A thesis submitted to the Faculty of Science, University of the Witwatersrand,
Johannesburg in fulfilment of the requirements for the degree of Doctor of
Philosophy. Johannesburg, 28 May 2018. / Since their discovery, protease inhibitors continue to be an essential component of
antiretroviral treatment for human immunodeficiency virus type 1 (HIV-1). However, the
development of resistance to protease inhibitors remains one of the most significant challenges
in the fight for sustained viral suppression in those infected with HIV-1. Studies show that
specific mutations arising within the HIV-1 gag and protease genes can lead to the
development of resistance. In this research, a South African HIV-1 subtype C Gag-protease
variant (W1201i) was investigated. This variant was considered due to the presence of a
mutation and insertion (N37T↑V), located within the hinge region of the protease enzyme.
Moreover, the variant displayed the following polymorphisms: Q7K, I13V, G16E, M36T,
D60E, Q61E, I62V and M89L. Genotyping of W1201i Gag revealed a previously unreported
MSQAG insertion between the CA/p2 and p2/NC cleavage sites. Additionally, a mutation and
insertion (I372L↑M), and multiple polymorphisms (S369N, S371N, I373M and G377S) were
discovered within the p2/NC cleavage site. Single-cycle phenotypic assays were performed to
determine the drug susceptibility and replication capacity of the variant. The results show that
the mutations present in the N37T↑V protease conferred a replicative advantage and reduced
susceptibility to lopinavir, atazanavir and darunavir. Interestingly, the mutations in W1201i
Gag were found to modulate both replication capacity and protease inhibitor susceptibility.
In silico studies were performed to understand the physical basis for the observed variations.
Molecular dynamics simulations showed that the N37T↑V protease displayed altered dynamics
around the hinge and flap region and highlighted the amino acids responsible for the observed
fluctuations. Furthermore, induced fit docking experiments showed that the variant bound the
iv
protease inhibitors with fewer favourable chemical interactions than the wild-type protease.
Collectively, these data elucidate the biophysical basis for the selection of hinge region
mutations and insertions by the HI virus and show that protease, as well as Gag, needs to be
evaluated during resistance testing. / EM2018
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/25929 |
| Date | January 2018 |
| Creators | Zondagh, Jake |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | Online resource (139 leaves), application/pdf |
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