Development of an HIV-1 integrase enzyme strand transfer assay

Show simple item record Qasim, Muhammad 2011-11-23T05:09:20Z 2011-11-23T05:09:20Z 2011-11-23
dc.description.abstract The Human Immunodeficiency Virus type 1 (HIV-1) integrase is an essential enzyme required for viral replication. Integrase forms part of an ensemble of proteins known as the preintegration complex and functions by a two-step process. Firstly, the cleaving of the 3’ ends of the viral cDNA genome, known as 3’-end processing. The second step is the insertion of these ends into host DNA by esterification, known as strand transfer. There is no human homologue to integrase which makes it an ideal drug target. However, the strand transfer inhibitor raltegravir is currently the only antiretroviral treatment available that inhibits integrase. The aims of this study were two-fold: firstly to characterise a cohort of South African patients so as to determine the viability of introducing raltegravir as a new treatment option, and secondly, to set up high-throughput integrase inhibitor screening assays (testing integrase enzymatic functionality). An HIV-1 subtype C specific RT-PCR and PCR assay was established for integrase genotyping using 51 integrase inhibitor-naïve patient plasma samples and 22 antiretroviral drug-naive primary viral isolates from South Africa. Seventy-one of the 73 samples were classified as HIV-1 subtype C and two samples were unique AC and CG recombinants in integrase. Amino acid sequence analysis revealed there were no primary mutations (Y143R/C/H, Q148H/R/K, and N155H/S) associated with reduced susceptibility to the integrase inhibitor raltegravir. However, one sample had the T97A mutation, three samples had the E157Q and V165I mutations, and the majority of samples contained the polymorphic mutation, V72I. The expected finding of no major integrase mutations conferring resistance to integrase inhibitors suggests that this new antiretroviral drug class will be effective in our region where HIV-1 subtype C predominates. However, the impact of E157Q and other naturally occurring polymorphisms warrants further phenotypic investigation. The integrase sequence of viral isolate, FV3, was closest to the consensus sequence, and thus chosen for preintegration complex isolation for use in strand transfer assays. Isolation of preintegration complexes following FV3 infections of several cell lines was unsuccessful as determined by western blot analysis. Subsequently, the focus was changed to isolation of HIV-1 subtype B recombinant integrase and its functional evaluation. Expression of native integrase (INwt) and soluble integrase (INsol) was induced in E. coli, and both proteins were purified by nickel chelating chromatography. The purified recombinant proteins were used to develop three assays to test for strand transfer activity, of which two were successfully established. Furthermore, only INsol showed strand transfer activity in the high-throughput microtitre plate assay and scintillation proximity assay (SPA)-bead strand transfer assay. Activity of INsol was shown to be inhibited by the control compound, chicoric acid with an IC50 of 101.5nM in the high-throughput microtitre plate assay, whereas INsol activity as well as a dose response to chicoric acid with an IC50 of 248.5nM was recorded in the SPA-bead strand transfer assay. Visualization of radiolabelled enzymatic products of strand transfer by polyacrylamide gel electrophoresis of urea sequencing gels was unsuccessful. Overall, the high-throughput microtitre plate and SPA-bead strand transfer assays have been successfully established in our laboratories, and are available to screen compound libraries for potential antiretroviral drug candidates targeting integrase strand transfer. en_US
dc.language.iso en en_US
dc.title Development of an HIV-1 integrase enzyme strand transfer assay en_US
dc.type Thesis en_US

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