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Background:  HIV-1 plasma viremia after initiation of potent ART shows a 2- or 3-phase exponential decay, which likely reflects differences in the life-span of cells producing virus at high, intermediate, or low levels. To characterize such cells and their viral RNA expression in vivo, we combined limiting dilution analysis with real-time PCR.

Methods:  Serial dilutions of PBMC from 2 HIV-infected patients were obtained before and during ART (lopinavir/zidovudine/lamivudine [LPV/AZT/3TC]) (day 0, week 2, 4, 8, 12, 24, 36, 48). Unspliced (UsRNA) and multiply spliced HIV-RNA (MsRNA) were determined in total cell extracts and extracellular virion-associated RNA was assayed independently in cell-fractions cleared of intracellular RNA. Real-time RT-PCR using primers and RNA standards, matched to bulk PBMC HIV-RNA obtained at day 0, reached sensitivities < 2 copies. A 2-step algorithm was used to define classes of HIV-infected cells: Initial identification of cell classes was based on significant differences between mean HIV-RNA load per HIV-RNA-positive cell at succeeding time points. Subsequently 50% endpoints and average HIV-RNA contents were recalculated.

Results:  HIV-RNA-positive PBMC could be stratified into 3 classes:  cells expressing low (1 to 10 copies UsRNA), intermediate (1 to 400 copies UsRNA, 1 to 40 copies MsRNA), and high levels of HIV-RNA (40 to 1200 copies UsRNA, 45 to 3500 copies MsRNA). The latter class was rare prior to ART, rapidly declined after its initiation (t-½ ≤ 2 to 4 days) and became undetectable after 2 to 4 weeks. Intriguingly, the decay of these cells and their initial frequencies was almost identical to cells expressing high virion-related RNA levels (10 to 200c) suggesting identity for these cell categories, most likely representing productively infected cells. In contrast, cells with low or intermediate HIV-RNA levels, after an initial decline (t-½ = 0.5 to 6.6 days), persisted on ART (plasma viremia < 20 copies/mL).

Conclusions:  Combination of limiting dilution with patient-matched quantitation of different HIV-RNA allowed to characterize a specific RNA expression pattern of productively HIV-infected cells. Clearance was rapid after initiation of ART, in contrast to the persistence of cells exhibiting attenuated, non-productive RNA expression patterns, most likely representing latently infected cells transcribing HIV-RNA at low levels. Applying this novel method to larger cohorts of HIV-infected patients may allow us to refine our understanding of the population dynamics of HIV-1-infected cells in vivo.

Keywords: Viral Transcription; Viral Population Dynamics; Latency