Analyses of HIV proteases variants at the threshold of viability reveals relationships between processing efficiency and fitness

Abstract

Investigating the relationships between protein function and fitness provides keys for understanding biochemical mechanisms that underly evolution. Mutations with partial fitness defects can delineate the threshold of biochemical function required for viability. We utilized a previous deep mutational scan of HIV-1 protease (PR) to identify variants with 15-45% defects in replication and analyzed the biochemical function of eight variants (L10M, L10S, V32C, V32I, A71V, A71S, Q92I, Q92N). We purified each variant and assessed the efficiency of peptide cleavage for three cut sites (MA-CA, TF-PR, PR-RT) as well as a gel-based analyses of processing of purified Gag. The cutting activity of at least one site was perturbed relative to WT protease for all variants, consistent with cutting activity being a primary determinant of fitness effects. We examined the correlation of fitness defects with cutting activity of different sites. MA-CA showed the weakest correlation (R2=0.02) with fitness, suggesting relatively weak coupling with viral replication. In contrast, cutting of the TF-PR site showed the strongest correlation with fitness (R2=0.53). Cutting at the TF-PR site creates a new PR protein with a free N-terminus that is critical for activity. Our findings indicate that increasing the pool of active PR is rate limiting for viral replication making this an ideal step to target with inhibitors.

Systematic analyses of the resistance potential of drugs targeting SARS-CoV-2 main protease

“…Subsequently, compensatory mutations arise that rescue the enzyme defects caused by primary mutations; occasionally such mutations appear to also contribute to drug resistance both in HIV and SARS-CoV-2 in cell culture (Iketani et al 2022b; Zhou et al 2022; Kozisek et al 2007; Ragland et al 2017). In principle, mutations that increase enzymatic activity should provide a small growth advantage in the presence of drugs, and this might be amplified for viral proteases such as M pro which must cleave themselves out of polyproteins to generate additional active protease molecules (Schneider-Nachum et al 2021). The highest level of drug resistance observed in HIV protease and in SARS-CoV-2 M pro appears to arise from a combination of primary mutations that disrupt drug binding and compensatory mutations that increase enzyme activity.…”

Sequence dependencies and biophysical features both govern cleavage of diverse cut-sites by HIV protease

“…The wide range of substrate cleavage rates led to the suggestion that the order of cleavage may be important for viral maturation [20, 21]. Recent studies [18, 22] indicate that the cutting rate of many sites can be altered dramatically without compromising fitness. However, the relationship between viral fitness and cutting efficiency has not been directly assessed for most cut sites.…”

Sequence dependencies and biophysical features both govern cleavage of diverse cut‐sites by HIV protease

“…19,20 The wide range of substrate cleavage rates led to the suggestion that the order of cleavage may be important for viral maturation. 21,22 Recent studies 19,23 indicate that the cutting rate of many sites can be altered dramatically without compromising fitness. However, the relationship between viral fitness and cutting efficiency has not been directly assessed for most cut sites.…”