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.
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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12977-016-0298-1) contains supplementary material, which is available to authorized users.
HIV Gag; HIV protease; Protease inhibitors; Late domain; Anti-retroviral drugs; ESCRT
An HIV-1 tropism test is recommended prior to CCR5 antagonist administration to exclude patients harboring non-R5 virus from treatment with this class of antiretrovirals. HIV-1 tropism determination based on proviral DNA (pvDNA) may be useful in individuals with plasma viral RNA suppression. We developed a genotypic tropism assay for pvDNA and assessed its performance in a retrospective analysis of samples collected longitudinally.
We randomly selected paired plasma/PBMC samples from the Women’s Interagency HIV Study with plasma viral load ≥5,000 cp/mL at time 1 (T1), undetectable viral load maintained for ≥1 year and CD4+ >200 cells/μL at time 2 (T2). pvDNA was isolated from cryopreserved PBMCs. Sequences were analyzed in triplicate from 49/50 women, with tropism assigned using the geno2pheno (g2p) algorithm. The median time between T1 and T2 was 4.1 years. CXCR4-using virus was detected in 24% of the RNA samples and 33% of the pvDNA samples at T1, compared to 37% of the pvDNA samples at T2. Concordance between plasma RNA and pvDNA tropism was 88% at T1 and 80% at T2. The g2p scores for RNA (T1) vs DNA (T1, T2) were strongly correlated (Spearman rho: 0.85 (T1); 0.78 (T2)). In women with evidence of tropism switch at T2 (either R5 to non-R5 or non-R5 to R5), there was a correlation between change in tropism and time. Mean pvDNA viral load decreased by 0.4 log10 copies/106 cells between T1 and T2 (p < 0.0001), but this decrease was not significantly associated with tropism status.
We demonstrated that pvDNA tropism in women with HIV-1 suppression is concordant with prior RNA tropism results, even after a median time of >4 years. pvDNA tropism testing may be useful to determine eligibility of patients with viral suppression to switch to a CCR5-antagonist based regimen as well as for research purposes.
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The online version of this article (doi:10.1186/s12981-015-0055-x) contains supplementary material, which is available to authorized users.
HIV; Tropism; pvDNA
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.
Epigenetics of HIV-1 proviral DNA; Integrated HIV-1 DNA in PBMC’s from infected individuals; Wide spectrum of infection outcome; Bisulfite sequencing; Methylation analysis of integrated HIV-1 genomes; Predominance of unmethylated CpG’s in PBMC’s; Escape from proviral DNA methylation; Fluctuation of CpG methylation in one LTNP individual
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.
HIV-1 coreceptor tropism testing is used to evaluate eligibility for CCR5 antagonist therapy. However, HIV-1 RNA-based tests are not suitable for virologically suppressed patients, therefore the use of proviral DNA tropism testing has been investigated. We describe a novel proviral DNA-based genotypic tropism assay and compare its performance to that of a sensitive HIV-1 RNA-based genotypic test.
Tropism was determined using HIV-1 plasma RNA and proviral DNA from 42 paired samples from patients with plasma viral loads ≥1000 HIV-1 RNA copies/mL. Proviral DNA sample types included whole blood, separated peripheral blood mononuclear cells resuspended in phosphate-buffered saline and peripheral blood mononuclear cells resuspended in spun plasma. The HIV-1 envelope V3 region was PCR-amplified, sequenced in triplicate, and analyzed for tropism with the geno2pheno algorithm using a 10% false-positive rate (FPR).
Amplicons were obtained from proviral DNA and plasma RNA in 41/42 samples. Tropism predictions were highly concordant (93%–98%) between proviral DNA and plasma RNA, regardless of the proviral DNA isolation method. Non-R5 proviral DNA results were obtained for 100% of patients with detectable non-R5 plasma HIV-1 RNA results. Geno2pheno FPRs for proviral DNA and plasma RNA were highly correlated (Spearman rho = 0.86).
Our findings demonstrate that proviral DNA tropism determinations from whole blood or peripheral blood mononuclear cells were highly concordant with plasma HIV-1 RNA tropism determinations. This assay may be useful for screening virologically suppressed patients for CCR5-antagonist eligibility and for research purposes.
HIV-1 diagnostic tests; HIV-1 tropism; HIV-1 proviral tropism
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.
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.
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.
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.
