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 [ICs] 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 PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs ( < 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. Large-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a virus's position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease affects the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and a virus with recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotypes were indistinguishable between the recoded virus and the WT virus, suggesting that the HIV-1 strain with synonymously recoded protease and the WT virus are equally robust and evolvable.

HIV-1 Lethality and Loss of Env Protein Expression Induced by Single Synonymous Substitutions in the Virus Genome Intronic-Splicing Silencer

“…Prior studies have documented the relevance of codon usage on virus phenotypes and life cycles (2). Synonymous substitutions were shown to have strong impacts on HIV-1 gag, pol, and env expression and the virus phenotype (5,7,12,13). Synonymous codon changes can produce unexpected outcomes by changing the structure of the mRNA or the protein.…”

“…The designed mutant was considered lethal when p24 was not detected by 30 days posttransfection. Virus titration was performed in MT-4 cells, and values were expressed in terms of the tissue culture dose for 50% infectivity (TCID 50 ), as previously described (13).…”

Impact of Synonymous Genome Recoding on the HIV Life Cycle

“…Under the presence of a viral inhibitor, the number of mutations found in the synonymous-recoded virus is higher than those generated in the WT virus. Moreover, these mutations differ from both viral mutant spectra, indicating that the sequence space influences the development of inhibitor resistances (Nevot et al, 2018).…”

Synonymous genome recoding: a tool to explore microbial biology and new therapeutic strategies

“…More recently, synonymous virus genome recoding has been used to investigate how the HIV-1 position in sequence space defines its mutant spectrum. To this end, a synthetic virus was generated carrying a reengineered protease sequence including 13% of synonymous mutations (137). WT and recoded viruses were propagated in the presence of HIV-1 protease inhibitors (PIs).…”