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Journal of Virology, June 2003, p. 6589-6600, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6589-6600.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Structural Basis for Distinctions between Substrate and Inhibitor Specificities for Feline Immunodeficiency Virus and Human Immunodeficiency Virus Proteases

Ying-Chuan Lin, Zachary Beck, Garrett M. Morris, Arthur J. Olson, and John H. Elder^*

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Received 17 January 2003/ Accepted 23 March 2003

We used feline immunodeficiency virus (FIV) protease (PR) as a mutational framework to define determinants for the observed substrate and inhibitor specificity distinctions between FIV and human immunodeficiency virus (HIV) PRs. Multiple-substitution mutants were constructed by replacing the residues in and around the active site of FIV PR with the structurally equivalent residues of HIV-1 PR. Mutants included combinations of three critical regions (FIV numbering, with equivalent HIV numbering in superscript): I37³²V in the active core region; N55⁴⁶M, M56⁴⁷I, and V59⁵⁰I in the flap region; and L97⁸⁰T, I98⁸¹P, Q99⁸²V, P100⁸³N, and L101⁸⁴I in the 90s loop region. Significant alterations in specificity were observed, consistent with the involvement of these residues in determining the substrate-inhibitor specificity distinctions between FIV and HIV PRs. Two previously identified residues, I35 and I57 of FIV PR, were intolerant to substitution and yielded inactive PRs. Therefore, we attempted to recover the activity by introducing secondary mutations. The addition of G62⁵³F and K63⁵⁴I, located at the top of the flap and outside the active site, compensated for the activity lost in the I57⁴⁸G substitution mutants. An additional two substitutions, D105⁸⁸N and N88⁷⁴T, facilitated recovery of activity in mutants that included the I35³⁰D substitution. Determination of K_i values of potent HIV-1 PR inhibitors against these mutants showed that inhibitor specificity paralleled that of HIV-1 PR. The findings indicate that maintenance of both substrate and inhibitor specificity is a function of interactions between residues both inside and outside the active site. Thus, mutations apparently peripheral to the active site can have a dramatic influence on inhibitor efficacy.

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* Corresponding author. Mailing address: Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-8270. Fax: (858) 784-2750. E-mail: jelder{at}scripps.edu.

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Journal of Virology, June 2003, p. 6589-6600, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6589-6600.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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