NUCLEOTIDE SUBSTITUTIONS WITHIN U5 ARE CRITICAL FOR EFFICIENT REVERSETRANSCRIPTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 WITH A PRIMER BINDING-SITE COMPLEMENTARY TO TRNA(HIS)

Sequence analysis of integrated proviruses of human immunodeficiency v irus type 1 (HIV-1) which utilize tRNA(His) to initiate reverse transc ription [virus derived from pHXB2(His-AC-TGT)] revealed five additiona l nucleotide substitutions in the U5 and primer binding site (PBS) reg ions (ATGAC for CCTGT at nucleotides 152, 160, 174, 181, and 200, resp ectively) (Z. Zhang et al., Virology 226:306-317, 1996). We constructe d a mutant proviral genome [pHXB2(His-AC-GAC)] which contained the ATG AC substitutions to test if they represented a necessary adaptation by the virus for use of tRNA(His) to initiate reverse transcription. Vir uses from pHXB2(His-AC-TGT) and pHXB2(His-AC-GAC) were infectious. Seq uence analysis of the U5 and PBS regions of integrated provirus from a cell culture infected with virus derived from pHXB2(His-AC-TGT) revea led a G-to-A change in CCTGT at nucleotide 181 after limited in vitro culture, suggesting that this nucleotide change represented an adaptat ion by the virus to efficiently utilize tRNA(His) to initiate reverse transcription. To further address this possibility, we used a specific mutation in reverse transcriptase (RT), a methionine-to-valine change in the highly conserved YMDD amino acid motif of HIV-1 RT (M184V), wh ich has been shown in previous studies to influence the fidelity and a ctivity of the enzyme. The M184V RT mutation was cloned into pHXB2(His -AC-GAC) and pHXB2(His-AC-TGT). Virus derived from pHXB2(His-AC-GAC) , vith M184V RT had slightly delayed replication compared to the virus f rom pHXB2(His-AC-GAC) with wild-type RT; in contrast, virus from pHXB2 (His-AC-TGT) with M184V RT was severely compromised in replication. Us ing an endogenous reverse transcription-PCR assay to analyze the rever se transcription of viruses obtained after transfection, we found that viruses derived from pHXB2(His-AC-GAC) with the, wild-type RT were sl ightly faster in the initiation of reverse transcription than viruses with M184V RT. The initiation of reverse transcription was delayed in viruses derived from pHXB2(His-AC-TGT) with, wild-type RT and M184V RT compared to viruses derived from pHXB2(His-AC-GAC). Finally, sequence analysis of U5 and PBS regions of proviruses from pHXB2(His-AC-GAC) w ith, wild-type RT revealed considerably more nucleotide substitutions than in viruses derived from pHXB2(His-AC-GAC) containing the M184V mu tation in RT after extended in vitro culture. Our studies point to a r ole for these additional nucleotide substitutions in U5 as an adaptati on by the virus to utilize an alternative tRNA to initiate reverse tra nscription.