HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput.

Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.

Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure.DOI: http://dx.doi.org/10.7554/eLife.09115.001

No significant differences were revealed in levels of residual plasma HIV-1 RNA, total HIV-1 DNA or intracellular markers of ongoing virus replication (unspliced and multiply spliced HIV-1 RNA and 2-LTR circles) between treatment groups.

bEpisomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. With the persistence of a viral HIV-1 reservoir despite the use of combination antiretroviral therapy (cART) (1), there is a strong need for sensitive tools to characterize the viral reservoir and its dynamics. Recent data indicate that episomal unintegrated circular HIV-1 DNA (two-long terminal repeat [2-LTR] circles) serves as a marker for viral replication (2-4).DNA isolation procedures for 2-LTR recovery were recently compared (5). Although 2-LTR circles were recovered more efficiently by total DNA isolation than by plasmid DNA isolation in vitro, the small number of patients (n ϭ 3) assessed impeded an in vivo evaluation at the lower end of detection. This is especially important, because of the small abundance of 2-LTR circles in patients on cART (6, 7). PCR inhibition by excessive DNA load limits the input material per reaction of total genomic DNA (gDNA) (8), while plasmid DNA isolation which specifically isolates episomal DNA from the bulk chromosomal DNA allows input from larger amounts of cells.Here, two sample processing procedures were compared to quantify 2-LTR episomal HIV-1 DNA by using droplet digital PCR (ddPCR) in peripheral blood mononuclear cells (PBMCs) from a large number of HIV-1-infected patients. This comparison was made by ddPCR, which provides direct absolute quantification with a sensitivity similar to or better than that of real-time quantitative PCR (qPCR), as shown in studies assessing low levels of plasma HIV-1 RNA (9), cell-associated HIV-1 RNA (10), total viral DNA (8, 11-13), and 2-LTR circles (8, 13). In addition, absolute quantification by ddPCR provides a direct measure to assess template loss in pre-PCR processing steps.A modified plasmid DNA isolation method was optimized, using a reference plasmid (pSIF1-H1) (see Fig. S1 in the supplemental material), further referred to as pSIF, spiked in the samples before DNA isolation for normalization to cell equivalents (see Methods and Results in the supplemental material for in-depth optimization and Fig. S2 in the supplemental material for the workflow). The pSIF plasmid was quantified by detection of the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (see Methods and Table S1 in the supplemental material), further referred to as the pSIF assay.Quantification of pSIF in plasmid DNA and RPP30 in gDNA isolates revealed a higher number of cell equivalents per ddPCR replicate in plasmid DNA than in genomic DNA isolates, i.e., 6.1-fold and 12.7-fold more cell equivalents in the dilution series and in the patient-derived samples (n ϭ 59), respectively (see Results in the supplemental material). 2-LTR quantities were normaliz...