HIV-1 integrates into resting CD4+ T cells even at low inoculums as demonstrated with an improved assay for HIV-1 integration

Abstract

Human Immunodeficiency Virus Type 1 (HIV-1) establishes a latent reservoir early in infection that is resistant to the host immune response and treatment with highly active antiretroviral therapy (HAART). The best understood of these reservoirs forms in resting CD4(+) T cells. While it remains unclear how reservoirs form, a popular model holds that the virus can only integrate in activated CD4(+) T cells. Contrary to this model, our previous results suggest that HIV-1 can integrate directly into the genomes of resting CD4(+) T cells. However, a limitation of our previous studies was that they were conducted at high viral inoculum and these conditions may lead to cellular activation or saturation of restriction factors. In the present study, we tested if our previous findings were an artifact of high inoculum. To do this, we enhanced the sensitivity of our integration assay by incorporating a repetitive sampling technique that allowed us to capture rare integration events that occur near an Alu repeat. The new technique represents a significant advance as it enabled us to measure integration accurately down to 1 provirus/well in 15,000 genomes--a 40-fold enhancement over our prior assay. Using this assay, we demonstrate that HIV can integrate into resting CD4(+) T cells in vitro even at low viral inoculum. These findings suggest there is no threshold number of virions required for HIV to integrate into resting CD4(+) T cells.

Quantification of Integrated HIV DNA by Repetitive-Sampling Alu-HIV PCR on the Basis of Poisson Statistics

“…2B). The slope coefficients close to 0.1 revealed that the absolute number of integrated copies measured in the repetitive-sampling strategy is 10-fold lower than the expected number in the integration standard; this fold difference is stable at all dilution points assessed and is consistent with published data showing that 10% of integration events are detected (16,17 ). A third standard curve with higher numbers of integrated copies was run to assess the upper limit of positive to negative wells in the 42-replicate setup.…”

“…Sorting of green fluorescent protein (GFP)-positive cells resulted in a population of cells with 1 proviral copy per cell (15)(16)(17). Peripheral blood mononuclear cells were isolated from 56 HIVinfected patients by density gradient centrifugation and frozen to Ϫ80°C at a cooling rate of 1°C/min in 90% fetal calf serum with 10% DMSO.…”

“…Over several years, the assay was further optimized, enabling its use in patient samples (7,8,(15)(16)(17)(18)(19)(20).…”

“…Hence, each integration will have a specific PCR efficiency depending on its distance from an Alu element. The repetitivesampling strategy, assessing multiple replicate reactions (Ͼ40) of the same sample, compensates for this integration specific bias (16 ). The HIV-only PCR (right) linearly amplifies integrated and unintegrated HIV DNA.…”

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

“…Although researchers do employ patient samples, when doing so, MHC mismatched CD4ϩ T cells are typically added to the cultures to allow expansion of the virus, so it is unclear whether allogeneic reactivity might contribute to virus activation. Viral particles enveloped with the CXCR4-tropic envelope integrate into the genomes of resting CD4 ϩ T cells and result in a latent infection, as early as 3 days after infection, which can be reactivated by various stimuli including IL-7 and CD3/CD28 stimulation (32)(33)(34)(35). PBMCs from whole blood were isolated by Ficoll gradient centrifugation and then subjected to negative selection to isolate resting CD4 ϩ T cells.…”