HIV-1; Alix; Tsg101; Nedd4; CA NTD; Viral particle maturation; Protease; Viral budding
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 (Tm) 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.
HIV-1 gp120 V3; denaturing heteroduplex tracking assay; Locked Nucleic Acid clamps; viral tropism
The human gene for CC chemokine receptor 5, a coreceptor for human immunodeficiency virus type 1 (HIV-1), affects susceptibility to infection. Most studies of predominantly male cohorts found that individuals carrying a homozygous deleted form of the gene, Δ32, were protected against transmission, but protection did not extend to Δ32 heterozygotes. The role played by this mutation in HIV-1 transmission to women was studied in 2605 participants in the Women's Interagency HIV Study. The Δ32 gene frequency was 0.026 for HIV-1–seropositive women and 0.040 for HIV-1–seronegative women, and statistical analyses showed that Δ32 heterozygotes were significantly less likely to be infected (odds ratio, 0.63 [95% confidence interval, 0.44–0.90]). The CCR5 Δ32 heterozygous genotype may confer partial protection against HIV-1 infection in women. Because Δ32 is rare in Africans and Asians, it seems plausible that differential genetic susceptibility, in addition to social and behavioral factors, may contribute to the rapid heterosexual spread of HIV-1 in Africa and Asia.
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 ~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.
HIV-1 drug resistance; HIV-1 recombination; HIV-1 tropism
The majority of natural history studies of human immunodeficiency virus (HIV) infection have immune and viral parameters in men. Data demonstrating that women have lower HIV-1 RNA levels than men at the same CD4 cell counts have raised the question of immunologic differences in HIV-seropositive women. This study describes levels and changes in phenotypic markers of immune maturity, function, and activation in the CD4 and CD8 cell subsets in HIV-seropositive and high-risk HIV-seronegative women. Our primary hypothesis was that activation levels would be significantly higher among illicit drug users. However, results showed that HIV-1 RNA level was the strongest predictor of marker level and that both HIV-1 RNA level and CD4 cell count were independently associated with CD4 activation, but illicit drug use was not. In summary, this study demonstrated that immune activation was a significant pathogenic feature in women and that activation was driven by HIV infection and not illicit drug use.
Both antiretroviral therapy and the human coreceptor polymorphism CCR2-V64I slow progression of human immunodeficiency virus type 1 (HIV-1) disease. To examine the effect of V64I on disease progression in patients receiving therapy, we determined CCR2 genotypes in the Women’s Interagency HIV Study cohort. We studied 2047 HIV-1–infected women, most of whom initiated treatment during the study. No association was seen between CCR2 genotype and either disease progression or therapeutic response, suggesting that the benefits of treatment most likely overshadow the salutary effects of the V64I polymorphism.
Antibodies that mediate human immunodeficiency virus (HIV)-specific antibody-dependent cell-mediated cytotoxicity (ADCC) are present in the cervical fluid of many HIV-positive women; however, the role that these antibodies play in host defense against HIV is not known. To understand the contribution of ADCC in cervical secretions as a protective mechanism against HIV, we evaluated ADCC titers in paired serum and cervical-lavage (CVL) samples from >300 HIV-1–positive women who participated in the multicenter Division of AIDS Treatment Research Initiative Study 009. The present study demonstrates that women with CVL ADCC activity had lower genital viral loads than did women with serum ADCC activity only. Women with CVL ADCC activity were likely to have HIV-1 gp120–specific immunoglobulin (Ig) G, but not IgA, in their cervical fluid. This finding suggests that specific IgG in cervical fluid can mediate ADCC activity that inversely correlates with genital viral load.
To investigate the viral features of long-term nonprogressive HIV-1 infection and the selection of viral genomes, we studied serial complete HIV-1 sequences obtained from a mother–child pair, both long-term nonprogressors. Analysis of four genomic sequences demonstrated that all viral genes were intact, lacking major deletions or premature stop codons to easily explain the slow disease progression. These data suggest that viral attenuation, if present, was caused by subtle sequence variations or virus–host interactions. Serial sequences from an HIV-1-infected mother–child pair afforded us the opportunity to examine the immune selection of HIV-1 sequences years after transmission between individuals. We demonstrated that the daughter's strains were most likely subjected to immunoselection or immunoediting according to the presence of novel MHC class I alleles that differed between mother and daughter. An analysis of nef-specific cytotoxic T-lymphocyte responses in the child, whose HIV-1 nef sequence differed from the maternal nef, supported this interpretation. This study highlights the potential of full genome analysis in the investigation of pathogenesis and immune selection during HIV-1 evolution.
