Previous studies using in vitro chemical and enzyme methods demonstrated that, in addition to the primer-binding site (PBS), two regions upstream of the PBS in U5 of HIV-1 RNA interact with tRNA(Lys,3) during the initiation of reverse transcription. One region corresponds to nucleotides 167-172 of U5, which are complementary to the anticodon region of tRNA(Lys,3) a second region corresponds to nucleotides 142-145 of U5, which interacts with nucleotides 43-46 of tRNA(Lys,3). To study the importance of these viral RNA- tRNA interactions in reverse transcription and viral replication, we mutated the two corresponding regions in the infectious HIV-1 proviral DNA (HXB2). Changing nucleotides 167-172 from GAAAAU to CCACAA (which is complementary to the anticodon of tRNA(His)) or changing nucleotides 142-144 from CCC to GGG did not affect protein expression or production of virus from transfected proviral DNAs. Analysis of these viruses revealed that, although all were infectious, the initial replication was delayed compared with wild-type virus. Using an endogenous reverse transcription-PCR assay, we found that the initiation of the reverse transcription in the mutant viruses was less efficient than that for the wild-type virus. Analysis of the proviral DNA sequences after 2 months of in vitro culture revealed that most progeny viruses derived from the mutant that contained the CCACAA motif had acquired nucleotide substitutions within and surrounding the CCACAA nucleotides. All the viruses recovered from the mutant that originally contained the GGG nucleotides reverted back to contain the wild-type CCC sequence. The majority of the proviral clones derived from virus containing the double mutations had gained additional mutations within the CCACAA and GGG motifs. The replication of the mutant viruses was now similar to that of the wild type. The results of these studies demonstrate that interactions between the tRNA and U5 are important for generation of an optimized initiation complex required for efficient reverse transcription.