Kohli, A.; (2008) The development of a HIV vector system to explore drug susceptibility and fitness of clinical isolates. Doctoral thesis , University of London.

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Abstract

Following the identification of a highly resistant protease containing a novel insertion (35QN), together with a series of additional gag mutations. We wished to explore the gag-protease interrelationships, and associated replication capacity deficit/compensation. A replication competent NL4-3 based HIV-1 vector system was developed allowing ligation of full length gag with or without protease derived from patient plasma virus. Drug susceptibility assays were undertaken using the MTT assay, and replication capacity assessed by a GHOST indicator cell line. Recombinant viruses containing the protease alone or protease with full length gag demonstrated reduced susceptibility to Pis. The recombinants containing the gag from the patient isolated virus were shown to have a much reduced replication capacity compared to wild-type, even when cloned with the cognate protease. However, the virus containing the patient virus derived gag and protease, demonstrated a 2-fold increase in resistance to some of the Pis tested. The production of high titred stocks was shown to require multiple reversions in gag. This occurred in both viruses containing this region derived from the original patient virus. The reversion pattern was shown to be dependant on whether or not the recombinant contained the highly resistant protease. The lack of reversions from the input genotype in the protease only virus isolate, suggest that this virus is not growth impaired, which was also confirmed by a growth comparison study. The interaction of protease with the gag demonstrates a co-dependence regarding the evolution under drug selection pressure. The removal of the pressure causes reversion of some, but not all of the gag mutations. The lack of reversion in the protease gene suggests that the virus mutates gag to accommodate the protease. The reversion pattern in gag was particularly evident in the p2 protein and or the p2/p7 cleavage site.

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