Pathways to HIV latency and reactivation in vitro
AuthorREZAEI, SIMIN DOKHT
AffiliationMicrobiology & Immunology
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr Simin Dokht Rezaei
Human immunodeficiency virus (HIV) infection remains a major global health issue. Antiretroviral drugs improve life expectancy and significantly reduce the rate of viral transmission; however, we are far from finding a cure for HIV. The major barrier to finding a cure is the persistence of the replication-competent yet transcriptionally silent latent reservoir. Current latency reversal agents (LRA) lack efficacy to eliminate all the latent proviruses from the reservoir. The response to the same LRAs is varied in latently infected cells ex vivo or in vitro. We hypothesised that HIV could generate different populations of latently infected cells that differ in HIV integration sites and response to reactivation by LRAs. We used a Nef-competent EGFP reporter virus to generate infection and to determine the latently infected cells in chemokine-treated CD4+ T cells in vitro. We first demonstrated that EGFP expression is dependent on viral integration and can be used to determine productively expressed and latently induced infected cells in our culture system. Infection and latency were established in both resting untreated and CCL19-treated CD4+ T cells in vitro. Addition of integrase inhibitor, raltegravir, at time of infection reduced the levels of EGFP expression in both T cell conditions, providing evidence that in our culture system EGFP expression is dependent on viral integration. There was a 4-fold reduction in EGFP expression in the CCL19-treated compared to the matched resting untreated cells. The reduction in the EGFP expression following addition of integrase inhibitor strongly suggested that incubating CD4+ T cells with CCL19 favors viral integration in vitro. We subsequently showed that the addition of IL-7 significantly increases the levels of latency in the chemokine-treated CD4+ T cells. Thus, we clearly showed that both resting and chemokine-treated CD4+ T cells are permissive to direct infection with HIV in vitro. However, the effect of CCL19 in the induction of latency is more pronounced with the addition of IL-7. We further asked whether the establishment of latency affects the response to reactivation by LRAs or T cell receptor (TCR) signalling. We used resting CD4+ T cells to establish infection in the pre-activation pathway and used activated T cells as a model for the establishment of infection in the post-activation pathway. Co-culturing EGFP- cells with allogeneic monocytes alone or in combination with an antibody against CD3 (aCD3); we showed a significant increase in EGFP expression from latently infected cells in the pre-activation latency model. Response to allogeneic monocytes in combination with signals derived from aCD3 significantly correlated with T cell proliferation and there was a minimal spontaneous EGFP expression from latently infected cells in this culture. In contrast, allogeneic monocytes alone or in combination with aCD3 reduced the EGFP expression from latently infected cells in the post-activation latency model. There was no correlation between T cell proliferation and viral expression. The level of spontaneous EGFP expression from latently infected cells was high, and the inhibition of EGFP expression by monocytes was dependent on the direct contact between monocytes and T cells. We further showed that the interaction between T cells and monocytes at time of infection induced spontaneous expression, providing evidence that monocyte-T cell interaction at an early time post infection maintains latency in activated T cells. By direct comparison of pre- and post-activation latency in vitro we, therefore, demonstrated that effective strategies to reverse latency would depend on how latency is established. We further profiled the HIV integration sites in pre- and post-activation latency models and showed a significant enrichment of the sites in genic, exon and intron; in sense direction in the introns of pre-activation latency compared to the post-activation models, suggesting preferential integration of proviral DNA in these locations. By indexing genes with integration sites with gene expression available for these genes in GEO dataset using RNA-Seq analysis, we found a set of genes that are not expressed during activation of T cells in response to TCR stimulation. This observation was found across all T cell subsets in the GEO datasets and suggests there is a common mechanism in T cells that allows for viral entry and integration in non-expressing genes. Our study has clearly shown that how latency is established is a critical factor affecting how latency is maintained or reversed in response to LRAs. Understanding the relationship between chromatin status of the genes that are never expressed during activation of T cells and establishment of infection or latency is of interest for designing strategies to induce the expression from latency or to permanently silence the virus.
KeywordsHIV; latency; cure
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