Development of an HIV-1 integrase enzyme strand transfer assay

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

The Human Immunodeficiency Virus type 1 (HIV-1) integrase is an essential \nenzyme required for viral replication. Integrase forms part of an ensemble of \nproteins known as the preintegration complex and functions by a two-step \nprocess. Firstly, the cleaving of the 3’ ends of the viral cDNA genome, known as \n3’-end processing. The second step is the insertion of these ends into host DNA \nby esterification, known as strand transfer. There is no human homologue to \nintegrase which makes it an ideal drug target. However, the strand transfer \ninhibitor raltegravir is currently the only antiretroviral treatment available that \ninhibits integrase. The aims of this study were two-fold: firstly to characterise a \ncohort of South African patients so as to determine the viability of introducing \nraltegravir as a new treatment option, and secondly, to set up high-throughput \nintegrase inhibitor screening assays (testing integrase enzymatic functionality). \nAn HIV-1 subtype C specific RT-PCR and PCR assay was established for integrase \ngenotyping using 51 integrase inhibitor-naïve patient plasma samples and 22 \nantiretroviral drug-naive primary viral isolates from South Africa. Seventy-one of \nthe 73 samples were classified as HIV-1 subtype C and two samples were unique \nAC and CG recombinants in integrase. Amino acid sequence analysis revealed \nthere were no primary mutations (Y143R/C/H, Q148H/R/K, and N155H/S) \nassociated with reduced susceptibility to the integrase inhibitor raltegravir. \nHowever, one sample had the T97A mutation, three samples had the E157Q and \nV165I mutations, and the majority of samples contained the polymorphic \nmutation, V72I. The expected finding of no major integrase mutations conferring \nresistance to integrase inhibitors suggests that this new antiretroviral drug class \nwill be effective in our region where HIV-1 subtype C predominates. However, \nthe impact of E157Q and other naturally occurring polymorphisms warrants \nfurther phenotypic investigation. The integrase sequence of viral isolate, FV3, \nwas closest to the consensus sequence, and thus chosen for preintegration complex isolation for use in strand transfer assays. Isolation of preintegration \ncomplexes following FV3 infections of several cell lines was unsuccessful as \ndetermined by western blot analysis. Subsequently, the focus was changed to \nisolation of HIV-1 subtype B recombinant integrase and its functional evaluation. \nExpression of native integrase (INwt) and soluble integrase (INsol) was induced in E. \ncoli, and both proteins were purified by nickel chelating chromatography. The \npurified recombinant proteins were used to develop three assays to test for \nstrand transfer activity, of which two were successfully established. Furthermore, \nonly INsol showed strand transfer activity in the high-throughput microtitre plate \nassay and scintillation proximity assay (SPA)-bead strand transfer assay. Activity \nof INsol was shown to be inhibited by the control compound, chicoric acid with an \nIC50 of 101.5nM in the high-throughput microtitre plate assay, whereas INsol \nactivity as well as a dose response to chicoric acid with an IC50 of 248.5nM was \nrecorded in the SPA-bead strand transfer assay. Visualization of radiolabelled \nenzymatic products of strand transfer by polyacrylamide gel electrophoresis of \nurea sequencing gels was unsuccessful. Overall, the high-throughput microtitre \nplate and SPA-bead strand transfer assays have been successfully established in \nour laboratories, and are available to screen compound libraries for potential \nantiretroviral drug candidates targeting integrase strand transfer.