The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

BACKGROUND: The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. RESULTS: Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. CONCLUSIONS: Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.

The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities.

BACKGROUND: HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. RESULTS: Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. CONCLUSIONS: The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.

Long-term antiretroviral therapy mitigates mortality and morbidity independent of HIV tropism: 18 years follow-up in a women's cohort.

OBJECTIVE: CXCR4 (X4)-tropic HIV-1 was found previously to herald CD4 + cell depletion and disease progression in individuals who were antiretroviral-naive or took combination antiretroviral therapy (cART) for less than 5 years. We updated this finding by investigating whether the deleterious effect of X4-tropic strains is mitigated by long-term cART. DESIGN: We examined morbidity and mortality in relation to HIV-1 tropism and cART in 529 participants followed up to 18 years in the Women's Interagency HIV Study; 91% were women of color. METHODS: Plasma-derived HIV-1 tropism was determined genotypically. RESULTS: We categorized participants according to the number of visits reported on cART after initiation. Group 1: three or less visits, 74% of these participants reporting no cART; group 2: at least four visits and less than 70% of visits on cART; group 3: at least 70% of visits on cART. AIDS mortality rates for participants in each group with X4 virus compared with those with R5 virus exclusively were, respectively: 62 vs. 40% ( P  = 0.0088); 23% vs. 22% [nonsignificant (NS)]; 7% vs. 14% (NS). Kaplan-Meier curves showed accelerated progression to AIDS death or AIDS-defining illness in participants with three or less cART visits and X4 viruses ( P  = 0.0028) but no difference in progression rates stratified by tropism in other groups. Logistic regression found that HIV-1 suppression for at least 10 semiannual visits (≥5 years total) mitigated X4 tropism's deleterious effect on mortality, controlling for maximal viral load, and CD4 + nadir. CONCLUSION: Long-term cART markedly mitigated the deleterious effect of X4 viruses on AIDS morbidity and mortality. Mitigation was correlated with duration of viral suppression, supporting HIV-1 suppression as a crucial goal.

Methods for viral RNA isolation and PCR amplification for sequencing of near full-length HIV-1 genomes.

HIV-1 in plasma represents the viral quasispecies replicating in the patient at any given time. Studies of HIV-1 viral RNA from plasma or other body fluids therefore reflect the virus present in real time. To obtain near full-length genomic sequences derived from virion RNA it is first necessary to carefully isolate and amplify the RNA.The procedure described below, involves viral RNA extraction, reverse transcription (RT) of the extracted RNA to produce cDNA copies, and PCR amplification of long HIV-1 gene fragments using site-specific, overlapping primers. The primers are based on subtype B HIV-1 strains, and plasma specimens are used in the procedures. However, the protocol can easily be adapted to other HIV-1 subtypes by modifying the primers to match the subtype of interest.

Association of High-Risk Human Papillomavirus with Genital Tract Mucosal Immune Factors in HIV-Infected Women.

PROBLEM: High-risk human papillomavirus (HR-HPV) is prevalent in HIV-infected women and may be associated with mucosal changes that promote HIV replication. METHOD OF STUDY: Innate immune molecules, antimicrobial activity, HIV RNA, and HPV DNA genotypes were measured in a cross-sectional study of 128 HIV-infected women categorized into HPV-16 (n = 8), other HR-HPV (n = 41), and non-HR-HPV controls (n = 79). RESULTS: Compared to controls, HR-HPV groups had higher plasma viral loads (P = 0.004), lower CD4 cells (P = 0.02), more genital tract HIV RNA (P = 0.03), greater number of different HPV types (P < 0.001), higher cervicovaginal lavage (CVL) IL-1α (P = 0.03) and human beta-defensin 2 (HBD2) (P = 0.049), and less anti-HIVB al activity (P = 0.03). HPV-16 remained significantly associated with higher HBD2 (P = 0.03), higher IL-1α (P = 0.009), and lower anti-HIVB aL activity (P = 0.03) compared to controls after adjusting for plasma viral load and CD4 T cell count. CONCLUSION: HR-HPV is associated with mucosal changes in HIV-infected women that could adversely impact genital tract health.

