Creation of the HIV-1 antisense gene asp coincided with the emergence of the pandemic group M and is associated with faster disease progression

: Despite being first identified more than three decades ago, the antisense gene asp of HIV-1 remains an enigma. asp is present uniquely in pandemic (group M) HIV-1 strains, and it is absent in all non-pandemic (out-of-M) HIV-1 strains and virtually all non-human primate lentiviruses. This suggests that the creation of asp may have contributed to HIV-1 fitness or worldwide spread. It also raises the question of which evolutionary processes were at play in the creation of asp. Here, we show that HIV-1 genomes containing an intact asp gene are associated with faster HIV-1 disease progression. Furthermore, we demonstrate that the creation of a full-length asp gene occurred via the evolution of codon usage in env overlapping asp on the opposite strand. This involved differential use of synonymous codons or conservative amino acid substitution in env that eliminated internal stop codons in asp, and redistribution of synonymous codons in env that minimized the likelihood of new premature stops arising in asp. Nevertheless, the creation of a full-length asp gene reduced the genetic diversity of env. The Luria-Delbruck fluctuation test suggests that the interrupted asp open reading frame (ORF) is the progenitor of the intact ORF, rather than a descendant under random genetic drift. Therefore, the existence of group-M isolates with a truncated asp ORF indicates an incomplete transition process. For the first time, our study links the presence of a full-length asp ORF to faster disease progression, thus warranting further investigation into the cellular processes and molecular mechanisms through which the ASP protein impacts HIV-1 replication, transmission, and pathogenesis.IMPORTANCEOverlapping genes engage in a tug-of-war, constraining each other's evolution. The creation of a new gene overlapping an existing one comes at an evolutionary cost. Thus, its conservation must be advantageous, or it will be lost, especially if the pre-existing gene is essential for the viability of the virus or cell. We found that the creation and conservation of the HIV-1 antisense gene asp occurred through differential use of synonymous codons or conservative amino acid substitutions within the overlapping gene, env. This process did not involve amino acid changes in ENV that benefited its function, but rather it constrained the evolution of ENV. Nonetheless, the creation of asp brought a net selective advantage to HIV-1 because asp is conserved especially among high-prevalence strains. The association between the presence of an intact asp gene and faster HIV-1 disease progression supports that conclusion and warrants further investigation.