JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.M200282200 on April 10, 2002

J. Biol. Chem., Vol. 277, Issue 25, 22345-22352, June 21, 2002
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Substitutions of Phe61 Located in the Vicinity of Template 5'-Overhang Influence Polymerase Fidelity and Nucleoside Analog Sensitivity of HIV-1 Reverse Transcriptase*

Timothy S. FisherDagger and Vinayaka R. Prasad§

From the Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461

Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe61 residue, located within the beta 3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe61 contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe right-arrow Ala, Leu, Trp, or Tyr) at Phe61. The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe61 mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideoxy,2'3'-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.

* This work was supported by Public Service Grant RO1 AI30861 (to V. R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Predoctoral Training Grant T32-GM07491.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Rm. GB 401, Bronx, NY 10461. Tel.: 718-430-2517; Fax: 718-430-8976; E-mail: prasad@aecom.yu.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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