Impact of HIV Gag mutations on protease inhibitor resistance
Thursday 17 January 2013
The introduction of HIV-1 protease inhibitors (PI) has been one of the key components in the success of antiretroviral combination therapy. The protease enzyme plays a crucial role in the viral life cycle and is required for the ordered cleavage of the viral precursor Gag and GagPol polyprotein, resulting in mature infectious virus particles. However, the selection of drug resistant viruses is a major problem causing therapy failure in a substantial proportion of HIV-1 infected patients.
During the last decade HIV-1 protease resistance has been documented in the target gene of the inhibitor, the viral protease. Recently, we have identified a novel HIV protease inhibitor resistance mechanism. We show that increased Gag polyprotein processing due to mutations in one particular Gag cleavage site (substrate of the viral protease) causes resistance in the background of wild type HIV-1 protease.
We have indications that this novel protease resistance pathway may have clinical significance. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical database isolates demonstrated the presence of these cleavage site mutations in clinical samples. Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in the absence of protease mutations.
In this study we want to investigate the impact of the Gag substrate on protease resistance in vivo. Therefore, we have selected 20 patients who were being treated and failed multiple PI containing drug regimens. From these patients we will analyse genotypic and phenotypic protease resistance. To do so, the viral Gag and protease genes from the pretherapy and therapy failure plasma samples will be amplified and sequenced. Furthermore the viral protease gene alone and the viral protease gene in combination with the viral gag gene will be cloned in an HIV-1 reference strain to investigate the impact of the viral gag substrate on protease inhibitor resistance. Protease inhibitor resistance will be determined by culturing the recombinant viruses in increasing levels of protease inhibitors. Finally, the mechanism of Gag-related protease inhibitor resistance will be investigated.
Cell culture, Viral culture, Elisa, Viral RNA/DNA isolation, PCR, real-time TaqMan PCR, Sequencing, Cloning
6 or 9 months
Dr M. Nijhuis, M.Nijhuis@umcutrecht.nl
Dr A. Wensing, A.M.J.Wensing@umcutrecht.nl
Website UMC Utrecht - Medical Microbiology