HIV-1 replication and pathogenesis in the human thymus

How HIV replicates and causes destruction of the thymus, and how to restore thymic function, are among the most important questions of HIV-1 pathogenesis and therapy in adult as well as pediatric patients. The thymus appears to function, albeit at reduced levels, throughout the life of adults, to respond to T cell depletion induced by HIV and to be suppressed by HIV. In this review, we summarize recent findings concerning HIV replication and pathogenesis in the human thymus, focusing on mechanistic insights gleaned from studies in the SCID-hu Thy/Liv mouse and human fetal-thymus organ culture (HF-TOC) models. First, we discuss HIV viral determinants and host factors involved in the replication of HIV in the thymus. Second, we consider evidence that both viral factors and host factors contribute to HIV-induced thymocyte depletion. We thus propose that multiple mechanisms, including depletion and suppression of progenitor cells, paracrine and direct lytic depletion of thymocytes, and altered thymocyte selection are involved in HIV-induced pathology in the thymus. With the SCID-hu Thy/Liv mouse and HF-TOC models, it will be important in the coming years to further clarify the virological, cell biological, and immunological mechanisms of HIV replication and pathogenesis in human thymus, and to correlate their significance in HIV disease progression.     

Concordance between HIV-1 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA

Objective

The aim of the study was to evaluate the use of proviral DNA as a source of viral genetic material for genotypic coreceptor tropism testing (GTT).

Methods

GTT consisted of bulk V3 sequencing followed by geno2pheno interpretation with the interpretative cut‐off [false positive rate (FPR)] set at 5 and 10%. GTT was performed for 165 patients with a viral load of >500 HIV‐1 RNA copies/mL on simultaneously collected plasma RNA and proviral DNA, and for 126 patients with a viral load of <500 copies/mL on current proviral DNA and pretreatment plasma RNA. Phenotypic tropism testing (PTT) results were available for 142 samples.

Results

In the simultaneous RNA/DNA comparison, concordance in prediction was 95.2% (at FPR 10%) and 96.4% (at FPR 5%). Six RNA‐R5/DNA‐X4 and two RNA‐X4/DNA‐R5 discordances were observed at an FPR of 10%, and six RNA‐R5/DNA‐X4 discordances were observed at an FPR of 5%. In the longitudinal RNA/DNA comparison, concordance was 88.1% (at FPR 10%) and 90.5% (at FPR 5%). Eight RNA‐X4/DNA‐R5 and seven RNA‐R5/DNA‐X4 discordances were seen at an FPR of 10%, and 10 RNA‐R5/DNA‐X4 and two RNA‐X4/DNA‐R5 discordances at an FPR of 5%. The overall concordance of RNA GTT with PTT was 82% (at FPR 10%) and 83% (at FPR 5%). The overall concordance of DNA GTT with PTT was 85% (at both 10 and 5% FPRs).

Conclusions

GTT produced highly concordant tropism predictions for proviral DNA and plasma RNA. GTT on proviral DNA offers a promising approach for tropism prediction in clinical practice, particularly for the assessment of treated patients with low or suppressed viraemia.     

Stromal cell-derived factor-1 genotype, coreceptor tropism, and HIV type 1 disease progression

This study used a well characterized cohort of human immunodeficiency virus type 1 (HIV-1)-infected hemophiliacs to define the relationship between the SDF1-3'A allele, the plasma HIV-1 coreceptor tropism, and the natural history of HIV-1 disease. Subjects heterozygous or homozygous for the SDF1-3'A allele experienced higher rates of decline in CD4+ T cell counts over time than did those without the allele (P=.009). Moreover, they had an increased risk of progression to acquired immunodeficiency syndrome and death, a relationship that persisted even when baseline plasma HIV-1 RNA levels and CD4+ T cell counts or CCR5 Delta 32 and CCR2-64I genotype were controlled for. This relationship was even stronger in a subgroup of subjects for whom tropism data were available. Subjects with the SDF1-3'A allele were also more likely to have detectable X4-tropic viruses (P=.012), and, when tropism was included in the survival analyses, the effect of the SDF1-3'A allele on disease progression was no longer significant. Therefore, the increased frequency of X4-tropic viruses in subjects carrying the SDF1-3'A allele may explain the observed adverse effect that this allele has on the natural history of HIV-1 disease.     

