The influence of retroviral codon usage on the acquisition of the tRNA used to prime reverse transcription

An essential step in retrovirus replication is the conversion of the genomic-length viral RNA into a DNA copy, a reaction which is catalyzed by reverse transcriptase and primed by a host cell tRNA. The nearly continuous association of tRNA with the various components of translation, as well as the severely limited time in which a tRNA molecule spends in an uncharged state, constitute seemingly formidable barriers to the acquisition and use of host cell tRNA as primers. The mechanism by which retroviruses acquire this implausible primer are unknown.

Naturally occurring isolates of most retrovirus species show a conserved use of specific tRNA species. As examples, human immunodeficiency virus type 1 (HIV-1) and avian leucosis virus (ALV) use tRNA^Lys,3 and tRNA^Trp, respectively. Laboratory strains of these viruses that are made to use alternate tRNAs as primers by site-directed mutagenesis of the primer-binding site (PBS) replicate more slowly than do the parent viruses, and tend to revert back to the wild-type sequence. We investigated a third, divergent retrovirus, murine leukemia virus (MuLV) for its tolerance of alternate PBS sequences and found that, as with HIV-1 and ALV, this virus exhibits strong preferences for certain sequences, with the wild-type PBS most preferred.

We also demonstrated that retroviruses prefer the use of translationally competent tRNA, indicating that primers are not obtained from an unconventional pool of tRNA but rather that primer acquisition is coordinated with translation. We noted a coincidence between the tRNA species preferred for use as primers by HIV-1 and MuLV and the codon usage near the end of the gag open reading frame (ORF), a region associated with ribosome stalling. We therefore modified HIV-1's codon usage in this region and found that tRNA^Lys1,2 -decoded codons strongly enhance the use of tRNA^Lys1,2 as a primer. This suggests that retroviruses obtain their primers from the subset of tRNA molecules exiting the ribosomes that are used in the translation of the viral RNA. Based on these genetic analyses of MuLV and HIV-1, the primer capture is probably coordinated with the translation of the Gag-Pol polyprotein.