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Journal of Virology, February 2002, p. 1359-1368, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1359-1368.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

Deborah S. Dauber,1 Rainer Ziermann,2,{dagger} Neil Parkin,2 Dustin J. Maly,3 Sami Mahrus,1 Jennifer L. Harris,4,{ddagger} Jon A. Ellman,3 Christos Petropoulos,2 and Charles S. Craik1,4*

Graduate Program in Chemistry and Chemical Biology,1 University of California, San Francisco, San Francisco, California, 94143; ViroLogic, Inc., South San Francisco, California, 94080,2 Department of Chemistry, University of California, Berkeley, Berkeley, California, 94720,3 Department of Pharmaceutical Chemistry4

Received 16 August 2001/ Accepted 29 October 2001

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV.

* Corresponding author. Mailing address: Department of Pharmaceutical Chemistry, Box 0446, UC San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0446. Phone: (415) 476-8146. Fax: (415) 502-8298. E-mail: craik{at}cgl.ucsf.edu.

{dagger} Present address: Bayer Diagnostics, Berkeley, CA 94702.

{ddagger} Present address: Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121.

Journal of Virology, February 2002, p. 1359-1368, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1359-1368.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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