Appraising the performance of genotyping tools in the prediction of coreceptor tropism in HIV-1 subtype C viruses

ABSTRACT: BACKGROUND: In human immunodeficiency virus type 1 (HIV-1) infection, transmitted viruses generally use the CCR5 chemokine receptor as a coreceptor for host cell entry. In more than 50% of subtype B infections, a switch in coreceptor tropism from CCR5- to CXCR4-use occurs during disease progression. Phenotypic or genotypic approaches can be used to test for the presence of CXCR4-using viral variants in an individual's viral population that would result in resistance to treatment with CCR5-antagonists. While genotyping approaches for coreceptor-tropism prediction in subtype B are well established and verified, they are less so for subtype C. METHODS: Here, using a dataset comprising V3 loop sequences from 349 CCR5-using and 56 CXCR4-using HIV-1 subtype C viruses we perform a comparative analysis of the predictive ability of 11 genotypic algorithms in their prediction of coreceptor tropism in subtype C. We calculate the sensitivity and specificity of each of the approaches as well as determining their overall accuracy. By separating the CXCR4-using viruses into CXCR4-exclusive (25 sequences) and dual-tropic (31 sequences) we evaluate the effect of the possible conflicting signal from dual-tropic viruses on the ability of a of the approaches to correctly predict coreceptor phenotype. RESULTS: We determined that geno2pheno with a false positive rate of 5% is the best approach for predicting CXCR4-usage in subtype C sequences with an accuracy of 94% (89% sensitivity and 99% specificity). Contrary to what has been reported for subtype B, the optimal approaches for prediction of CXCR4-usage in sequence from viruses that use CXCR4 exclusively, also perform best at predicting CXCR4-use in dual-tropic viral variants. CONCLUSIONS: The accuracy of genotyping approaches at correctly predicting the coreceptor usage of V3 sequences from subtype C viruses is very high. We suggest that genotyping approaches can be used to test for coreceptor tropism in HIV-1 group M subtype C with a high degree ofconfidence that they will identify CXCR4-usage in both CXCR4-exclusive and dual tropic variants.     

HIV-1 coreceptor usage, transmission, and disease progression

HIV-1 coreceptor usage is believed to play a critical role in pathogenesis. To initiate infection, HIV-1 interacts with two cell surface receptors; CD4 is the primary receptor and the beta-chemokine receptors CCR5 and CXCR4 usually serve as secondary receptors. HIV-1 strains transmitted in vivo generally use CCR5. Viruses that use CCR5 (R5 viruses) appear to be associated with relatively stable infection. Years after chronic infection is established, CXCR4 utilizing strains emerge in approximately 50% of infected individuals. Viruses that use the coreceptor CXCR4 (X4 viruses) are associated with rapid CD4+ cell decline and disease progression. However, the mechanism by which X4 viruses are associated with accelerated disease progression has never been properly elucidated. For example, the association between X4 virus and acceleration of HIV-1 disease progression has been ascribed to the expanded spectrum of CXCR4+ precursor cells susceptible to infection by X4 strains. It has also been postulated that the decline of the host immune system associated with clinical AIDS may allow X4 viruses to evolve and replicate freely in late-stage infection. Discriminating between these and other alternatives is central to increasing our understanding of the fundamental pathogenic processes involved in HIV-1 infection. In this article, we critically review those studies published over the last few years that purport to examine the relationship between HIV-1 coreceptor usage, transmission, CD4+ T-cell depletion, and disease progression.     

