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.

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Available from: Eric Delwart, Oct 07, 2015
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    • "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]. "
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    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
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    • "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]. "
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    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
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    • "Second round gp41 PCR products amplified from plasma were used directly in the heteroduplex assay as described elsewhere [8,59]. "
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    Retrovirology 03/2012; 9(1):22. DOI:10.1186/1742-4690-9-22 · 4.19 Impact Factor
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