Evaluation human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: Specificity and comparison to primer/template binding

A host cell-derived tRNA(3)(Lys) molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome, We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA(3)(Lys), and to address RT's putative role in the required and specific packaging of tRNA(3)(Lys) into the budding virus, Titration of RT with tRNA(3)(Lys) resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (K-d) Of 57.6 +/- 7.5 nM was derived. Titration of RT with Escherichia coli tRNA(2)(Glu), E. coli tRNA(2)(Tyr), E. coli tRNA(Lys), yeast tRNA(Phe), Or in vitro-synthesized human tRNA(3)(Lys) (no base modifications) resulted in similar fluorescence changes and K-d values as obtained for the natural tRNA(3)(Lys). The specific interaction between RT and tRNA(3)(Lys) during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT. Other factors during viral assembly must therefore cooperate in the packaging of tRNA(3)(Lys). The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding. The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNA(Phe) was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA. The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites, Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands. The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both tRNA and p/t binding. Reverse transcriptase was found to bind to the mutant tRNA 10-fold more tightly than to the unaltered tRNA (K-d = 4.5 +/- 1.0 and 44.6 +/- 6.6 nM, respectively). Further analyses revealed that the tighter affinity is probably due to a preferred p/t binding mode and not to one expected if separate tRNA and p/t binding regions are accessed simultaneously by the same molecule.