Epigenetic analysis of HIV-1 proviral genomes from infected individuals: predominance of unmethylated CpG's.

Efforts to cure HIV-1 infections aim at eliminating proviral DNA. Integrated DNA from various viruses often becomes methylated de novo and transcriptionally inactivated. We therefore investigated CpG methylation profiles of 55 of 94 CpG's (58.5%) in HIV-1 proviral genomes including ten CpG's in each LTR and additional CpG's in portions of gag, env, nef, rev, and tat genes. We analyzed 33 DNA samples from PBMC's of 23 subjects representing a broad spectrum of HIV-1 disease. In 22 of 23 HIV-1-infected individuals, there were only unmethylated CpG's regardless of infection status. In one long term nonprogressor, however, methylation of proviral DNA varied between 0 and 75% over an 11-year period although the CD4+ counts remained stable. Hence levels of proviral DNA methylation can fluctuate. The preponderance of unmethylated CpG's suggests that proviral methylation is not a major factor in regulating HIV-1 proviral activity in PBMC's. Unmethylated CpG's may play a role in HIV-1 immunopathogenesis.

Plasmodium falciparum Infection Does Not Affect Human Immunodeficiency Virus Viral Load in Coinfected Rwandan Adults.

BACKGROUND: Plasmodium falciparum infection has been reported to increase human immunodeficiency virus (HIV) viral load (VL), which can facilitate HIV transmission. We prospectively studied the impact of mild P falciparum coinfection on HIV VL in Rwanda. METHODS: We measured plasma HIV VL at presentation with malaria infection and weekly for 4 weeks after artemether-lumefantrine treatment in Rwandan adults infected with HIV with P falciparum malaria. Regression analyses were used to examine associations between malaria infection and HIV VL changes. Samples with detectable virus underwent genotypic drug-resistance testing. RESULTS: We enrolled 28 HIV-malaria coinfected patients and observed 27 of them for 5 weeks. Three patients (11%) were newly diagnosed with HIV. Acute P falciparum infection had no significant effect on HIV VL slope over 28 days of follow-up. Ten patients with VL <40 copies/mL at enrollment maintained viral suppression throughout. Seventeen patients had a detectable VL at enrollment including 9 (53%) who reported 100% adherence to ARVs; 3 of these had detectable genotypic drug resistance. CONCLUSIONS: Unlike studies from highly malaria-endemic areas, we did not identify an effect of P falciparum infection on HIV VL; therefore, malaria is not likely to increase HIV-transmission risk in our setting. However, routine HIV testing should be offered to adults presenting with acute malaria in Rwanda. Most importantly, we identified a large percentage of patients with detectable HIV VL despite antiretroviral (ARV) therapy. Some of these patients had HIV genotypic drug resistance. Larger studies are needed to define the prevalence and factors associated with detectable HIV VL in patients prescribed ARVs in Rwanda.

Molecular epidemiology of HIV type 1 subtypes in Rwanda.

HIV-1 infection is characterized by genetic diversity, with multiple subtypes and recombinant variants circulating, particularly in sub-Saharan Africa. During the Rwandan genocide, many women experienced multiple rapes and some became HIV-1 infected. We studied plasma and peripheral blood mononuclear cells (PBMCs) from 30 infected women comprising two exposure groups: those with numerous contacts, raped multiple times, and women with one lifetime sexual partner and no history of rape. Population-based sequences from gag, pol, and env genes were analyzed to determine HIV-1 subtypes and intersubtype recombination. Individual plasma-derived variants from 12 women were also analyzed. Subtype A was found in 24/30 (80%), intersubtype recombination (AC and AD) in 4/30 (13%), and subtypes C and D in 1/30 each. In two subjects, the pattern of HIV-1 recombination differed between plasma and PBMC-derived sequences. Intersubtype recombination was common, although there were no significant differences in subtype or recombination rates between exposure groups.

A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism.

