The beta 7-beta 8 loop of the p51 subunit in the heterodimeric (p66/p51) human immunodeficiency virus type 1 reverse transcriptase is essential for the catalytic function of the p66 subunit

The heterodimeric human immunodeficiency virus type I reverse transcriptase (HIV-1 RT) is composed of p66 and p51 subunits, p66 being the catalytic su bunit. Our earlier investigation on the role of p51 in the catalytic proces s has shown that the p51 subunit facilitates the loading of the p66 subunit onto the template primer (TP). We had postulated that the beta7-beta8 loop of the p51 subunit may be involved in opening the polymerase cleft of p66 for DNA binding [Pandey, V. N., et al. (1996) Biochemistry, 35, 2168]. We r eport here that deletion or alanine substitution of four residues of the be ta7-beta8 loop results in severe impairment of the polymerase function of t he heterodimeric enzyme. The enzyme activity was restored to the wild-type levels when the mutant p66 subunit was dimerized with the wild-type p51, su ggesting that the intact beta7-beta8 loop in the p51 subunit is indispensab le for the catalytic function of p66. Further, the template primer binding ability of the enzyme was significantly reduced upon deletion or alanine su bstitution in the beta7-beta8 loop. Interestingly, the loss of the TP bindi ng ability of the mutant p66 was restored upon dimerization with wild-type p5l. Examination of the glycerol gradient ultracentrifugation analysis reve aled that while the wild-type HIV-1 RT sediments as a dimeric protein, the mutant enzymes carrying deletion or alanine substitution in both the subuni ts sediment predominantly as monomeric proteins, suggesting their inability to form stable dimers. In contrast, mutant p66 dimerized with wild-type p5 1 (p66(Delta)/p51(WT) and p66(Ala)/p51(WT)) sedimented at the dimeric posit ion. Taken together, these results clearly implicate the importance of the beta7-beta8 loop of p51 in the formation of stable functional heterodimers.