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Development of an HIV-1 intergrase enzyme strand transfer assay

M.Sc.(Med.) (Molecular Medicine and Haematology), Faculty of Health Sciences, University of the Witwatersrand, 2011 / 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.

Identiferoai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/11197
Date30 January 2012
CreatorsFish, Muhammad Qasim
Source SetsSouth African National ETD Portal
LanguageEnglish
Detected LanguageEnglish
TypeThesis
Formatapplication/pdf

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