Human immunodeficiency virus pol open reading frame encodes a protease for the processing of the gag and gag-pol polyproteins during virion maturation. The HIV protease, like other retroviral protease is structurally and functionally analogous to carboxyl (acid) proteases such as pepsin. The mature HIV protease derives from the gag-pol polyprotein via self-cleavages. Due to its critical role in virus assembly and replication, virion particles with lesions in the protease are non- infectious. Hence, the HIV protease represents an ideal target for anti- viral compounds. Although a great deal of biochemical details and the three dimensional structure of the HIV protease are available, the exact mechanism through which HIV protease mature from the gag-pol precursor remains unknown. This proposal aims to: I. Investigate the biochemical mechanism of the autoprocessing reaction catalyzed by the HIV protease by (i) site-directed mutagenesis of the active site residue, (ii) formation of protease precursor homodimer and wild type/mutant heterodimers and (iii) an in vitro autoprocessing assay based on a bacterially derived lacZ-protease fusion protein precursor. II. Introduce amino acid substitutions in the protease and its cleavage sites to examine (i) the structure and function of the protease and its cleavage specificity; (ii) the order of proteolysis of the various cleavage sites of gag and gag-pol by the protease. III. Incorporate the protease and its NH2 and/or COOH cleavage site mutations into the infectious viral DNA to determine their effects on HIV assembly and replication. IV. Purify and characterize and the wild type and mutant HIV protease overproduced in E. coli. Our long range objective is to understand the molecular mechanism of autoprocessing and the proteolytic processing of the gag, RT and integrase by the protease to facilitate the rational design of its inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI029327-04
Application #
3144089
Study Section
Special Emphasis Panel (ARR (V1))
Project Start
1990-11-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1994-06-30
Support Year
4
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106