Temporal relationship between V1V2 variation,macrophage replication,and coreceptor adaptation during HIV-1 disease progression

BACKGROUND: Specific mutations in VPR and V2 potentially restrict HIV-1 replication in macrophages. Such restriction could potentially limit HIV replication in long-term non-progressors (LTNP), thus accounting for low viral load and delayed progression to AIDS. OBJECTIVE: To examine whether a specific VPR phenotype (truncated versus non-truncated) correlates with disease progression and whether elongated V2 restricts viral replication in macrophages or alters viral tropism. METHODS: Sequence analysis was carried for VPR and V1-V3 env from four rapid progressors (RPs), six late progressors (LPs), and three LTNPs in cohort of HIV-1-infected homosexual men. The replication kinetics of sequential isolates was examined in primary CD4 cells and macrophages and coreceptor usage was determined by GHOST infection assays. RESULTS: No differences were found in the VPR protein from RP and LTNP isolates. Analysis of the V2 region revealed that all RPs maintained similar V2 lengths (40 aa), whereas LPs and LTNPs acquired additional amino acids (2-13 aa) in the V2 region. Coreceptor specificity revealed that RP switch from CCR5 to multiple coreceptor usage, whereas LTNPs maintained R5 viruses. Sequential isolates from each group revealed comparable replication efficiencies in both T-cells and macrophages, regardless of the V2 length or coreceptor utilization. In addition, cross-section analysis of six LTNPs from Australia revealed extended V2 with consistent usage of CCR5 coreceptor. CONCLUSION: The present results suggest that acquisition of a V2 extension over time in HIV-1-infected LPs/LTNPs appears to correlate with maintenance of CCR5 usage among LTNPs. These findings may be important for a better understanding of the host interactions and disease progression.     

HIV-1 chemokine coreceptor utilization in paired cerebrospinal fluid and plasma samples: a survey of subjects with viremia

BACKGROUND: Chemokine receptors serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, influence cell tropism, and may critically determine central nervous system infection pathogenesis. Using an in vitro functional entry assay, we examined utilization of 2 principal coreceptors in cerebrospinal fluid (CSF) and plasma in 46 subjects. METHODS: Paired CSF and plasma samples were selected from subjects with a range of CD4 T cell counts. Amplified populations of env sequences were characterized as using CCR5 (R5), CXCR4 (X4), or both receptors (R5+X4). Individual clones derived from 3 subjects were analyzed for viral tropism and phylogeny. RESULTS: CSF and plasma pairs were mainly concordant for R5 (36/46) or R5+X4 (5/46) viruses. However, 5 pairs were discordant, 2 of which had the R5+X4 phenotype in CSF despite having the R5 phenotype in plasma. Although R5+X4 tropism was associated with advanced immunodeficiency, all 4 subjects with acquired immunodeficiency syndrome dementia complex had R5 tropism in CSF. Clones derived from R5+X4-tropic populations revealed mixtures of R5 and X4 viruses and viruses able to utilize either coreceptor, suggesting both virus exchange between compartments and autonomous CSF virus evolution. CONCLUSIONS: Although R5 viruses predominate in the CSF, HIV-1 populations able to utilize CXCR4 are also present. Discordant tropism in CSF and plasma may have implications for R5 inhibitor therapy.     

HIV-1 tropism and survival in vertically infected Ugandan infants

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) may utilize the CXCR4 coreceptor (X4 virus), the CCR5 coreceptor (R5 virus), or both (dual/mixed [DM] virus). We analyzed HIV-1 coreceptor tropism in Ugandan infants enrolled in the HIVNET (HIV Network for Prevention Trials) 012 trial. METHODS: Plasma or serum was analyzed using a commercial coreceptor tropism assay. HIV env subtype was determined by phylogenetic methods. RESULTS: Tropism results were obtained for 57 samples from infants collected 6-14 weeks after birth. Fifty-two infants had only R5 virus, and 5 had either X4 or DM virus. The mothers of those 5 infants also had X4 or DM virus. In infants, subtype D infection was associated with high-level infectivity in CCR5-bearing cells and also with the detection of X4 or DM strains. High-level infectivity in CCR5-bearing cells was associated with decreased infant survival, but infection with X4 or DM virus was not. HIV clones from infants with DM viral populations showed different patterns of coreceptor use. V3 loop sequence-based algorithms predicted the tropism of some, but not all, env clones. CONCLUSIONS: Complex patterns of HIV tropism were found in HIV-infected newborn infants. Subtype D infection was associated with X4 virus and with high-level replication in CCR5-bearing cells. High-level replication of R5 virus was associated with decreased infant survival.     

