginal CD13⁺ CD11c⁺ DCs rapidly and efficiently bound transmitted/founder (T/F) CCR5-tropic (R5) viruses, transported them through explanted vaginal mucosa, and transmitted them in trans to vaginal and blood lymphocytes. Vaginal myeloid DCs may play a key role in capturing and disseminating T/F R5 HIV-1 in vivo and are candidate “gatekeeper” cells in HIV-1 transmission.     

Human immunodeficiency virus type 1 induces apoptosis in CD4(+) but not in CD8(+) T cells in ex vivo-infected human lymphoid tissue

Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4(+) T cells along with death and/or dysfunction of CD8(+) T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4(+) and CD8(+) T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4(+) T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8(+) T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4(+) T cells, neither viral isolate induced apoptosis in CD8(+) T cells. Moreover, in both infected and control tissues we found similar numbers of CD8(+) T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3-anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4(+) T cells, the death of CD8(+) T cells apparently requires additional factors.     

Evaluation of cervical mucosa in transmission bottleneck during acute HIV-1 infection using a cervical tissue-based organ culture

Background

Although there are different strains of HIV-1 in a chronically infected individual, only one or limited virus strains are successfully transmitted to a new individual. The reason for this “transmission bottleneck” is as yet unknown.

Methodology/Principal Findings

A human cervical explant model was used to measure HIV-1 transmission efficiency of viral strains from chronic infections, and transmitter/founder variants. We also evaluated the genetic characteristics of HIV-1 variants in the inoculums compared to those transmitted across the cervical mucosa. Eight different HIV-1 isolates were used in this study, six chronic isolates and two transmitter/founder viruses. The transmission efficiency of the chronic and transmitter/founder virus isolates and the viral diversity of chronic isolates before and after viral transmission were assessed. The results indicate that transmitter/founder viruses did not display higher transmission efficiency than chronic HIV-1 isolates. Furthermore, no evidence for a difference in diversity was found between the inoculums and transmitted virus strains. Phylogenetic analysis indicated that the sequences of variants in the inoculums and those present in transmitted virus intermingled irrespective of co-receptor usage. In addition, the inoculum and transmitted variants had a similar pairwise distance distribution.

Conclusion

There was no selection of a single or limited number of viral variants during HIV-1 transmission across the cervical mucosa in the organ culture model, indicating that the cervical mucosa alone may not produce the transmission bottleneck of HIV-1 infection observed in vivo.     

Human immunodeficiency virus type 1 (HIV-1) non-B subtypes are similar to HIV-1 subtype B in that coreceptor specificity is a determinant of cytopathicity in human lymphoid tissue infected ex vivo

We sought to determine the relationship between virus-mediated CD4(+) T-lymphocyte cytopathicity and viral coreceptor preference among various human immunodeficiency virus type 1 (HIV-1) subtypes in an ex vivo-infected human lymphoid tissue model. Our data show that all R5 HIV-1 infections resulted in mild depletion of CD4(+) T lymphocytes, whereas all X4 HIV-1 infections caused severe depletion of CD4(+) T lymphocytes regardless of their subtype origin. Thus, at least for the viruses within subtypes A, B, C, and E that were tested, coreceptor specificity is a critical factor that determines the ability of HIV-1 to deplete CD4(+) T cells in human lymphoid tissue infected ex vivo.     

Transmitted/Founder and Chronic Subtype C HIV-1 Use CD4 and CCR5 Receptors with Equal Efficiency and Are Not Inhibited by Blocking the Integrin α4β7

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.     

Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo

Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.     

Rectal Transmission of Transmitted/Founder HIV-1 Is Efficiently Prevented by Topical 1% Tenofovir in BLT Humanized Mice

Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1_JRCSF or the MSM-derived transmitted/founder (T/F) virus HIV-1_THRO within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1_JRCSF and HIV-1_THRO, respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1_JRCSF and 0% (0/6; log rank p = 0.02) for HIV-1_THRO. This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides.     

