17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

GAG NC/P1 PROTEASE RESISTANCE MUTATIONS CAN CAUSE SELECTION OF ADDITIONAL NC/P1 CHANGES TO OPTIMIZE CLEAVAGE EFFICIENCY AND REPLICATIVE CAPACITY

Antivir Ther. 2008; 13(Suppl. 3):A52 (abstract no. 47)

NM van Maarseveen¹, PJ Schipper¹, D de Jong¹, CAB Boucher^1,2 and M Nijhuis^1
1Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands; ²Department of Virology, Erasmus Medical Centre, Rotterdam, The Netherlands

BACKGROUND: Substrate-based protease inhibitor (PI) resistance due to mutations in NC/p1 is caused by an enhanced processing of the gag protein. We investigated the effect of enhanced gag processing due to NC/p1 resistance mutations on replicative capacity (RC) and the consequences for evolution in the absence of PI pressure.

METHODS: A set of four recombinant viruses containing NC/p1 mutations conferring different levels of PI resistance were generated: HXB2^431V, HXB2^437V, HXB2^437T and HXB2^436E+437T. To investigate the effect of enhanced gag processing on RC, viral replication curves were generated. To investigate the potential evolutionary pathways in the absence of PI pressure, multiple individual in vitro evolution experiments were performed, after which complete Gag and protease were sequenced. From the viruses that were selected, RC, gag processing (quantitative immunoblot analysis) and PI susceptibility (MTT assay) were assessed.

RESULTS: Single NC/p1 mutants that displayed only a slight increase in PI resistance did not show an obvious change in RC compared with wild type. This was also reflected in the in vitro evolution experiments where the single NC/p1 mutants showed no signs of evolution, with the exception of the selection of A429K in 1/5 experiments for HXB2^431V. In contrast, the double NC/p1 mutant (HXB2^436E+437T), which displayed a clear increase in processing efficiency and PI resistance, also demonstrated a clear reduction in RC. Interestingly, in all evolution experiments, amino acid changes in/near the NC/p1 site were observed (2/5, -436E; 2/5, +435R; 1/5, +438R). These selected changes restored the processing efficiency and RC. Furthermore, it was observed that due to the normalization of gag processing efficiency, in parallel, PI susceptibility returned to wild-type level.

CONCLUSIONS: The results from this study clearly demonstrate that there is an optimum rate for HIV-1 gag cleavage. When enhanced gag processing due to PI resistance mutations in NC/p1 reduces RC, HIV-1 can modulate the NC/p1 sequence by selection of additional changes to restore gag cleavage and RC.

2008-06-10
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