Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers

Abstract

In human immunodeficiency virus (HIV)-infected individuals, the proportion of circulating mononuclear cells (PBMCs) which carry HIV provirus and the number of HIV proviral sequences per infected PBMC have been matters for conjecture. Using a double polymerase chain reaction which allows the detection of single molecules of provirus and a method of quantifying the provirus molecules, we have measured provirus frequencies in infected individuals down to a level of one molecule per 10' PBMCs. As a general rule, only a small proportion of PBMCs contain provirus (median value of samples from 12 patients, one per 8,000 cells), and most if not all of the infected cells carry a single provirus molecule. The frequency of provirus-carrying cells correlated positively both with the progression of the disease and with the success with which virus could be isolated from the same patients by cocultivation methods. Of seven asymptomatic (Centers for Disease Control stage H) patients, all but one contained one provirus molecule per 6,000 to 80,000 cells; of five Centers for Disease Control stage IV patients, all but one contained one provirus molecule per 700 to 3,300 cells. When considered in conjunction with estimates of the frequency of PBMCs that express viral RNA, our results suggest that either (i) the majority of provirus-containing cells are monocytes or (ii) most provirus-containing lymphocytes are transcriptionally inactive. We also present nucleotide sequence data derived directly from provirus present in vivo which we show is not marred by the in vitro selection of potential virus variants or by errors introduced by Taq polymerase. We argue from these data that, of the provirus present in infected individuals, the proportion which is defective is not high in the regions sequenced.

Comparison of Tissue Distribution, Persistence, and Molecular Epidemiology of Parvovirus B19 and Novel Human Parvoviruses PARV4 and Human Bocavirus

“…DNA extraction. Tissue samples were extracted as described elsewhere [24]. Extracted DNA was quantified by spectrophotometry at 260/280 nm.…”

“…Measurement of viral load and sequencing. Nested PCRs for the 3 virus targets were sensitive for single copies of the target sequence [12], enabling use of limiting dilution [24] for quantitation of positive samples. For the autopsy samples, initial screening was performed by use of 0.5 mg DNA from each sample, and positive samples were titrated further in 5-fold dilution steps until limiting dilution was observed in 4 or 8 replicate reactions; viral loads then were calculated from the frequencies of positive reactions at limiting dilution [25] and were expressed as copies per million cells.…”

Comparison of hepatitis B virus subtyping of d/y determinants by radioimmunoprecipitation assay and the polymerase chain reaction

“…Resuspended DNA was amplified essentially as described by Simmonds et al [1990], excluding dimethylsulphoxide and EDTA from the reaction. To reduce the risk of contamination and monitor performance, samples were amplified in batches of ten, each batch consisting of five samples and five controls numbered randomly.…”

A role for CD8+ T lymphocytes in the pathogenesis of AIDS

“…Current evidence shows that there is an extremely low level of HIV provirus present in the circulating PBL from HIV-infected persons, and that more than 99 °7o of the lymphocytes which contain provirus do not express the virus (HO et al , 1989;Schnittman et aL , 1989;Simmonds et al, 1990;Brinchmann etaL, 1991). Thus, we may be facing a situation in which the observed final increase in the percentage of infected CD4 + cells in AIDS patients is a consequence of clonal growth and not of virus replication and spread.…”