Investigation of human immunodeficiency virus type 1 (HIV-1) in the genital tract of women is crucial to the development of vaccines and therapies. Previous analyses of HIV-1 in various anatomic sites have documented compartmentalization, with viral sequences from each location that were distinct yet phylogenetically related. Full-length RNA genomes derived from different compartments in the same individual, however, have not yet been studied. Furthermore, although there is evidence that intrapatient recombination may occur frequently, recombinants comprising viruses from different sites within one individual have rarely been documented. We compared full-length HIV-1 RNA sequences in the plasma and female genital tract, focusing on a woman with high HIV-1 RNA loads in each compartment who had been infected heterosexually and then transmitted HIV-1 by the same route. We cloned and sequenced 10 full-length HIV-1 RNA genomes from her genital tract and 10 from her plasma. We also compared viral genomes from the genital tract and plasma of four additional heterosexually infected women, sequencing 164 env and gag clones obtained from the two sites. Four of five women, including the one whose complete viral sequences were determined, displayed compartmentalized HIV-1 genomes. Analyses of full-length, compartmentalized sequences made it possible to document complex intrapatient HIV-1 recombinants that were composed of alternating viral sequences characteristic of each site. These findings demonstrate that the genital tract and blood harbor genetically distinct populations of replicating HIV-1 and provide evidence that recombination between strains from the two compartments contributes to rapid evolution of viral sequence variation in infected individuals.
There is substantial evidence for ongoing replication and evolution of human immunodeficiency virus type 1 (HIV-1), even in individuals receiving highly active antiretroviral therapy. Viral evolution in the presence of antiviral therapy needs to be considered when developing new therapeutic strategies. Phylogenetic analyses of HIV-1 sequences can be used for this purpose but may give rise to misleading results if rates of intrapatient evolution differ significantly. To improve analyses of HIV-1 evolution relevant to studies of pathogenesis and treatment, we developed a Bayesian hierarchical model that incorporates all available sequence data while simultaneously allowing the phylogenetic parameters of each patient to vary. We used this method to examine evolutionary changes in HIV-1 coreceptor usage in response to treatment. We examined patients whose viral populations exhibited a shift in coreceptor utilization in response to therapy. CXCR4 (X4) strains emerged in each patient but were suppressed following initiation of new antiretroviral regimens, so that CCR5-utilizing (R5) strains predominated. By phylogenetically reconstructing the evolutionary relationship of HIV-1 obtained longitudinally from each patient, it was possible to examine the origin of the reemergent R5 virus. Using our Bayesian hierarchical approach, we found that the reemergent R5 virus detectable after therapy was more closely related to the predecessor R5 virus than to the X4 strains. The Bayesian hierarchical approach, unlike more traditional methods, makes it possible to evaluate competing hypotheses across patients. This model is not limited to analyses of HIV-1 but can be used to elucidate evolutionary processes for other organisms as well.
To initiate infection, HIV-1 requires a primary receptor, CD4, and a secondary receptor, principally the chemokine receptor CCR5 or CXCR4. Coreceptor usage plays a critical role in HIV-1 disease progression. HIV-1 transmitted in vivo generally uses CCR5 (R5), but later CXCR4 (X4) strains may emerge; this shift heralds CD4+ cell depletion and clinical deterioration. We asked whether antiretroviral therapy can shift HIV-1 populations back to R5 viruses after X4 strains have emerged, in part because treatment has been successful in slowing disease progression without uniformly suppressing plasma viremia. We analyzed the coreceptor usage of serial primary isolates from 15 women with advanced disease who demonstrated X4 viruses. Coreceptor usage was determined by using a HOS-CD4+ cell system, biological and molecular cloning, and sequencing the envelope gene V3 region. By constructing a mathematical model to measure the proportion of virus in a specimen using each coreceptor, we demonstrated that the predominant viral population shifted from X4 at baseline to R5 strains after treatment. Multivariate analyses showed that the shift was independent of changes in plasma HIV-1 RNA level and CD4+ cell count. Hence, combination therapy may lead to a change in phenotypic character as well as in the quantity of HIV-1. Shifts in coreceptor usage may thereby contribute to the clinical efficacy of anti-HIV drugs.