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.
Highlights
The phenomenal intrapatient variation of human immunodeficiency virus type 1 (HIV-1) genome needs no introduction [1,2,3,4,5,6,7]
As the present study focussed on cells with Fluorescence in situ hybridization (FISH)-positive proviruses such cells were not scored
While the values for Z are large compared to those derived from tissue culture, a recent report in a very different setting, peripheral blood mononuclear cells from HIV-infected Elite suppressors identified an unintegrated/integrated DNA ratios of between 10/1 to up to 10,000/1 [39]
Summary
The phenomenal intrapatient variation of human immunodeficiency virus type 1 (HIV-1) genome needs no introduction [1,2,3,4,5,6,7]. Recombination is present at all levels of HIV genetics [11,12]. Within an infected individual recombinant genomes show up in network analyses of HIV sequences [13,14,15,16]. Macaques inoculated simultaneously with SIVmac239Dvpx or Dvpr and with SIVmac239Dnef, the emergence of wild-type virus was detected in blood in as little as 2 weeks post-inoculation [17,18]. Some strains in widespread circulation are clearly composites of at least 2–3 other clades [19,20,21,22]
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