Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA molecule that are required for interaction with the RT protein remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified RT to mutant forms of tRNA. The anticodon loop could be mutated without eliminating RT recognition. However, mutations in the TC stem were found to partially interfere with RT binding, and D arm mutants were completely inactive in RT binding. Interestingly, binding of the RT protein to tRNA facilitates the subsequent annealing of template strand to the 3`-terminus of the tRNA molecule. Consistent with this finding, we demonstrate that mutant HIV-1 virions lacking the RT protein do contain a viral RNA genome without an associated tRNA primer. We also found that a preformed primer tRNA-template complex is efficiently recognized by RT protein in vitro. Extension of the template molecule over the TC loop did result in complete inhibition of RT binding, suggesting the presence of additional recognition elements in the TC loop. These results, combined with a comparative sequence analysis of tRNA species present in HIV-1 virions and RNA motifs selected in vitro for high affinity RT binding, suggest that RT recognizes the central domain of the tRNA tertiary structure, which is formed by interaction of the D and TC loops.

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