Antiretroviral drug resistance, HIV-1 tropism, and HIV-1 subtype among men who have sex with men with recent HIV-1 infection

OBJECTIVE: Antiretroviral drug treatment may be complicated in individuals infected with antiretroviral drug-resistant or non-subtype B HIV-1 strains. HIV-1 tropism may also affect disease progression. We analyzed antiretroviral drug resistance, HIV-1 subtype, and HIV-1 tropism among 195 men who have sex with men from six major cities in the United States, using samples collected within 6 months of HIV-1 seroconversion (1999-2003). METHODS: HIV-1 genotyping was performed using the ViroSeq HIV-1 Genotyping System. HIV-1 tropism was determined using a commercial assay. HIV-1 subtyping was performed by phylogenetic analysis of pol region sequences. RESULTS: Thirty-one (15.9%) of the men had evidence of antiretroviral drug resistance. Seven (3.6%) men had multi-class resistance, including three (1.5%) with resistance to all three antiretroviral drug classes. We found no statistically significant association of antiretroviral drug resistance with demographic factors, sexual practices, self-reported sexually transmitted infections, use of recreational drugs, or use of antiretroviral drug post-exposure prophylaxis. All samples were HIV-1 subtype B. Four men had CXCR4-using HIV-1 strains. One man with a CXCR4-using strain also had antiretroviral drug resistance. CONCLUSIONS: Antiretroviral drug resistance is relatively common among recently infected men who have sex with men in the United States. CXCR4-using strains were detected in a small number of these infections, which were all subtype B HIV-1.     

HAART治疗前后HIV-1细胞嗜性分析

目的 分析7例患者在高效抗逆转录病毒治疗(HAART)前和治疗后,艾滋病病毒Ⅰ型(HIV-1)细胞嗜性的改变及影响因素. 方法 采用BD公司的流式细胞仪,通过绝对计数法测定CD 4 T淋巴细胞计数和CD 8 T淋巴细胞计数,荧光标记物为BD TriTEST CD3FITC/CD4PE/CD45PerCP.采用核酸序列扩增实验(NASBA)测定病毒载量(生物梅里埃公司提供仪器和试剂).套式聚合酶链反应(Nested-PCR)对gp120V3环区基因进行扩增、测序分析,并确定HIV-1细胞嗜性. 结果 在7例患者中,有3例发生细胞嗜性转换,2例是从R5到X4嗜性的转换,1例是X4到R5嗜性的转换.在7例患者中,病毒细胞嗜性转换与患者病毒载量、CD 4、CD 8 T淋巴细胞之间无相关性. 结论 HIV-1细胞嗜性的转换与疾病进展的关系不确定;HAART治疗对嗜性转换的作用还不明确.     

Primary HIV-1 infection: Diagnosis,pathogenesis, and treatment

Primary HIV-1 infection represents the time when the virus is first disseminating throughout the body and induces host immune responses. Diagnosing this stage of disease requires an understanding of who is at risk, the clinical manifestations of primary infection, and how the diagnosis is made. Identifying these individuals allows for counseling to prevent further transmission to others and the potential benefits associated with early antiretroviral therapy. Moreover, studying these individuals provides important insight into the biology of HIV-1 transmission and immunopathogenesis.     

HIV-1 coreceptor use in triple-class treatment-experienced patients: baseline prevalence, correlates, and relationship to enfuvirtide response

OBJECTIVE: We wished to assess, in heavily treatment-experienced patients, the prevalence of and baseline characteristics associated with HIV-1 coreceptor use and their relationship to responses to enfuvirtide treatment. METHODS: Samples were obtained from participants in phase 3 studies of enfuvirtide. Multiple logistic regression and analysis of covariance were performed on data for baseline coreceptor use, virological and immunological response, and changes in coreceptor use during treatment. RESULTS: Baseline envelopes were phenotyped for 724 patients; 50% harbored R5 strains, 48% harbored dual/mixed (D/M) strains, and 2% harbored X4 strains. D/M strains were associated with significantly lower CD4(+) cell counts but comparable viral loads, compared with R5 strains (P=.0005). Virological and immunological responses to enfuvirtide-based treatment showed no correlation with baseline coreceptor use. Changes in virus tropism from D/M to R5 strains during treatment were common, particularly in patients who received enfuvirtide (27%, vs. 14% who received no enfuvirtide; P<.05). CONCLUSION: At baseline, D/M strains were associated with lower CD4(+) cell counts but similar viral loads, compared with R5 strains, and were common across CD4(+) cell count strata. The comparable virological and immunological responses and bias toward shifts from D/M to R5 strains in patients who received enfuvirtide support its use in triple-class treatment-experienced patients and its study as a therapeutic partner for coreceptor-binding inhibitors.     