Phenotypic and genotypic comparisons of CCR5- and CXCR4-tropic human immunodeficiency virus type 1 biological clones isolated from subtype C-infected individuals

Individuals infected with human immunodeficiency virus type 1 (HIV-1) subtype C infrequently harbour X4 viruses. We studied R5 and X4 biological clones generated from HIV-1 subtype C-infected individuals. All subtype C R5 viruses demonstrated slower profiles of replication on CD4⁺ lymphocytes in comparison to subtype B viruses, whereas subtype C X4 viruses replicated with comparable efficiency to subtype B X4 viruses. No differences were identified in CC or CXC chemokine inhibitions (RANTES and SDF-1α, respectively) between subtype C and subtype B viruses. Immature dendritic cells were shown in coculture experiments to similarly enhance the infection of subtype C and subtype B R5 as well as X4 viruses. By amino acid sequence analysis, we showed that the R5 and X4 subtype C gp120 envelope gene alterations were similar to those for a switching subtype B virus, specifically with respect to the V3 charge and envelope N-linked glycosylation patterns. By phylogenetic analysis, we showed that one patient was infected with HIV-1 C′ and the other was infected with HIV-1 C" and that one of the patients harbored a virus that was a recombinant in the gp120 env gene between an R5 and an X4 virus, with the resultant virus being R5. No differences were identified between the long terminal repeat regions of the subtype C R5 and X4 biological clones. These results indicate that even though R5 subtype C viruses are restrictive for virus replication, the R5-to-X4 phenotype switch can occur and does so in a manner similar to that of subtype B viruses.     

Association of Nef with p21-activated kinase 2 is dispensable for efficient human immunodeficiency virus type 1 replication and cytopathicity in ex vivo-infected human lymphoid tissue

Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4⁺ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis.     

Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif

HIV-1 Vif assembles the Cul5-EloB/C E3 ubiquitin ligase to induce proteasomal degradation of the cellular antiviral APOBEC3 proteins. Detailed structural studies have confirmed critical functional domains in Vif that we have previously identified as important for the interaction of EloB/C, Cul5, and CBFβ. However, the mechanism by which Vif recognizes substrates remains poorly understood. Specific regions of Vif have been identified as being responsible for binding and depleting APOBEC3G and APOBEC3F. Interestingly, we have now identified distinct yet overlapping domains that are required for HIV-1 Vif-mediated G2/M-phase cell cycle arrest and APOBEC3H degradation, but not for the inactivation of APOBEC3G or APOBEC3F. Surprisingly, Vif molecules from primary HIV-1 variants that caused G2/M arrest were unable to inactivate APOBEC3H; on the other hand, HIV-1 Vif variants that could inactivate APOBEC3H were unable to induce G2/M arrest. All of these Vif variants still maintained the ability to inactivate APOBEC3G/F. Thus, primary HIV-1 variants have evolved to possess distinct functional activities that allow them to suppress APOBEC3H or cause G2 cell cycle arrest, using mutually exclusive interface domains. APOBEC3H depletion and G2 arrest are apparently evolutionary selected features that cannot co-exist on a single Vif molecule. The existence and persistence of both types of HIV-1 Vif variant suggests the importance of APOBEC3H suppression and cell cycle regulation for HIV-1''s survival in vivo.     

Uterine epithelial cell regulation of DC-SIGN expression inhibits transmitted/founder HIV-1 trans infection by immature dendritic cells

Background

Sexual transmission accounts for the majority of HIV-1 infections. In over 75% of cases, infection is initiated by a single variant (transmitted/founder virus). However, the determinants of virus selection during transmission are unknown. Host cell-cell interactions in the mucosa may be critical in regulating susceptibility to infection. We hypothesized in this study that specific immune modulators secreted by uterine epithelial cells modulate susceptibility of dendritic cells (DC) to infection with HIV-1.

Methodology/Principal Findings

Here we report that uterine epithelial cell secretions (i.e. conditioned medium, CM) decreased DC-SIGN expression on immature dendritic cells via a transforming growth factor beta (TGF-β) mechanism. Further, CM inhibited dendritic cell-mediated trans infection of HIV-1 expressing envelope proteins of prototypic reference. Similarly, CM inhibited trans infection of HIV-1 constructs expressing envelopes of transmitted/founder viruses, variants that are selected during sexual transmission. In contrast, whereas recombinant TGF- β1 inhibited trans infection of prototypic reference HIV-1 by dendritic cells, TGF-β1 had a minimal effect on trans infection of transmitted/founder variants irrespective of the reporter system used to measure trans infection.

