Human Immunodeficiency Virus Type 1 Evolution In Vivo tracked by DNA Heteroduplex Mobility Assays

Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305-5402.
Journal of Virology (Impact Factor: 4.44). 11/1994; 68(10):6672-83.
Source: PubMed


High mutation rates and strong selective pressures imposed on human immunodeficiency viruses in vivo result in the formation of pools of genetic variants known as quasispecies. DNA heteroduplex mobility and tracking analyses were used to monitor the generation of HIV sequence diversity, to estimate quasispecies complexity, and to assess the turnover of genetic variants to approach an understanding of the relationship between viral quasispecies evolution in vivo and disease progression. Proviral DNA pools were nearly homogeneous soon after sexual transmission. The emergence and clearance of individual variants then occurred at different rates in different individuals. High quasispecies complexity was found in long-term-infected, asymptomatic individuals, while rapid CD4+ cell decline and AIDS were often, but not always, associated with lower quasispecies complexity. Proviral genetic variation was often low following in vitro culture, because of the outgrowth of one or a few variants that often became more abundant only later as proviruses in peripheral blood mononuclear cells. These studies provide insight into the dynamics of human immunodeficiency virus sequence changes in vivo and illustrate the utility of heteroduplex analysis for the study of phenomena associated with rapid genetic changes.

Download full-text


Available from: Eric Delwart,
  • Source
    • "Nested PCR products of the env gene were analyzed by the heteroduplex tracking assay (HTA) to determine the amount of virus produced in the dual infection/competition experiments [39]. The same genomic region (env C2-V3) was PCR-amplified from different subtype-specific HIV-1 env clones [subtype A RW020 and SF170, subtype C BR025, subtype E TH22 and CAR7] [41] for use as DNA probes. However, in this case, the E80 primer was radiolabeled with T4 polynucleotide kinase and 2uCi of 5′-[γ32P] ATP, prior to amplification, as described [39]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (n = 9, 32.1%), C (n = 1, 3.6%), and CRFs (n = 18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous "CRF" B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events.
    PLoS ONE 04/2014; 9(4):e92084. DOI:10.1371/journal.pone.0092084 · 3.23 Impact Factor
  • Source
    • "The phenomenal intrapatient variation of human immunodeficiency virus type 1 (HIV-1) genome needs no introduction [1], [2], [3], [4], [5], [6], [7]. The absence of proofreading mechanisms associated with the reverse transcriptase, the high recombination rate and high turnover are the main factors [8], [9], [10]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the nucleus of HIV-1 infected cells, unintegrated HIV-1 DNA molecules exist in the form of one and two LTR circles and linear molecules with degraded extremities. In tissue culture they are invariably more numerous than the provirus, the relative proportion of integrated to unintegrated forms varies widely from ∼1:1 to 1:10 and even over 1:100. In vivo, this ratio is unknown. To determine it, single nuclei from two infected patients with a known provirus copy number were microdissected, HIV DNA was amplified by nested PCR, cloned and individual clones sequenced. Given the extraordinary sequence complexity, we made the assumption that the total number of distinct sequences approximated to real number of amplifiable HIV-1 DNA templates in the nucleus. We found that the number of unintegrated DNA molecules increased linearly with the proviral copy number there being on average 86 unintegrated molecules per provirus.
    PLoS ONE 05/2012; 7(5):e36246. DOI:10.1371/journal.pone.0036246 · 3.23 Impact Factor
  • Source
    • "Second round gp41 PCR products amplified from plasma were used directly in the heteroduplex assay as described elsewhere [8,59]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: HIV-1 superinfection occurs at varying frequencies in different at risk populations. Though seroincidence is decreased, in the negative partner of HIV-discordant couples after joint testing and counseling in the Zambia Emory HIV Research Project (ZEHRP) cohort, the annual infection rate remains relatively high at 7-8%. Based on sequencing within the gp41 region of each partner's virus, 24% of new infections between 2004 and 2008 were the result of transmission from a non-spousal partner. Since these seroconvertors and their spouses have disparate epidemiologically-unlinked viruses, there is a risk of superinfection within the marriage. We have, therefore, investigated the incidence and viral origin of superinfection in these couples. Superinfection was detected by heteroduplex mobility assay (HMA), degenerate base counting of the gp41 sequence, or by phylogenetic analysis of the longitudinal sequences. It was confirmed by full-length env single genome amplification and phylogenetic analysis. In 22 couples (44 individuals), followed for up to five years, three of the newly infected (initially HIV uninfected) partners became superinfected. In each case superinfection occurred during the first 12 months following initial infection of the negative partner, and in each case the superinfecting virus was derived from a non-spousal partner. In addition, one probable case of intra-couple HIV-1 superinfection was observed in a chronically infected partner at the time of his seroconverting spouse's initial viremia. Extensive recombination within the env gene was observed following superinfection. In this subtype-C discordant couple cohort, superinfection, during the first year after HIV-1 infection of the previously negative partner, occurred at a rate similar to primary infection (13.6% [95% CI 5.2-34.8] vs 7.8% [7.1-8.6]). While limited intra-couple superinfection may in part reflect continued condom usage within couples, this and our lack of detecting newly superinfected individuals after one year of primary infection raise the possibility that immunological resistance to intra-subtype superinfection may develop over time in subtype C infected individuals.
    Retrovirology 03/2012; 9(1):22. DOI:10.1186/1742-4690-9-22 · 4.19 Impact Factor
Show more