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 digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence 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 and renatured after SDS-PAGE; the proteins are then probed with the primer analogues P-32-labeled d(T)16 or P-32-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 congruent-to 300. 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 corresponding expressed protein, p35, and found that this protein binds to tRNA(Lys,3), demonstrating that residues 273-302 are able to confer nucleic acid binding to the binding-negative C-terminal segment spanning residues 303 congruent-to 560. Further, an additional domain fragment 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].