| Summary: | Dissertation submitted to the Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of
Master of Science in Medicine.
Johannesburg, 2011 === The 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.
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