Changes in codon-pair bias of human immunodeficiency virus type 1 have profound effects on virus replication in cell culture

Background: Human immunodeficiency virus type 1 (HIV-1) has a biased nucleotide composition different from human genes. This raises the question of how evolution has chosen the nucleotide sequence of HIV-1 that is observed today, or to what extent the actual encoding contributes to virus replication capacity, evolvability and pathogenesis. Here, we applied the previously described synthetic attenuated virus engineering (SAVE) approach to HIV-1. Results: Using synonymous codon pairs, we rationally recoded and codon pair-optimized and deoptimized different moieties of the HIV-1 gag and pol genes. Deoptimized viruses had significantly lower viral replication capacity in MT-4 and peripheral blood mononuclear cells (PBMCs). Varying degrees of ex vivo attenuation were obtained, depending upon both the specific deoptimized region and the number of deoptimized codons. A protease optimized virus carrying 38 synonymous mutations was not attenuated and displayed a replication capacity similar to that of the wild-type virus in MT-4 cells and PBMCs. Although attenuation is based on several tens of nucleotide changes, deoptimized HIV-1 reverted to wild-type virulence after serial passages in MT-4 cells. Remarkably, no reversion was observed in the optimized virus. Conclusion: These data demonstrate that SAVE is a useful strategy to phenotypically affect the replicative properties of HIV-1.