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TitleInhibition of Human Immunodeficiency virus replication through small RNA-induced gene silencing of HIV-1 Tat specific factor 1
AuthorGreen, Victoria Andress
Subjectanti-HIV therapies
SubjectHIV-1 genetics
AbstractPh.D., Faculty of Health Sciences, University of the Witwatersrand, 2011
AbstractThe HIV-­‐1 pandemic continues unabated. Although treatments exist that can substantially alleviate the morbidity and mortality associated with HIV, there is still a need for improved anti-­‐HIV treatments that reduce toxicities and administration frequency and mediate sustained inhibition of viral replication. Given the high mutability and variability of the virus, a strategy that is garnering increasing focus is the targeting of host factors that the virus requires to replicate, so-­‐called HIV-­‐dependency factors (HDFs). It is hoped this will reduce the emergence of viral drug resistance. A number of genome-­‐wide screens have been performed to identify HDFs, although many remain to be validated, particularly in relevant cells lines. An objective of this thesis was to validate three host factors as HDFs, in both TZM-­‐bl reporter and T cell-­‐derived cell lines, and to examine their potential as anti-­‐HIV-­‐1 therapeutic targets through exploitation of the cellular gene silencing pathway, RNA interference (RNAi). These were HIV-­‐1 Tat specific factor 1 (HTATSF1), DEAD (Asp-­‐Glu-­‐Ala-­‐Asp) box polypeptide 3, X-­‐ linked (DDX3X) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 (SMARCB1), selected because they had been previously implicated in HIV-­‐ 1 pathogenesis. The well-­‐characterised HDF, PC4 and SFRS1 interacting protein 1 (PSIP1)/lens epithelium-­‐derived growth factor (LEDGF)/p75, was included in the study as a positive control. Cassettes expressing short hairpin RNAs (shRNAs) targeting the four host proteins were generated, although shRNAs did not suppress endogenous ddx3x mRNA levels. The ability of shRNAs to inhibit HIV-­‐1 replication in the reporter cell line, TZM-­‐bl, was examined. These HeLa-­‐ derived cells are permissive for R5-­‐tropic HIV-­‐1 infection and contain an integrated luciferase gene driven by the viral promoter. shRNAs mediated a dose-­‐dependent inhibition of luciferase activity in cells infected with a HIV-­‐1 subtype B molecular clone and, although production of the viral protein p24 was unaltered, infectious particle production was decreased in cells treated with a shRNA suppressing HTATSF1. Little effect was observed with a shRNA targeting SMARCB1, suggesting that this may not function as an HDF under these conditions. No effect on infectious particle production was seen with the shRNA targeting PSIP1, which was a result of the long half-­‐ life of this protein, highlighting a limitation of using such reporter systems for HDF validation. Importantly, shRNAs were not associated with any cytotoxic effects in TZM-­‐bl cells. Whether HTATSF1 is a potential therapeutic target was interrogated further in the more relevant T cell-­‐derived SupT1 cell line. Lentiviruses were used to generate populations where >90% had one copy of the integrated shRNA expression cassette. Replication of the subtype B molecular clone p81A-­‐4 was significantly inhibited in the shH1-­‐expressing SupT1 cell line, which targets HTATSF1, for over 14 days post-­‐infection, although inhibition was not as pronounced asthat observed in the shP1-­‐expressing SupT1 cell line, which targets PSIP1. In contrast to a previous report, no change in the ratio of unspliced to singly-­‐ or multiply-­‐spliced HIV-­‐1 transcripts were detected in shH1-­‐expressing SupT1 cells, suggesting that HTATSF1 does not function as a splicing cofactor in this system. A slight rebound in p24 levels at 14 days post-­‐infection was accompanied by increased HTATSF1 expression and a decrease in the percentage of cells with transgene expression in the population. In addition, there was a slight decrease in shH1-­‐derived guide strand expression, but no change in transcription rates of the htatsf1 gene, suggesting that cells within the population with shH1 expression and HTATSF1 suppression may have a growth disadvantage. Thus, although this work demonstrates for the first time that HTATSF1 functions as an HDF in T cell-­‐derived SupT1 cells, it may not constitute a viable therapeutic target. A second objective of this thesis was to examine the feasibility of transcriptional gene silencing (TGS) of HDFs as an anti-­‐HIV strategy. TGS is a small RNA-­‐induced gene silencing pathway that operates through chromatin remodelling with the potential to mediate long-­‐term silencing of gene expression. Thus, its application may reduce the frequency of drug administration and associated toxicities. Short interfering RNAs (siRNAs) targeting the htatsf1 promoter were able to reduce target mRNA expression, which was accompanied by decreased htatsf1 transcription rates in HEK293T cells, suggesting silencing via a TGS mechanism. The htatsf1 silencing inhibited infectious HIV-­‐1 particle production from TZM-­‐bl cells. This work provides proof of principle that TGS induction at a HDF may inhibit HIV-­‐1 replication. siRNAs targeting the ddx3x promoter did not induce TGS. To examine whether gene susceptibility to TGS may be influenced by promoter architectures, 49 promoter features were examined for enrichment in genes at which small RNA-­‐induced TGS has been reported. Initially, the TGS group was compared to a random set of 2,000 promoters and then all other promoters in the genome. To control for gene activation, two further analyses were performed comparing the TGS group features to those from promoters active in the THP-­‐1 cell line and housekeeping genes. Whilst difficult to ascribe differences between the TGS group and the control groups to anything beyond a variation in the proportion of active genes within each group, there was enrichment for certain promoter features that are independent of activity; the TGS group was characterised by broad transcription start regions, high CpG content and a single expression profile. Moreover, the fraction of promoters with reported non-­‐coding RNA overlap was greater in the TGS group than the control groups. Thus, there is some evidence that a number of promoter features are associated with TGS susceptibility. It is hoped this novel analysis will facilitate selection of future TGS targets, including HDFs. In summary, the work presented in this thesis paves the way for development of improved anti-­‐HIV therapies involving HDF-­‐targeted TGS-­‐based gene therapies that mediate sustained inhibition of the virus.