Retroviral proteases are enzymes that cleave gag and gag-pol precursor polyproteins into functional proteins of mature viral particles, an event necessary for infectivity of virus particles. Therefore, characterization of these proteases and development of specific inhibitors should lead to control of infectivity. Recent evidence suggests that the HIV protease belongs to the aspartic proteinase family and may be a dimmer.
The specific aims of this project are: (1) to isolate HIV protease, (2) to characterize its substrate specificity as an aspartic proteinase, (3) to test its interaction with known aspartic proteinase inhibitors, (4) to develop screening methods for tests of candidate de novo inhibitors, and (5) to characterize its structural properties, in order to investigate additional means of inhibiting the protease. The protease will be isolated from infected cell lysates and from an E. coli recombinant expression system using affinity to pepstatin-Sepharose, or to antibody resin, or by conventional means. The amino- and carboxyl-terminal sequences of the isolated protease will be determined to establish the precise boundaries of the protease gene in the viral genome. An assay based on proteolysis of the gag precursor will be developed. Subsequently, substrate specificity toward gag sequences and to aspartic proteinase substrate and inhibitors will be tested in order to determine how related the catalytic mechanism of the protease may be to other proteases. The active site subsite specificity will be mapped by chemically mutating the known gag protein sequence and determining affinities of these analogs to the protease. Physical properties such as the effect of various conditions on enzymatic activity and stability will be tested. These studies will allow us to gain a better understanding of the substrate specificity and inhibitory spectrum of the HIV protease and should allow us to ultimately design even more efficient inhibitors which could possibly be used in vivo to inactivate viral action.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Abbott Laboratories
Department
Type
DUNS #
City
North Chicago
State
IL
Country
United States
Zip Code
60064
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