MAPPING OF NUCLEIC-ACID BINDING IN PROTEOLYTIC DOMAINS OF HIV-1 REVERSE-TRANSCRIPTASE

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the d igestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain frag ment was mapped by both immunoblotting and N-terminal amino acid seque nce analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane an d renatured after SDS-PAGE; the proteins are then probed with the prim er analogues P-32-labeled d(T)16 or P-32-labeled tRNA(Lys,3). A V8 pro tease domain fragment spanning residues 195 to approximately 300 (p12) , which was found earlier to be UV cross-linked to the primer in intac t RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed bindin g to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 congruent-to 30 0. By contrast, a C-terminal domain fragment termed p30(303 congruent- to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed. We purified the corr esponding expressed protein, p35, and found that this protein binds to tRNA(Lys,3), demonstrating that residues 273-302 are able to confer n ucleic acid binding to the binding-negative C-terminal segment spannin g residues 303 congruent-to 560. Further, an additional domain fragmen t corresponding to residues 1 congruent-to 230 (p29) was found to have nucleic acid-binding capacity. These results indicate that RT nucleic acid binding occurs in at least two domains in the N-terminal half of p66. The results appear in good agreement with the model of template- primer bound to the p66/p51 heterodimer, proposed by Kohlstaedt et al. [(1992) Science 256, 1780-1789].