3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Generation of soluble, catalytically active covalent HIV-1 subtype C integrase-DNA complexes to identify novel strand transfer inhibitors(2012) Beyleveld, Grant JamesThe HIV-1 integrase (IN) enzyme is an integral part of the viral replication cycle and has no known human homologues, making it an ideal target for antiretroviral therapy. To date, only one inhibitor of IN strand transfer activity (Raltegravir, IsentressTM) is available for human use. However, the inevitable emergence of antiretroviral drug resistance requires ongoing research into new/novel therapies. There are currently no assays to screen for IN inhibitors against HIV-1 subtype C in South Africa (and worldwide), therefore, the overall objective of this study was to generate and characterize locally relevant, soluble, functional recombinant HIV-1 subtype C IN proteins for use in strand transfer assays. Recombinant integrase genes, including a soluble HIV-1 subtype C mutant (05ZAFV6 with C56S, C65S, W131D, F185D and C280S) and HIV-1 subtype C Y143C mutant (05ZAFV6 soluble with Y143C) were designed, generated and cloned in frame into pET15b. Optimal bacterial expression conditions for the expression of these constructs as well as an HIV-1 subtype C wild type (05ZAFV6), subtype B wild type (NL4-3), and subtype B soluble (NL4-3 with F185K and C280S; as controls) IN, in E.coli BL21 cells were determined. All five recombinant IN were successfully purified using nickel affinity chromatography, and subsequently used to establish a strand transfer assay to assess their activity and their response to two well-known integrase inhibitors, L-Chicoric acid and Raltegravir. All five recombinant IN proteins were found to be biologically active, with INY143C (116.67%) showing equivalent activity to INBwt (117.37%), while INCsol (52.96%) was the lowest. The IC50 values of L-Chicoric acid were higher than the expected values for all five recombinant IN, with the subtype B and C IN solubility mutations contributing to an increased resistance to inhibition by L-Chicoric acid. The dose responses to Raltegravir for INCwt and INBsol were as expected, with IC50’s in line with published data, and the INY143C mutant (known mutation conferring resistance to Raltegravir) was resistant to inhibition of strand transfer activity at all Raltegravir concentrations tested except the highest (50 μM). Finally, methods to complex the INY143C mutant to thiolated-DNA were evaluated, however definitive data could not be obtained. Future work should focus on optimization of the purification and characterization of the IN-DNA complexes. Overall, this study has led to the establishment of functional strand transfer assays based on HIV-1 subtype C recombinant IN proteins, and established a framework for screening of novel HIV-1 subtype C IN inhibitors.