HIV-1 in genital tract and plasma of women: compartmentalization of viral sequences, coreceptor usage, and glycosylation

Worldwide, 90% of HIV-1 infections are transmitted heterosexually. Because the genital mucosa are the sites of initial contact with HIV-1 for most exposed individuals, study of the virus from the genital tract is critical for the development of vaccines and therapeutics. Previous analyses of HIV-1 in various tissues have documented compartmentalization of viral genomes. Whether compartmentalization was associated with viral phenotypic differences or immune status, however, was not well understood. We compared HIV-1 gp120 env sequences from the genital tract and plasma of 12 women. Eight women displayed compartmentalized HIV-1 RNA genomes, with viral sequences from each site that were clearly discrete, yet phylogenetically related. The remaining four exhibited env sequences that were intermingled between the two sites. Women with compartmentalized HIV-1 genomes had higher CD4+ cell counts than those displaying intermingled strains (P = 0.02). Intrapatient HIV-1 recombinants comprising sequences that were characteristic of both sites were identified. We next compared viral phenotypes in each compartment. HIV-1 coreceptor usage was often compartmentalized (P 0.01). The number of N-linked glycosylation sites, associated with neutralization resistance, also differed between compartments (P < 0.01). Furthermore, disparities between the density of gp120 glycosylations in each compartment correlated with higher CD4+ counts (P = 0.03). These data demonstrate that the genital tract and plasma can harbor populations of replicating HIV-1 with different phenotypes. The association of higher CD4+ cell counts with compartmentalization of viral genomes and density of gp120 glycosylations suggests that the immune response influences the development of viral genotypes in each compartment. These findings are relevant to the prevention and control of HIV-1 infection.     

Dual tropism of HIV-1 IIIB for chimpanzee lymphocytes and monocytes.

In humans, macrophages serve as a major reservoir of human immunodeficiency virus (HIV-1) in the infected host and may play a role in the pathogenesis of the disease. In HIV-1-infected chimpanzees, however, virus could not be recovered from cells of the monocyte/macrophage lineage, leaving the question of macrophage tropism of HIV-1 in this species unresolved. The data reported that HIV-1 IIIB shows dual tropism and is infectious for both chimpanzee monocytes and lymphocytes in vitro. Viral replication in chimpanzee monocytes was clearly demonstrated by infection of allogeneic phytohemagglutinin (PHA) blasts in vitro and by electron microscopy (EM). EM revealed HIV particles associated with 10-15% of the HIV-1 IIIB-infected chimpanzee monocytes. Viral particles budding from the monocyte surface in the typical crescent form were noted as well. This is in contrast to the human situation, where monocytotropic HIV strains preferentially bud into and accumulate in cytoplasmic vacuoles. These results indicate that both lymphocytes and cells of the monocyte/macrophage lineage replicate virus in the chimpanzee; the cell tropism of viral strains, however, is different in chimpanzees and humans.     

The role of Vpr in HIV-1 pathogenesis

The HIV-1 vpr gene is conserved among the human (HIV-1, HIV-2) and simian immunodeficiency viruses (SIV). HIV-1 vpr encodes a 96-amino acid, 14 kDa protein (Vpr). Research from a number of laboratories in the last decade has shown that Vpr performs multiple functions, including the induction of cell cycle arrest in the G(2) phase, transactivation of the viral promoter, nuclear import of preintegration complexes, and induction of apoptosis in the infected cell. More recent studies have attempted to elucidate the cellular targets that Vpr utilizes in order to perform the above functions. This review presents the latest findings about the pathogenic events triggered by Vpr, the cellular pathways involved, and the molecular and cellular consequences of the action of Vpr in the context of HIV-1 infection.