Synonymous Codon Pair Recoding of the HIV-1 <i>env</i> Gene Affects Virus Replication Capacity

Synonymous codon pair deoptimization is an efficient strategy for virus attenuation; however, the underlying mechanism remains controversial. Here, we optimized and deoptimized the codon pair bias (CPB) of the human immunodeficiency virus type 1 (HIV-1) envelope (<i>env</i>) gene to investigate the influence of <i>env</i> synonymous CPB recoding on virus replication capacity, as well as the potential mechanism. We found that <i>env</i> CPB deoptimization did not always generate attenuation, whereas CPB optimization attenuated virus replication in MT-4 cells. Furthermore, virus attenuation correlated with reduced Env protein production but not with decreased viral RNA synthesis. Remarkably, in our model, increasing the number of CpG dinucleotides in the 5′ end of <i>env</i> did not reduce the replication capacity of HIV-1. These results indicate that factors other than CPB or CpG content may have impacted the viral fitness of the synonymously recoded study variants. Our findings provide evidence that CPB recoding-associated attenuation can affect translation efficiency. Moreover, we demonstrated that an increased number of CpGs in the 5′ end of HIV-1 <i>env</i> is not always associated with reduced virus replication capacity.