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ItemPathways to HIV latency and reactivation in vitroRezaei, Simin Dokht ( 2018)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.
ItemPermanent silencing of HIV transcription using triplex forming oligonucleotides: a novel strategy for an HIV cureLiu, Haoming ( 2018)Combination antiretroviral therapy (cART) for HIV infection has significantly reduced morbidity and mortality, however, treatment is lifelong. The main barrier to a cure for HIV is the persistence of long lived latently infected T-cells. Virus can integrate in the host genome and be transcriptionally silenced however, upon reactivation of transcription virus can re-emerge from these latently infected cells. In individuals on ART, reactivation of virus goes undetected but once ART is stopped, reactivation of virus leads to virus replication and rebound. One strategy to eliminate virus rebound after cessation of ART is to permanently silence HIV transcription. Here we explore an alternative approach to silence HIV transcription in CD4+ T cells using triplex formation oligonucleotides (TFO). We hypothesize that TFOs can bind irreversibly to the integrated provirus in a sequence specific manner with limited off-target effects. We assessed TFO activity against the green fluorescent protein (GFP) and HIV in vitro by using uninfected and latently infected cell lines and determined the effects of gold nanoparticles to enhance nuclear localization.
ItemFollowing the HIV-1 RNA footprint in cells with latent provirus: reversing silent infection through TatLee, Michelle ( 2018)Globally, the HIV-1 epidemic remains robust and the size of the infected population continues to grow, particularly in sub-Saharan Africa. Although viral suppression is achieved through administration of cART, therapy is lifelong. A compartment of cells that carry HIV-1 in a transcriptionally inactive state, but which retains replicative potential, persists in infected individuals and re-emerges to seed infection when treatment is interrupted. Use of latency reversing agents for perturbation of this reservoir has been shown to be ineffective in the clinical context. This stresses the need for the development of more refined approaches to reactivate latent infection. Multiple layers of repression are present in the cell and at the latent HIV-1 promoter. A central aspect of HIV replication that is blocked during latency is the process of transcriptional elongation. In productive infection, this stage of transcription is enhanced by the action of the viral protein, Tat. Hence, reactivation of latency may be possible through inducing the expression of Tat in a latently infected cell. From the pre-existing DNA template in cells with silenced proviral genomes, tat sequences can be transcribed by a process independent of the 5’ LTR. Generation of readthrough transcripts containing sequences of both human and viral origin is a consequence of HIV-1’s propensity to integrate into introns of transcriptionally active genes. These transcripts are an HIV-1 RNA footprint that may provide the means for expression of Tat in latently infected cells. Here, the HIV-1 RNA footprint in the CCL19-induced primary cell model of HIV-1 latency and in ex vivo samples from individuals on suppressive therapy was studied using target enrichment and next-generation sequencing technologies. Chimeric cellular:tat mRNAs were detected in the primary cell model that had some stretches of tat sequence incorporated in a variety of different configurations. A subset of these have the potential to translate Tat in their original unfragmented forms. In addition, as expected for the generation of readthrough transcripts, HIV-1 integration was predominantly parallel to the human gene, although a small proportion was attributed to the convergent orientation. No cellular:tat mRNAs were detected in the ex vivo samples, however, 3’ LTR activation and the use of the splice donor 1 (SD1) site were the major mechanisms leading to the generation of chimeras. The restrictive context of incorporation of tat sequences into a chimeric cellular:HIV transcript would impede translation through canonical 5’ cap-dependent ribosome scanning modalities. An internal ribosome entry site (IRES) located within Tat encoding sequences has been described and its properties in the context of chimeric cellular:tat mRNA was investigated. Robust, but low-level expression of Tat from an IRES-dependent mechanism was observed using luciferase-based assay systems, and a correspondingly weak reactivation of viral production from the J-Lat10.6 T-cell line model of HIV-1 latency was detected. In addition, SRP14 and HMGB3, two cellular RNA-binding proteins, are putative co-factors of Tat IRES translation detected by affinity purification-mass spectrometry, were shown to be positive and negative regulators of Tat expression respectively and may have roles in the regulation of HIV replication. In cells with quiescent proviruses this novel pathway of Tat expression could be targeted as part of a more biologically relevant combinatorial strategy for reversing latency.