Doherty Institute - Research Publications
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ItemHuman Immunodeficiency Virus (HIV)-1 Integration Sites in Viral Latency(SPRINGER, 2015-03)The persistence of human immunodeficiency virus type 1 (HIV-1) in latent reservoirs is a major barrier to HIV cure. Reservoir establishment depends on low viral expression that may be related to provirus integration sites (IS). In vitro, in cell lines and primary T cells, latency is associated with specific IS through reduced viral expression mediated by transcriptional interference by host cellular promoters, reverse orientation, and the presence of specific epigenetic modifiers. In primary T cell models of latency, specific IS are associated with intracellular viral antigen expression that is not directly related to cell activation. In contrast, in patient CD4+ T cells, there is enrichment for IS in genes controlling cell cycle and survival and in some clonally expanded T cell subpopulations. Multiple insertion sites within some specific genes may suggest that integrated HIV can increase the host's T cell survival.
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ItemHIV integration sites in latently infected cell lines: evidence of ongoing replication(BIOMED CENTRAL LTD, 2017-01-13)BACKGROUND: Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. RESULTS: We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. CONCLUSION: Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.
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ItemUnderstanding Factors That Modulate the Establishment of HIV Latency in Resting CD4+T-Cells In Vitro(PUBLIC LIBRARY SCIENCE, 2016-07-06)Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability.