Abstract
The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires cellular tRNALys,3as a primer and occurs at a site in the viral RNA genome, designated as the primer binding site (PBS), which is complementary to the 3′-terminal 18 nucleotides of tRNALys,3. We previously described an HIV-1 virus [designated as HXB2(His-AC)], which contained a sequence within the U5 region complementary to the anticodon region of tRNAHisin addition to a PBS complementary to the 3′-terminal 18 nucleotides of the tRNAHis. That virus maintained a PBS complementary to tRNAHisafter extendedin vitroculture (Wakefieldet 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 HXB2(His-AC) back into the wild-type genome (pHXB2) resulted in an infectious virus, designated as HXB2(His-AC-gac), which again stably maintained a PBS complementary to tRNAHis. 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 replicated in CD4+Sup T1 cells similarly to the wild-type virus. Comparison 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 clones 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 viruses incorporated a similar subset of cellular tRNAs, with tRNALys,3being the predominant tRNA found within virions. There was no selective enrichment for tRNAHiswithin virions of HXB2(His-AC-gac) virus which selectively use tRNAHisto initiate reverse transcription. The results 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 tRNAHisto initiate reverse transcription.
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