Abstract
BackgroundWe recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency.ResultsHigh levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the nef open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat.ConclusionsIn this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with ex vivo findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent in vitro model of HIV latency.
Highlights
We recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7
We have previously demonstrated that latent infection can be established in resting memory CD4+ T-cells in vitro following incubation with the chemokines CCL19 and CCL21, CXCL9 and CXCL10 and CCL20 [11,12]
Latency is established in CCL19-treated CD4+ T-cells following single round infection, and there is no evidence of spontaneous productive infection We infected CCL19-treated CD4+ T-cells with wild type (WT) NL4.3 and NL4.3Δenv to determine if spreading infection contributed to the high levels of integrated HIV observed following infection of CCL19-treated CD4+ T-cells
Summary
We recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. We have previously demonstrated that latent infection can be established in resting memory CD4+ T-cells in vitro following incubation with the chemokines CCL19 and CCL21 (ligands for CCR7), CXCL9 and CXCL10 (ligands for CXCR3) and CCL20 (ligand for CCR6) [11,12]. These chemokines are important for T-cell migration and recirculation between blood and tissue [13,14,15], and we have proposed that the addition of chemokines in vitro to resting CD4+ T-cells may model chemokine rich micro-environments such as lymphoid tissue [11,16]. Reactivation of virus from in vitro models of HIV latency should closely mimic ex vivo findings from patient derived CD4+ T-cells
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