14th Annual Conference of the British HIV Association 23–25 April 2008, Belfast

GAG-PROTEASE INTERRELATIONSHIPS IN DRUG RESISTANCE AND VIRAL FITNESS

HIV Med 2008; 9(Suppl. 1):3 (abstract no. O9)

CM Parry, A Kohli and D Pillay
University College London, UK, and Health Protection Agency, London, UK

BACKGROUND: Resistance to protease inhibitors leads to reduced fitness and mutations within gag can reconstitute fitness. However commonly used replication capacity assay systems incorporate only partial gag sequence (typically part of p7, p1 and p6) therefore the full effects of gag with its corresponding protease cannot be explored. We have developed a single-cycle virus assay to explore gag-protease interrelationships of plasma derived full-length gag and protease genes.

METHODS: We identified a plasma derived gag-protease sequence from a highly drug experienced patient containing multiple and novel mutations in gag and protease. We inserted gag alone, protease alone or gag and protease genes into our single cycle virus system. Drug susceptibility and replication capacity (RC) have been determined.

RESULTS: Mutations in protease included L24I, L33F, 35QNins, M36L, M46I, I54V, K55R, R57K, I64V, L76V and V82A, and in gag an insertion (116TQins) in p17 Matrix, and cleavage site mutations at p2-p7 (five amino acid changes), p7-p1, p1-p6 and a partial duplication of the PTAPP motif in p6. The resulting recombinant virus demonstrated resistance to protease inhibitors. Expressing the mutant protease with wild-type gag lead to a 95% reduction in RC. Replication was fully recovered by coexpressing protease with the corresponding patient-derived gag gene, and further experiments identified the regions of gag responsible for this compensation.

CONCLUSIONS: Many existing RC assays are limited in an ability to fully explore protease gag interrelationships -important in light of increasing evidence of gag effects on drug susceptibility and replication. We demonstrate a complete restoration of RC for a highly mutated protease by full-length gag, and are able to better identify the determinants of viral fitness.

2008-04-23
O9

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