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

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 per cent defects in replication and analysed 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, and PR-RT) as well as 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 (R ² = 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 (R ² = 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.