Impact of p2/NC cleavage site polymorphisms on HIV-1 subtype C viral fitness.
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Date
2012
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Abstract
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.
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Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2012.
Keywords
Viruses., Molecular virology., Virology., Theses--Virology.