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J. Biol. Chem., Vol. 281, Issue 11, 7421-7428, March 17, 2006

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Structural Determinants of Slippage-mediated Mutations by Human Immunodeficiency Virus Type 1 Reverse Transcriptase^*

Monica E. Hamburgh¹, Kenneth A. Curr², Melissa Monaghan³, Vasudev R. Rao, Snehlata Tripathi, Bradley D. Preston^¶, Stefan Sarafianos^||, Eddy Arnold^||, Thomas Darden^**, and Vinayaka R. Prasad⁴

From the Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, the Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, the ^¶Department of Pathology, University of Washington, Seattle, Washington 98195, the ^||Center for Advanced Biotechnology and Medicine (CABM) and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, and the ^**Laboratory of Structural Biology, National Institutes of Health, NIEHS, Research Triangle Park, North Carolina 27707

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu⁸⁹ side chain. We investigated the role of Glu⁸⁹ in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu⁸⁹ can influence the slippage of both strands. Glu⁸⁹ substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu⁸⁹ on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu⁸⁹ and Lys¹⁵⁴, which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.

Received for publication, October 19, 2005 , and in revised form, January 18, 2006.

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

¹ Supported by National Institutes of Health Institutional Training Grant NIGMS T32-GM07491. Data are from a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University. Present address: Residency Program, Pediatrics, Rhode Island Hospital/Brown University, Providence, RI.

² Present address: 1680 Owens St., Gladstone Institute of Virology and Immunology, San Francisco, CA 94158.

³ Present address: Center for Biotechnology at Stony Brook, Stony Brook, NY 11794.

⁴ To whom correspondence should be addressed: 1300 Morris Park Ave., Rm. GB 401, Bronx, NY 10461. Tel.: 718-430-2517; Fax: 718-430-8976; E-mail: prasad{at}aecom.yu.edu.

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