HIV-1 protease evolvability is affected by synonymous nucleotide recoding

ABSTRACT One unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the levels of development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair-reengineered protease sequence including 38 (13%) synonymous mutations. The WT and MAX viruses showed indistinguishable replication in MT-4 cells or peripheral blood mononuclear cells (PBMCs). Both viruses were subjected to serial passages in MT-4 cells, with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (50% inhibitory concentrations [IC₅₀s] of 14.6 ± 5.3 and 21.2 ± 9 nM, respectively, for ATV and 5.9 ± 1.0 and 9.3 ± 1.9, respectively, for DRV). Ultradeep sequence clonal analysis revealed that both viruses harbored previously described mutations conferring resistance to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in the WT and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in the MAX virus. Remarkably, the G48L and L89I substitutions are rarely found in vivo in PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P < 0.0001), suggesting a higher selective pressure for change in this recoded virus. Our results indicate that the HIV-1 protease position in sequence space delineates the evolution of its mutant spectrum. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to those of the WT virus.