Changes in Human Immunodeficiency Virus Type 1 Gag at Positions L449 and P453 Are Linked to I50V Protease Mutants In Vivo and Cause Reduction of Sensitivity to Amprenavir and Improved Viral Fitness In Vitro

Abstract

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1F) and P453L (p1/p6 PP5L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single-and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity Ϸ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

Generation of Infectious Feline Immunodeficiency Virus (FIV) Encoding FIV/Human Immunodeficiency Virus Chimeric Protease

“…However, efficient processing at the NC-p2 junction to produce mature NC and p2 is required for FIV infectivity. The processing efficiency of the equivalent HIV-1 NC-p1-p6 cleavage junctions has been extensively examined, and proper temporal cleavage at these sites correlates with viral fitness, replication capacity, and drug resistance (11,40,45,47,49,52). The findings of the current study reinforce the notion that the cleavage and maturation of NC-p2 in FIV and of the equivalent NC-p1-p6 in HIV-1 offer a new potential therapeutic target.…”

Quantifying the Impact of Bacterial Fitness and Repeated Antimicrobial Exposure on the Emergence of Multidrug-Resistant Gram-Negative Bacilli

“…Numerous studies supporting the acquisition of fitness-compensatory mutations have been published and recently reviewed by Maisner-Patin and Andersson [1,23]. Among the most clinical relevant reductions in fitness cost associated with antimicrobial resistance include isoniazid-resistant Mycobacterium tuberculosis [33], protease inhibitors-resistant HIV-1 strains [22], azole-resistant Candida albicans [7], and clarythromycin-resistant Helicobacter pylori [4].…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…In an analysis of a clinical sequence database, the L449F and P453L mutations at the p1/p6 cleavage site in Gag were frequently observed in combination with the major protease inhibitor resistance mutation I50V, although the two cleavage site mutations were not observed together in individual clones derived from patient samples (107). These mutations each partially improved the replication fitness of HIV-1 strains containing the I50V mutation and increased the degree of protease inhibitor resistance; no effect on protease inhibitor resistance was seen when these mutations were introduced into a drug-sensitive laboratory strain (107).…”

“…These mutations each partially improved the replication fitness of HIV-1 strains containing the I50V mutation and increased the degree of protease inhibitor resistance; no effect on protease inhibitor resistance was seen when these mutations were introduced into a drug-sensitive laboratory strain (107). In the absence of these mutations, the I50V mutant accumulated uncleaved p1/p6 protein; this abnormality was partially corrected by the addition of one of the cleavage site mutations (107). Those studies also confirmed that purified protease containing the I50V mutation cleaved a mutant p1/p6 cleavage site more efficiently than a wild-type cleavage site (107).…”

“…In the absence of these mutations, the I50V mutant accumulated uncleaved p1/p6 protein; this abnormality was partially corrected by the addition of one of the cleavage site mutations (107). Those studies also confirmed that purified protease containing the I50V mutation cleaved a mutant p1/p6 cleavage site more efficiently than a wild-type cleavage site (107). Positive and negative associations between specific cleavage site and major protease-inhibitor resistance mutations were also observed in another study of treatment-experienced patients (170).…”