Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011 / The 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.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Background: Despite the effort of scientific research on HIV therapies and to reduce the rate of HIV infection, AIDS remains one of the major causes of death in the world and mostly in sub-Saharan Africa. To date, neither a cure nor an HIV vaccine had been found and the disease can only be managed by using High Active Antiretroviral Therapy (HAART) if detected early. The need for an effective early diagnostic and non-toxic treatment has brought about the necessity for the discovery of additional HIV diagnostic methods and treatment regimens to lower mortality rates. Antimicrobial Peptides (AMPs) are components of the first line of defense of prokaryotes and eukaryotes and have been proven to be promising therapeutic agents against HIV. Methods: With the utility of computational biology, this work proposes the use of profile search methods combined with structural modeling to identify putative AMPs with diagnostic and anti-HIV activity. Firstly, experimentally validated anti-HIV AMPs were retrieved from various publicly available AMP databases, APD, CAMP, Bactibase and UniProtKB and classified according to super-families. Hidden Markov Model (HMMER) and Gap Local Alignment of Motifs (GLAM2) profiles were built for each super-family of anti- HIV AMPs. Putative anti-HIV AMPs were identified after scanning genome sequence databases using the trained models, retrieved AMPs, and ranked based on their E-values. The 3-D structures of the 10 peptides that were ranked highest were predicted using 1-TASSER. These peptides were docked against various HIV proteins using PatchDock and putative AMPs showing the highest affinity and having the correct orientation to the HIV -1 proteins gp120 and p24 were selected for future work to establish their function in HIV therapy and diagnosis. Results: The results of the in silica analysis showed that the constructed models using the HMMER algorithm had better performances compare to that of the models built by the GLAM2 algorithm. Furthermore, the former tool has a better statistical and probability explanation compared to the latter tool. Thus only the HMMER scanning results were considered for further study. Out of 1059 species scanned by the HMMER models, 30 putative anti-HIV AMPs were identified from genome scans with the family-specific profile models after the elimination of duplicate peptides. Docking analysis of putative AMPs against HIV proteins showed that from the 10 best performing anti-HIV AMPs with the highest E-scores, molecules 1,3, 8, and 10 firmly bind the gp120 binding pocket at the VIN2 domain and the point of interaction between gp120 and T cells, with the 1st and 3rd highest scoring anti-HIV AMPs having the highest binding affinities. However, all 10 putative anti-HIV AMPs bind to the N-terminal domain of p24 with large surface interaction, rather than the C-terminal. Conclusion: The in silica approach has made it possible to construct computational models having high performances, and which enabled the identification of putative anti-HIV peptides from genome sequence scans. The in silica validation of these putative peptides through docking studies has shown that some of these AMPs may be involved in HIV/AIDS therapeutics and diagnostics. The molecular validation of these findings will be the way forward for the development of an early diagnostic tool and as a consequence initiate early treatment. This will prevent the invasion of the immune system by blocking the VIN2 domain and thus designing of a successful vaccine with broad neutralizing activity against this domain.