Conclusions/Significance

Our results provide the first direct evidence for uterine epithelial cell regulation of dendritic cell transmission of infection with reference and transmitted/founder HIV-1 variants. These findings have immediate implications for designing strategies to prevent sexual transmission of HIV-1.     

An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.     

Inhibition of HIV-1 infection in ex vivo cervical tissue model of human vagina by palmitic acid; implications for a microbicide development

Background

Approximately 80% of all new HIV-1 infections are acquired through sexual contact. Currently, there is no clinically approved microbicide, indicating a clear and urgent therapeutic need. We recently reported that palmitic acid (PA) is a novel and specific inhibitor of HIV-1 fusion and entry. Mechanistically, PA inhibits HIV-1 infection by binding to a novel pocket on the CD4 receptor and blocks efficient gp120-to-CD4 attachment. Here, we wanted to assess the ability of PA to inhibit HIV-1 infection in cervical tissue ex vivo model of human vagina, and determine its effect on Lactobacillus (L) species of probiotic vaginal flora.

Principal Findings

Our results show that treatment with 100–200 µM PA inhibited HIV-1 infection in cervical tissue by up to 50%, and this treatment was not toxic to the tissue or to L. crispatus and jensenii species of vaginal flora. In vitro, in a cell free system that is independent of in vivo cell associated CD4 receptor; we determined inhibition constant (Ki) to be ∼2.53 µM.

Significance

These results demonstrate utility of PA as a model molecule for further preclinical development of a safe and potent HIV-1 entry microbicide inhibitor.     

Interactions between human immunodeficiency virus type 1 and vaccinia virus in human lymphoid tissue ex vivo

Vaccinia virus (VACV) has been attracting attention recently not only as a vector for various vaccines but also as an immunization tool against smallpox because of its potential use as a bioterrorism agent. It has become evident that in spite of a long history of studies of VACV, its tissue pathogenesis remains to be fully understood. Here, we investigated the pathogenesis of VACV and its interactions with human immunodeficiency virus type 1 (HIV-1) in the context of human lymphoid tissues. We found that ex vivo-cultured tonsillar tissue supports productive infection by the New York City Board of Health strain, the VACV strain of the Dryvax vaccine. VACV readily infected both T and non-T (B) lymphocytes and depleted cells of both of these subsets equally over a 12-day period postinfection. Among T lymphocytes, CD8(+) cells are preferentially depleted in accordance with their preferential infection: the probability that a CD8(+) T cell will be productively infected is almost six times higher than for a CD4(+) T cell. T cells expressing CCR5 and the activation markers CD25, CD38, and HLA-DR are other major targets for infection by VACV in lymphoid tissue. As a consequence, VACV predominantly inhibits the replication of the R5(SF162) phenotype of HIV-1 in coinfected tissues, as R5-tropic HIV-1 requires activated CCR5(+) CD4(+) cells for productive infection. Human lymphoid tissue infected ex vivo by VACV can be used to investigate interactions of VACV with other viruses, in particular HIV-1, and to evaluate various VACV vectors for the purpose of recombinant vaccine development.     

HIV-1初始传播病毒Vpr基因遗传变异对诱导G2期阻滞及细胞凋亡的影响

人免疫缺陷病毒(HIV-1)急性感染过程中,病毒的遗传多样性显著减少,往往只有一株或几株病毒可以建立有效感染,这种病毒被称为初始传播病毒(Transmitted/Founder virus)。病毒蛋白R(Vpr)是HIV-1的辅助蛋白之一,在病毒复制过程中起重要作用。研究初始传播病毒Vpr基因遗传变异与生物学特征对于阐明病毒建立感染的关键环节具有重要意义。文章利用流式细胞术分析了C亚型HIV-1初始传播病毒株与慢性感染株MJ4的 Vpr蛋白诱导细胞G₂期阻滞和细胞凋亡的能力。结果显示,初始传播病毒ZM246和ZM247的Vpr诱导细胞G₂期阻滞和细胞凋亡的能力显著高于慢性感染株MJ4 Vpr。氨基酸序列分析表明,初始传播病毒Vpr在第77、85和94位上存在高频突变。研究结果提示初始传播病毒可能在病毒感染早期,通过Vpr基因的遗传突变,提升病毒诱导细胞停滞G₂期和细胞凋亡的能力,进而促进病毒在宿主体内的复制和传播。