HIV/AIDS is a global pandemic that poses a great threat especially in Sub-Saharan Africa where the highest population of those infected with the virus is found. It has far reaching medical, socio-economic and scientific implications. The HIV-1 protease enzyme is a prime therapeutic target that has been exploited in an effort to reduce morbidity and mortality. However problems arise from drug toxicity and drug-resistant mutations of the protease which is a motivation for research for new, safer and effective therapies. Evidence exists to show that there are significant genomic differences in Subtype B and C that have a negative effect on the intrinsic binding of inhibitors. It is imperative to look at all perspectives from epidemiological, molecular to the pharmacological ones so as to achieve rational design of therapeutic agents. This study involved the use of in silico structural analysis of the effects of mutations in the active site. The data was provided by the National Institute of Communicable Diseases consisting of HIV-1 Subtype C protease sequences of 29 infants exhibiting drug-resistance to ritonavir and lopinavir. The major active site mutations causing drug resistance identified in this study were M46I, I54V and V82A using the Stanford HIV database tool. Homology modeling without extra restraints produced models with improved quality in comparison to those with restraints. MetaMQAPII results differed when models were visualized as dimers giving erroneous modeled regions in comparison to monomers. A broader study with a larger dataset of HIV-1 subtype C protease sequences is required to increase statistical confidence and in order to identify the pattern of drug resistant mutations. Homology modeling without extra restraints is preferred for calculating homology models for the HIV-1 subtype C. Further investigations needs to be done to ascertain the accuracy of validation results for dimers from MetaMQAPII as it is designed for evaluation of monomers.

M.Sc. / Proteinases are involved in many cellular reactions involving protein degradation, such as degradation of storage proteins and protein degradation during senescence processes. Their action can be inhibited by proteinase inhibitors. Information is still limited about the regulation of these inhibitors in plants and their possible interaction with proteinases under stress conditions. To obtain a better understanding of the physiological role of a proteinase inhibitor in plants under stress, the expression of a papaya cysteine proteinase inhibitor (cystatin) and its relation to proteinase expression was investigated in more detail. For this purpose, expression of the inhibitor was studied in papaya plants exposed to different physiological stress conditions, such as high/low temperature, and treatment with selected chemicals, such as glutathione, OTC (L-2- Oxothiazolidine-4-carboxylate), bestatin ([(2S, 3R)-3-amino-2-hydroxy-4-phenyl butanoylj-L-leu) and 2.4-D (2,4-dichiorophenoxyacetic acid). Using detection tools like activity gel electrophoresis, immunoblotting and enzymatic assays, the production of the cystatin under stress was monitored in different papaya explants, such as roots, leaves and embryos. Inhibitor production increased under different stress conditions when compared to untreated controls. However, this increase was not dramatic in any of the stresses applied. Exact quantification of the increase by using immunoblotting as the only specific tool to determine cystatin expression, was difficult. Neither activity gel electrophoresis nor enzymatic assays were successful to further quantify the exact cystatin levels. Higher cystatin expression was accompanied with a decrease in proteinase activity. Transgenic tobacco plants carrying the gene for a rice cystatin had a significantly lower cysteine proteinase activity when compared to non-transgenic tobacco plants after prolonged cold stress. Furthermore, protein degradation and leaf yellowing as a consequence of cold treatment were prevented in transgenic plants. An attempt to obtain a transformed papaya plant to study silencing of cystatin expression under stress was unsuccessful. In this study, the protective role of a cystatin in cold stress was described for the first time.

Despite the current interventions to avert contagions and AIDS-related deaths, sub-Saharan Africa is still the region most severely affected by the HIV/AIDS pandemic, where clade C is the dominant circulating HIV-1 strain. The pol-encoded HIV-1 protease enzyme has been extensively exploited as a drug target. Protease inhibitors have been engineered within the framework of clade B, the commonest in America, Europe and Australia. Recent studies have attested the existence of sequence and catalytic disparities between clades B and C proteases that could upset drug susceptibilities. Emergence of drug-resistant associated mutations and combinatorial explosions due to recombination thwarts the attempt to stabilize the current highly active antiretroviral therapy (HAART) baseline. The project aimed at identifying the structural and molecular mechanisms hired by mutants to affect the efficacies of both FDA approved and Rhodes University (RU)-synthesized inhibitors, in order to define how current and or future drugs ought to be modified or synthesized with the intent of combating drug resistance. The rationale involved the generation of homology models of the HIV-1 sequences from the South African infants failing treatment with two protease inhibitors: lopinavir and ritonavir (as monitored by alterations in surrogate markers: CD4 cell count decline and viral load upsurge). Consistent with previous studies, we established nine polymorphisms: 12S, 15V, 19I, 36I, 41K, 63P, 69K, 89M, and 93L, linked to subtype C wild-type; some of which are associated with protease treatment in clade B. Even though we predicted two occurrence patterns of M46I, I54V and V82A mutations as V82A→I54V→M46I and I54V→V82A→M46V, other possibilities might exist. Mutations either caused a protracted or contracted active site cleft, which enforced differential drug responses. The in silico docking indicated susceptibility discordances between clades B and C in certain polymorphisms and non-polymorphisms. The RU-synthesized ligands displayed varied efficacies that were below those of the FDA approved protease inhibitors. The flaps underwent a wide range of structural motions to accommodate and stabilize the ligands. Computational analyses unravelled the need for these potential drugs to be restructured by (de novo) drug engineers to improve their binding fits, affinities, energies and interactions with multiple key protease residues in order to target resilient HIV-1 assemblages. Accumulating evidences on contrasting drug-choice interpretations from the Stanford HIVdb should act as an impetus for the customization of a HIVdb for the sub-Saharan subcontinent.