Abstract
BackgroundMutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V).ResultsHere, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one.The observed decrease in RC for the double NC/p1 mutant (HXB2436E+437T) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant.ConclusionsThe results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.
Highlights
Mutations in the substrate of Human Immunodeficiency Virus type-1 (HIV-1) protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and compensate for defects in viral replicative capacity (RC) due to a drug resistant protease
We investigated the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and replicative capacity by performing a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V)
Effect of NC/p1 cleavage site mutations on viral RC and Gag processing A set of four HIV-1 HXB2 recombinant virus clones containing NC/p1 resistance mutations described in the literature and conferring different levels of PI resistance was generated: HXB2431V; HXB2436E+437T; HXB2437T and HXB2437V (Figure 1a &1b) [18]
Summary
Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. The evolution of PI resistance has been characterized as a stepwise process in which amino acid changes in the substrate-binding pocket or at more distant sites in the viral PR are selected initially These amino acid changes directly or indirectly reduce the affinity of the viral PR for the inhibitor, thereby causing PI resistance. These amino acid changes affect the binding of the viral PR to its natural substrate, the Gag and GagPol polyproteins, and as a consequence many of these PI resistant variants display a reduced replicative capacity (RC) as compared to wild-type virus [5,6,7,8]. Within the Gag polyprotein, compensatory changes have frequently been observed in the C-terminal region, in particular in the NC/p1 and p1/p6 cleavage sites [8,10,11,12,13,15,16,17]
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