Interplay between Single Resistance-Associated Mutations in the HIV-1 Protease and Viral Infectivity, Protease Activity, and Inhibitor Sensitivity

Abstract

Resistance-associated mutations in the HIV-1 protease modify viral fitness through changes in the catalytic activity and altered binding affinity for substrates and inhibitors. In this report, we examine the effects of 31 mutations at 26 amino acid positions in protease to determine their impact on infectivity and protease inhibitor sensitivity. We found that primary resistance mutations individually decrease fitness and generally increase sensitivity to protease inhibitors, indicating that reduced virion-associated protease activity reduces virion infectivity and the reduced level of per virion protease activity is then more easily titrated by a protease inhibitor. Conversely, mutations at more variable positions (compensatory mutations) confer low-level decreases in sensitivity to all protease inhibitors with little effect on infectivity. We found significant differences in the observed effect on infectivity with a pseudotype virus assay that requires the protease to cleave the cytoplasmic tail of the amphotropic murine leukemia virus (MuLV) Env protein. Additionally, we were able to mimic the fitness loss associated with resistance mutations by directly reducing the level of virion-associated protease activity. Virions containing 50% of a D25A mutant protease were 3-to 5-fold more sensitive to protease inhibitors. This level of reduction in protease activity also resulted in a 2-fold increase in sensitivity to nonnucleoside inhibitors of reverse transcriptase and a similar increase in sensitivity to zidovudine (AZT), indicating a pleiotropic effect associated with reduced protease activity. These results highlight the interplay between enzyme activity, viral fitness, and inhibitor mechanism and sensitivity in the closed system of the viral replication complex.

Inference of epistatic effects leading to entrenchment and drug resistance in HIV-1 protease

“…This work builds upon a large literature, ranging from experimental work (Chang and Torbett 2011;Henderson et al 2012) and statistical analyses of covarying pairs of mutations (Wu et al 2003;Rhee et al 2007) to more advanced statistical models of patterns of mutations at many positions (such as Potts models) (Haq et al 2009(Haq et al , 2012Butler et al 2016), to strengthen our understanding of the emergent properties of drug resistance in HIV-1 protease. We demonstrate that, Position a Consensus ME LE ME LE ME LE…”

“…Experimentally derived values for either melting temperature (T m ) or viral infectivity via replicative capacity (RC) were mined from the results presented in Muzammil et al (2003), van Maarseveen et al (2006), Chang and Torbett (2011), Louis et al (2011), andHenderson et al (2012). A CSV file of the resulting mined data can be found in Supplementary Data 2, Supplementary Material online.…”

“…We have extracted results for viral replicative capacity in which 29 single protease mutants were studied by Henderson et al (2012) and an additional small set of more complex sequence variants (van Maarseveen et al 2006) that were tested relative to the wildtype sequence. As with the stability measurements, we find that the relative Potts energy correlates well with infectivity (r ¼ À0:64; P < 10 À5 ), shown in figure 3B.…”

Multi-step inhibition explains HIV-1 protease inhibitor pharmacodynamics and resistance

“…This cleavage activates the fusion potential of MLV-E. When Env-defective HIV-1 proviruses are pseudotyped with MLV-E, the HIV-1 protease cleaves p15 and renders the envelope fusogenic (12,46). We hypothesized that PIs might inhibit the entry of MLV-E-pseudotyped particles by preventing this essential cleavage.…”

“…While the interaction of PIs with the target enzyme is well understood at the structural and biochemical level (9)(10)(11)(12), it remains unclear where in the virus life cycle the inhibition of virus maturation becomes manifest. Virus maturation is generally considered to be important for early postentry steps including uncoating and reverse transcription (13)(14)(15).…”