Human immunodeficiency virus type 1 (HIV-1) is characterized by sequence variability. The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism) and pathogenesis. This report describes a novel denaturing heteroduplex tracking assay (HTA) to analyze the genetic variation of HIV-1 V3 DNA. It improved upon previous non-denaturing HTA approaches to distinguish HIV-1 CCR5 and CXCR4 tropic viruses in mixed populations. The modifications included the use of a single-stranded fluorescent probe based on the consensus V3 sequence of HIV-1 CCR5 tropic viruses, Locked Nucleic Acid (LNA) "clamps" at both ends of heteroduplex DNA, and denaturing gel electrophoresis using Mutation Detection Enhancement (MDE(®)) as matrix. The analysis demonstrated that the LNA "clamps" increased its melting temperature (T(m)) and the thermal stability of heteroduplex DNA. The partially denaturing gel used a defined concentration of formamide, and significantly induced mobility shifts of heteroduplex DNA that was dependent on the number and patterns of DNA mismatches and insertions/deletions. This new technique successfully detected tropisms of 53 HIV-1 V3 clones of known tropism, and was able to separate and detect multiple V3 DNA variants encoding tropisms for CCR5 or CXCR4 in a mixture. The assay had the sensitivity to detect 0.5% minority species. This method may be useful as a research tool for analysis of viral quasispecies and for genotypic prediction of HIV-1 tropism in clinical specimens.

Recombination between variants from genital tract and plasma: evolution of multidrug-resistant HIV type 1.

Multidrug-resistant (MDR) HIV-1 presents a challenge to the efficacy of antiretroviral therapy (ART). To examine mechanisms leading to MDR variants in infected individuals, we studied recombination between single viral genomes from the genital tract and plasma of a woman initiating ART. We determined HIV-1 RNA sequences and drug resistance profiles of 159 unique viral variants obtained before ART and semiannually for 4 years thereafter. Soon after initiating zidovudine, lamivudine, and nevirapine, resistant variants and intrapatient HIV-1 recombinants were detected in both compartments; the recombinants had inherited genetic material from both genital and plasma-derived viruses. Twenty-three unique recombinants were documented during 4 years of therapy, comprising ~22% of variants. Most recombinant genomes displayed similar breakpoints and clustered phylogenetically, suggesting evolution from common ancestors. Longitudinal analysis demonstrated that MDR recombinants were common and persistent, demonstrating that recombination, in addition to point mutation, can contribute to the evolution of MDR HIV-1 in viremic individuals.

Evolution and recombination of genes encoding HIV-1 drug resistance and tropism during antiretroviral therapy.

Characterization of residual plasma virus during antiretroviral therapy (ART) is a high priority to improve understanding of HIV-1 pathogenesis and therapy. To understand the evolution of HIV-1 pol and env genes in viremic patients under selective pressure of ART, we performed longitudinal analyses of plasma-derived pol and env sequences from single HIV-1 genomes. We tested the hypotheses that drug resistance in pol was unrelated to changes in coreceptor usage (tropism), and that recombination played a role in evolution of viral strains. Recombinants were identified by using Bayesian and other computational methods. High-level genotypic resistance was seen in approximately 70% of X4 and R5 strains during ART. There was no significant association between resistance and tropism. Each patient displayed at least one recombinant encompassing env and representing a change in predicted tropism. These data suggest that, in addition to mutation, recombination can play a significant role in shaping HIV-1 evolution.

Persistent HIV RNA shedding in semen despite effective antiretroviral therapy.

Effective antiretroviral therapy (ART) may reduce HIV sexual transmission by lowering genital HIV levels. A prospective study of men starting ART (n = 25) demonstrated rapid, substantial reductions in semen HIV RNA. However, despite an undetectable blood viral load, isolated semen HIV shedding was detected at more than one visit in 12 of 25 (48%) participants, with semen HIV RNA levels exceeding 5000 copies/ml in four of 25 (16%). Isolates were drug-sensitive, and this phenomenon was not associated with semen drug levels or regimen.

Transition from long-term nonprogression to HIV-1 disease associated with escape from cellular immune control.

