GENETIC-EVIDENCE OF THE INTERACTION BETWEEN TRNA(LYS,3) AND U5 FACILITATING EFFICIENT INITIATION OF REVERSE TRANSCRIPTION BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

Previous studies using in vitro chemical and enzyme methods demonstrat ed that, in addition to the primer-binding site (PBS), two regions ups tream of the PBS in U5 of HIV-1 RNA interact with tRNA(LyS,3) during t he initiation of reverse transcription, One region corresponds to nucl eotides 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 t he importance of these viral RNA-tRNA interactions in reverse transcri ption 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 aff ect protein expression or production of virus from transfected provira l DNAs. Analysis of these viruses revealed that, although all were inf ectious, the initial replication was delayed compared with wild-type v irus. Using an endogenous reverse transcription-PCR assay, we found th at the initiation of the reverse transcription in the mutant viruses w as less efficient than that for the wild-type virus. Analysis of the p roviral DNA sequences after 2 months of in vitro culture revealed that most progeny viruses derived from the mutant that contained the CCACA A motif had acquired nucleotide substitutions within and surrounding t he 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 fr om virus containing the double mutations had gained additional mutatio ns within the CCACAA and GGG motifs, The replication of the mutant vir uses was now similar to that of the wild type. The results of these st udies demonstrate that interactions between the tRNA and U5 are import ant for generation of an optimized initiation complex required for eff icient reverse transcription.