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Journal of Virology, September 2000, p. 8390-8401, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Mutants of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase Resistant to Nonnucleoside Reverse Transcriptase Inhibitors Demonstrate Altered Rates of RNase H Cleavage That Correlate with HIV-1 Replication Fitness in Cell Culture

Richard H. Archer,1 Carrie Dykes,1 Peter Gerondelis,1,2 Amanda Lloyd,1 Philip Fay,1,3,4 Richard C. Reichman,1,2 Robert A. Bambara,2,3,4 and Lisa M. Demeter1,2,4,*

Departments of Medicine,1 Microbiology and Immunology,2 Biochemistry and Biophysics,3 and the Cancer Center,4 University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received 4 January 2000/Accepted 7 June 2000

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three- to fourfold slowing of both DNA 3'-end-directed and RNA 5'-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803-5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3'-end- and RNA 5'-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.

* Corresponding author. Mailing address: University of Rochester Infectious Diseases Unit, 601 Elmwood Ave., Box 689, Rochester, NY 14642. Phone: (716) 275-4764. Fax: (716) 442-9328. E-mail: Lisa_Demeter{at}urmc.rochester.edu.

Journal of Virology, September 2000, p. 8390-8401, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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