A novel HIV-1 genome that stably utilizes tRNAHis rather than tRNALys,3 to initiate reverse transcription was used to study features for the interaction between the tRNA and viral RNA genome. In addition to a primer binding site (PBS) complementary to tRNAHis, this virus contains a six-nucleotide sequence in U5 complementary to the anticodon-loop of tRNAHis and three additional substitutions: U174-to-G, G181-to-A, and U200-to-C [HXB2(His-AC-GAC)]. Mutations in these three nucleotides resulted in viruses with three different genotypes: one group maintained a PBS complementary to tRNAHis with restored G174A181C200 or G174A181U200 configurations, one group reverted to a PBS complementary to tRNALys,3, and one group contained two or more PBSs complementary to different tRNAs on the same viral genome. Characterization of a previously identified virus with additional C152-to-A and C160-to-U substitutions [HXB2(His-AC-A152U160-GAC)] revealed that this virus maintained a PBS complementary to tRNAHis, whereas a mutant HXB2(His-AC-U152A160-GAC) reverted after culture to contain dual PBS complementary to tRNALys,3 and tRNAHis, respectively. Our results demonstrate that regions in U5 act in concert with the PBS to promote use of the tRNA primer for initiation of reverse transcription. These results are discussed with respect to structural models for the U5-PBS interactions with tRNA.