Research is proposed to study the protease of human immunodeficiency virus (HIV). These studies will involve in the enzyme properties, enzyme specificity, catalytic apparatus and mechanism, inhibitor studies, and the activation mechanisms of the precursor of HIV protease. These studies will permit a greater understanding on the roles of the HIV protease in the in vivo propagation of the HIV. More specifically, these studies are designed to provide new information on the molecular mechanisms and specificity in the proteolytic processing of polyprotein precursor of gag-pol genes. The major rationales and approaches of this research are as follows: (a) HIV protease gene will be synthesized and the enzyme expressed in E. coli. This will enable the purification of large amount of the enzyme for specificity and mechanism studies. (b) HIV protease and other retroviral proteases have many characteristics of the aspartic protease family. From the previous studies of the applicant, it can be hypothesized that the active form of the HIV protease has a homodimer structure. This hypothesis will be used to test the active site structure, study enzyme kinetic and mechanism, and design effective inhibitors. (c) The ability of retroviral proteases to self-excise from their polyprotein precursors is also similar to the autocatalyzed activation mechanism of the zymogens of aspartic proteases studied by the applicant. Thus, the processing kinetics and mechanisms of a """"""""miniprecursor"""""""" of HIV protease will be studied using the activation mechanisms of aspartic protease zymogen as model. The """"""""miniprecursors"""""""" of HIV protease will be designed, synthesized, and expressed to obtain the proteins from these studies. (d) The gene of the protease from Rous sarcoma virus (RSV, a retrovirus) has already been cloned and expressed in applicant's laboratory. RSV protease will be used in the above studies to confirm the HIV findings and to indicate whether these findings can be generalized to other retroviral enzymes.
