NUCLEOTIDE-SEQUENCES WITHIN THE U5 REGION OF THE VIRAL-RNA GENOME ARETHE MAJOR DETERMINANTS FOR AN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TO MAINTAIN A PRIMER BINDING-SITE COMPLEMENTARY TO TRNA(HIS)

The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires cellular tRNA(Lys.3) as primer an d occurs at a site in the viral RNA genome, designated as the primer b inding site (PBS), which is complementary to the 3'-terminal 18 nucleo tides of tRNA(Lys.3). We previously described an HIV-1 virus [designat ed as HXB2(His-AC)], which contained a sequence within the U5 region c omplementary to the anticodon region of tRNA(His) in addition to a PBS complementary to the 3'-terminal 18 nucleotides of the tRNA(His). Tha t virus maintained a PBS complementary to tRNA(His) after extended in vitro culture (Wakefield et al., J. Virol. 70, 966-975, 1996). In the present study, we report that subcloning a 200-base-pair DNA fragment encompassing the U5 and PBS regions from an integrated provirus of HXB 2(His-AC) back into the wild-type genome (pHXB2) resulted in an infect ious virus, designated as HXBa(His-AC-gec), which again stably maintai ned a PBS complementary to tRNA(His). DNA sequence analysis of the 200 -base-pair region revealed only three nucleotide changes from HXB2(His -AC): a T-to-G change at nucleotide 174, a G-to-A change at nucleotide 181, and a T-to-C change at nucleotide 200. The new mutant virus repl icated in CD4(+) Sup T1 cells similarly to the wild-type virus. Compar ison of the nucleotide sequence of nucleocapsid gene of the wild-type and HXB2 (His-AC-gac) virus revealed no differences Although we found numerous mutations in the reverse transcriptase gene in proviral clone s derived from HXB2 (His-AC-gac), no common mutations were found among the 13 clones examined. Comparison of the virion-associated tRNAs of HXB2(His-AC-gac) with those of the wild type revealed that both viruse s incorporated a similar subset of cellular tRNAs, with tRNA(Lys.3) be ing the predominant tRNA found within virions. There was no selective enrichment tor tRNA(His) within virions of HXB2(His-AC-gac) virus whic h selectively use tRNA(His) to initiate reverse transcription. The res ults of these studies suggest that the U5 and PBS regions in the viral RNA genome are important determinants for HXB2(His-AC) viruses in the selective use of tRNA(His) to initiate reverse transcription. (C) 199 6 Academic Press, Inc.