Complementarity between 3 ' terminal nucleotides of tRNA and primer binding site is a major determinant for selection of the tRNA primer used for initiation of HIV-1 reverse transcription

The initiation of reverse transcription of human immunodeficiency virus typ e 1 (HIV-1) exclusively utilizes tRNA(Lys.3) as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNA(Ile) or tRN A(His), to Initiate reverse transcription only if the primer binding site ( PBS) was made complementary to the 3' terminal 18 nucleotides of the cognat e tRNA. However, upon in vitro culture, the viruses with a PBS complementar y to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNA(Lys.3). To investigate the process of reversion, we have construct ed defective proviral genomes that contain a PBS complementary to tRNA(Ile) or tRNA(His). The genomes contain the gene for xanthine-guanosine phosphor ibosyl transferase (gpt) in place of env. Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G ) results in viruses that undergo a single round of HIV-1 infection; succes sful infections are scored as cells resistant to the drug mycophenolic acid . Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNA(Ile) or tRNA(His) is three- to fivefold less e fficient in replication as measured by production of drug-resistant cell co lonies compared to the wild-type virus. These viruses predominantly used th e cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNA (Lys.3). Using an HIV-1 provirus with a PBS complementary to yeast tRNA(Phe ), We established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNA(Phe). The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarit y between the tRNA primer and the PBS. (C) 1999 Academic Press.