Subtype C accounts for the majority of HIV infections and in South Africa, is the dominant subtype. The Gag cleavage sites of subtype C viruses show a high degree of natural variation compared to subtype B and group M sequences, with the p2/NC site having the highest degree of variation among all cleavage sites and between all subtypes. This study therefore aimed to determine the functional effect of this variation on viral fitness. A library of drug naïve subtype C sequences were screened using computational analysis to predict binding affinity between HIV protease and the Gag substrate at the p2/NC site. Ligands with high predicted affinity had hydrophobic cleavage sites with substantial diversity at positions P5-P3. Lower ranking ligands were mostly similar to the consensus subtype C. Three ligands were selected for fitness assays from each the high ranking and low ranking groups. Chimeric viruses expressing selected cleavage sites were generated by site directed mutagenesis. Replication capacity assays of these viruses showed moderate differences in fitness but failed to demonstrate a correlation with computational estimates of binding affinity. Enzymes assays were performed to further investigate substrate preferences and the binding mechanism of protease. To this end, recombinantly expressed HIV-1 protease was tested against a range of substrates the matching the p2/NC cleavage sites used in the replication capacity assay. Results of the enzyme assay did not correlate with either the computation studies or the replication capacity assay results, suggesting a sequence independent binding and recognition mechanism of HIV-1 protease. Taken together the results suggest that processing of Gag is determined by tertiary folding of the polyprotein and not amino acid sequence at the cleavage site. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2012.

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The replication of CD4+ T cell-tropic retroviruses such as the human immunodeficiency virus-1 (HIV-1) and the feline immunodeficiency virus (FIV) is intricately linked to host cell signalling and activation status. This intimate relationship between the viruses and their respective hosts plays crucial roles in the pathogenesis of each virus. Focusing on FIV, this thesis examined several questions relating to virus replication and cell signalling. Firstly, the possible cell stimulatory effect when FIV Env binds to its primary receptor CD134 was investigated and results suggested that FIV does not trigger CD134 signalling. Next the activation status of susceptible CD4+ T cells was manipulated to study FIV latency and it was shown that by removing exogenous supplement of the cytokine interleukin-2 (IL-2) from MYA-1 feline CD4+ T cells 24 hours before infection, productive FIV replication is down-regulated such that only a very low level of ongoing virus replication can be detected among the IL-2-depleted cells with a sensitive qPCR assay. The phorbol esters PMA and Prostratin can stimulate high level of productive infection from these IL-2-depleted MYA-1 cells and this is mediated by a protein kinase C (PKC) dependent mechanism. Furthermore, productive replication of FIV in the presence of IL-2 is blocked by stimulation of PKC with phorbol esters, which is analogous to findings of similar experiments with HIV-1. However, inhibition of viral replication is not at the level of viral entry, contrary to the findings of HIV-1 studies. The mechanism behind the Prostratin-mediated inhibition of FIV remains elusive. It was also discovered that ‘early’ and ‘late’ strains of FIV responded differently to cell signalling manipulation and an attempt was made to map the viral genome region(s) responsible. Preliminary data showed that both env and the 5’UTR may mediate the inter-strain differences in replication dynamics. Overall this thesis shows the complete dependency of FIV on host cell signalling, in particular optimum PKC activation to achieve productive viral replication. This may reflect the tropism of the virus. The similarities between HIV-1 and FIV replication and latency support the notion of using FIV and the cat as a model for HIV-1 latency in the development of novel therapeutic measures to eradicate hidden HIV-1 from the host. However, more research is required to fully characterise the differences between HIV-1 and FIV biology.

Herpes simplex virus (HSV-1) ICP27 is a multifunctional immediate early (IE) protein, required for expression of several early and late genes and acts at transcriptional and posttranscriptional levels. ICP27 contains both a nuclear localization signal and a nuclear export signal and shuttles between the nucleus and the cytoplasm. It interacts directly with various cellular proteins including REF and cellular mRNA export receptor TAP to export viral mRNAs. However other transport mechanisms may also be employed. All transport across the nuclear envelope must occur via the nuclear pore complex, which consists of proteins called nucleoporins (NUPs). NUPs act to regulate the transport cargos by interaction with mRNA bound proteins and transport receptors, karyopherins. To further explore the transport requirements for ICP27 and viral mRNA export, we investigated its direct interaction with nucleoporins and show that ICP27 interacts directly with nucleoporins tested, in vitro and in wild type and mutant virus infected cells and from RNase I treated and untreated cell extracts indicating that this interaction is not mediated via RNA. Implications of this interaction is discussed in this thesis.

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