Transition from long-term nonprogressive infection to progressive HIV-1 disease presents an opportunity to investigate pathogenesis in a defined immunogenetic background. We studied a male long-term nonprogressor (LTNP) who remained asymptomatic and viremic and had normal CD4 T-cell counts without antiretroviral therapy for >18 years and then experienced a transition to disease progression. We analyzed the complete HIV-1 genomic RNA sequence from plasma and cellular immune responses to predefined human leukocyte antigen-matched autologous viral peptides spanning the viral genome, before and after progression. Serial viral sequences did not seem attenuated and consistently utilized coreceptor CCR5. LTNP status was associated with elongated V2 domains and broad low-level T-cell immune responses targeting several regions of the viral genome. The transition to progressive disease was associated with the acquisition of viral mutations conferring escape from CD8 T-cell responses. Multiple changes in HIV-1 sequence and loss of immune response over time most likely contributed to the transition from LTNP status to progressive disease. These data are relevant to vaccine design and identification of the correlates of protection from disease progression.

HIV-1 drug resistance in variants from the female genital tract and plasma.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) drug-resistance mutations may arise in a fraction of viral variants, and these variants may differ between compartments, including the genital tract and blood. METHODS: We studied 14 women with detectable HIV-1 in both the genital tract and plasma despite antiretroviral treatment. We obtained HIV-1 RNA sequences from 280 unique viral variants and then determined the resistance genotype and the predicted phenotype (Virtual Phenotype; Virco BVBA) of each variant. RESULTS: Eight patients (57%) displayed mutations conferring high-level HIV-1 drug resistance. Although we observed differences in specific mutations among viral variants, 13 of the 14 women showed highly concordant HIV-1 genotypic and predicted phenotypic resistance patterns in the 2 compartments. In 1 patient, resistance mutations appeared only in plasma; all variants in her genital tract, which displayed a low viral load, were susceptible. CONCLUSIONS: These data suggest that, for the majority of women, determination of HIV-1 drug resistance in the plasma will approximate the drug-resistance pattern in the genital tract. Analysis of individual variants enabled us to identify minority species bearing distinctive linked mutations, which may serve as a source of novel resistance genotypes. These data are relevant to clinical management and the evolution of drug resistance.

Long-lasting CCR5 internalization by antibodies in a subset of long-term nonprogressors: a possible protective effect against disease progression.

Exposure to HIV-1 does not necessarily result in infection and progression toward disease, thus suggesting that the control of viral infection may be achieved. Antibodies to CCR5 have been detected in HIV-exposed but uninfected subjects (ESNs); thus, these antibodies could be involved in HIV protection. To assess whether anti-CCR5 antibodies may also contribute to slow HIV disease progression, we searched for anti-CCR5 antibodies in 497 subjects, including 85 long-term nonprogressors (LTNPs), 70 progressors, 135 HIV(+) patients treated with highly active antiretroviral therapy (HAART), and 207 seronegative donors. We found anti-CCR5 antibodies in a fraction of the LTNPs(23.5%) but not in the other populations studied (P < .001). These antibodies recognized a conformational epitope within the first extramembrane loop of CCR5, and they induced a stable and long-lasting downregulation of CCR5 on the surface of T lymphocytes, which inhibited HIV entry. In addition, CD4(+) lymphocytes from LTNPs having anti-CCR5 antibodies are resistance to R5 strains of HIV-1. Follow-up studies showed that the loss of anti-CCR5 antibodies occurred in some subjects, and this loss was significantly associated with a progression toward disease, whereas subjects who retained anti-CCR5 Abs maintained their LTNP status. Induction of anti-CCR5 Abs could be relevant to vaccine design and therapeutics.

Long-term survivors in Nairobi: complete HIV-1 RNA sequences and immunogenetic associations.

To investigate African long-term survivors (LTSs) infected with non-subtype B human immunodeficiency virus type 1 (HIV-1), we obtained full-length HIV-1 RNA sequences and immunogenetic profiles from 6 untreated women enrolled in the Pumwani Sex Worker Cohort in Nairobi, Kenya. There were no discernible sequence changes likely to cause attenuation. CCR2-V64I, an immunogenetic polymorphism linked to LTSs, was detected in 4 women, all of whom carried the HLA B58 allele. Further investigation of 99 HIV-1-infected Nairobi women found an association between CCR2-V64I and HLA B58 (P=.0048). Studying the interaction among immunogenetics, immune responses, and viral sequences from all HIV-1 subtypes may increase our understanding of slow HIV-1 disease progression.

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.