A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

The effects of HIV-1 infection, viral particle production, and proviral integration site on CD4+ T cell proliferation

The latent reservoir of HIV is the major barrier to cure. Stimulation of latently-infected T cells results in the nuclear translocation of key transcription factors that are required for both cellular activation and reactivation of the latent provirus. These two transcriptional programs result in opposing cell fates, cell division and survival or virion production and cell death. For HIV-infected individuals on long-term ART, most CD4+ T cells in the latent reservoir are clonal, having arisen from proliferation. Consequently, clonal expansion of infected CD4+ T cells is the major mechanism of HIV persistence, though the proliferation dynamics of infected cells remain poorly characterized. Specifically, it is unknown how proviral intactness, the production of viral particles, and the proviral integration site affect T cell fate. The focus of this thesis was to characterize the fate of individual latently-infected T cells following cellular activation, which may help to describe the mechanics of total reservoir persistence and decline. To this end, we rigorously developed and validated a novel high-throughput assay which isolated individual latently-infected cells and determined their fate after optimal ex vivo stimulation. Experimental results revealed highly attenuated proliferation of infected cells, especially if they harbored an intact HIV provirus. Near full-length sequencing results confirmed an ex vivo proliferative selection against infected cells with an intact HIV provirus. Infected cell clones that produced high numbers of viral particles carried a proliferative disadvantage, though >80% of infected cell clones produced no viral particles. Infected cells that proliferated well tended to harbor a defective HIV provirus, produce few or no viral particles, and/or harbor a provirus integrated in a cancer- or proliferation-associated gene. We propose that additional mechanisms contribute to infected CD4+ T cell proliferation dynamics, including intracellular viral protein expression and in vivo selection over years on ART. Clinically, these experiments suggest that anti-proliferative agents may affect uninfected T cells much more than T cells with intact HIV proviruses. These findings furthered our understanding of reservoir dynamics and provide an opportunity to target a unique vulnerability of HIV-infected T cells in future efforts to decrease the size of the latent reservoir

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection