Human immunodeficiency virus uses tRNALys,3 as primer for reverse transcription in HeLa‐CD4+ cells

Abstract

Significant amounts of different tRNA molecules are present in retroviral particles, but one specific tRNA species functions as primer in reverse transcription. It is generally believed that the HIV-1 virus uses the tRNALys,3 molecule as primer. This is based on sequence complementarity between the 3' end of tRNALys,3 and the primer-binding site (PBS) on HIV-1 genomic RNA. Recent biochemical analyses indicated that tRNALys,3 is indeed incorporated into viral particles. Interestingly, tRNALys,3 could not be detected in virions produced by HeLa-CD4+ cells [(1992) Biochem. Biophys. Res. Commun. 185, 1105-1115]. In order to test whether alternative tRNA molecules can function as primer in HIV replication, we performed a series of experiments based on the observation that tRNA primer sequences are inherited by the viral progeny. We cultured HIV-1 for prolonged periods of time in HeLa-CD4+ cells, but did not detect sequence changes in the PBS region. Furthermore, we found PBS-mutants to be replication incompetent, again suggesting that HIV-1 solely uses tRNALys,3 as primer. Most importantly, we obtained revertants of one such PBS-mutant, which had restored a wild-type PBS sequence. This tRNALys,3-mediated repair demonstrates a general requirement for this primer in HIV-1 reverse transcription