THE GAG PRECURSOR CONTAINS A SPECIFIC HIV-1 PROTEASE CLEAVAGE SITE BETWEEN THE NC (P7) AND P1 PROTEINS

The predicted protease cleavage site (p7/p1; [J. Virol. 66 (1992) 1856 -1865]) within the nucleocapsid precursor protein (p15) of human immun odeficiency virus, type 1, was confirmed using an in vitro assay emplo ying recombinant HIV-1 protease and a chemically synthesized 72 amino acid polypeptide containing the p7 and p1 protein domains of the nativ e gag polyprotein. The cleavage occurred between amino acid 55 (N) and amino acid 56 (F) of the polypeptide, as determined by N-terminal seq uencing. The hydrolysis was optimal at pH 6.0 and at high salt concent ration. The kinetic parameters K(m), k(cat) and k(cat)/K(m) were 99 mu M (+/- 8), 0.152 s-1 (+/- 0.002) and 1.56 mM-1 . s-1 (+/- 0.11), respe ctively. Reconstituted as well as denatured polypeptides were cleaved at approximately the same rate, demonstrating that the conformation of the p7 protein, as a result of the Zn2+-binding, had no significant e ffect on the rate of hydrolysis of the p7/p1 cleavage.