A novel interaction of tRNA(Lys,3) with the feline immunodeficiency virus RNA genome governs initiation of minus strand DNA synthesis

Complementarity between nucleotides at the 5' terminus of tRNA(Lys,3) and t he U5-IR loop of the feline immunodeficiency virus RNA genome suggests a no vel intermolecular interaction controls initiation of minus strand synthesi s in a manner analogous to other retroviral systems. Base pairing of this t RNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome co ntaining full-length or 5'-deleted variants of tRNA(Lys,3) hybridized to th e primer-binding site. A major pause in RNA-dependent DNA synthesis occurre d 14 nucleotides ahead of the primer-binding site with natural and syntheti c tRNA(Lys,3) primers, indicating it was not a consequence of tRNA base mod ifications. The majority of the paused complexes resulted in dissociation o f the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant wh ose 5' nucleotides are deleted relieved pausing at this position and subseq uently allowed high level DNA synthesis. Additional experiments with tRNA-D NA chimeric primers were used to localize the stage of minus strand synthes is at which the tRNA-viral RNA interaction was disrupted. Finally, replacin g nucleotides of the feline immunodeficiency virus US-IR loop with the (A), sequence of its human immunodeficiency virus (HIV)-1 counterpart also reli eved pausing, but did not induce pausing immediately downstream of the prim er-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequence s immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.