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.

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.

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.

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.