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J Biol Chem, Vol. 274, Issue 39, 27666-27673, September 24, 1999

New Human Immunodeficiency Virus, Type 1 Reverse Transcriptase (HIV-1 RT) Mutants with Increased Fidelity of DNA Synthesis
ACCURACY, TEMPLATE BINDING, AND PROCESSIVITY

Baek Kim, Jennifer C. Ayran^¶, Sarah G. Sagar^¶, Elinor T. Adman, Shannon M. Fuller, Nancy H. Tran, and Jeffrey Horrigan

From the  Department of Microbiology and Immunology, University of Rochester, Rochester, New York 14642, the ^¶ Departments of Pathology, and  Biological Structure, University of Washington, Seattle, Washington 98195

Infidelity of DNA synthesis by human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) is a presumptive determinant of HIV-1 hypervariability and is incompletely understood at the mechanistic and structural levels. Amino acid substitution at only three residues, including Asp-76 (Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) Biochemistry 37, 5831-5839), is known to increase fidelity. We report here that substitution at Arg-78 can also increase accuracy. Mutant R78A RT showed reduced primer extension in misincorporation assays lacking a complementary dNTP and exhibited a 9-fold decrease in mutation frequency in the M13mp2 lacZ forward mutation assay. Previous structural studies indicate that Arg-78 and Asp-76 lie in a region that interacts with template nucleotides. Interestingly, R78A RT exhibited 6- to 8-fold decreases in binding affinity (K_d) for RNA and DNA templates relative to wild type RT. In contrast, D76V RT, which also increases fidelity (Kim et al., 1996), showed a 6- to 7-fold increased affinity. The processivity of R78A RT on both RNA and DNA templates was substantially reduced relative to wild type RT, whereas the processivity of D76V RT was increased. We discuss relationships of fidelity, template binding, and processivity in these and other HIV RT mutants.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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