The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes
tRNALys,3 as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as
tRNAIle or
tRNAHis, to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3' terminal 18
nucleotides of the cognate
tRNA. However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to
tRNALys,3. To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to
tRNAIle or
tRNAHis. The genomes contain the gene for
xanthine-
guanosine phosphoribosyl 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; successful
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
tRNAIle or
tRNAHis is three- to fivefold less efficient in replication as measured by production of
drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the 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
tRNALys,3. Using an HIV-1 provirus with a PBS complementary to yeast
tRNAPhe, we established a single-round
infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast
tRNAPhe. The results of our studies suggest that the mechanism for selection of the
tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the
tRNA primerthe PBS.