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Journal of Virology, July 1999, p. 5803-5813, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The P236L Delavirdine-Resistant Human Immunodeficiency Virus Type 1 Mutant Is Replication Defective and Demonstrates Alterations in both RNA 5'-End- and DNA 3'-End-Directed RNase H Activities

Peter Gerondelis,1,2 Richard H. Archer,1 Chockalingam Palaniappan,3,dagger Richard C. Reichman,1,2 Philip J. Fay,1,3,4 Robert A. Bambara,2,3,4 and Lisa M. Demeter1,2,4,*

Departments of Medicine,1 Microbiology and Immunology,2 and Biochemistry and Biophysics3 and Cancer Center,4 University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received 28 December 1998/Accepted 19 April 1999

The nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) delavirdine (DLV) selects in vitro for the human immunodeficiency virus type 1 (HIV-1) RT mutation P236L, which confers high-level resistance to DLV but not other NNRTIs. Unexpectedly, P236L has developed infrequently in HIV-1 isolates obtained from patients receiving DLV; K103N is the predominant resistance mutation observed in that setting. We characterized the replication fitness of viruses derived from pNL4-3 containing P236L or K103N in both H9 and primary human peripheral blood mononuclear cell cultures infected in parallel with the two mutants. In the absence of DLV, p24 production by wild-type virus occurred more rapidly and to higher levels than with either mutant; P236L consistently demonstrated a two- to threefold decrease in p24 relative to K103N. At low levels of DLV, growth of wild-type virus was severely inhibited, and K103N replicated two- to threefold more efficiently than P236L. At high concentrations of DLV, P236L replication and K103N replication were both inhibited. Recombinant RTs containing K103N or P236L were analyzed for DNA polymerization on heteropolymeric RNA templates and RNase H degradation of RNA-DNA hybrids. Neither mutant demonstrated defects in polymerization. K103N demonstrated normal RNA 5'-end-directed RNase H cleavage and slowed DNA 3'-end-directed RNase H cleavage compared to wild-type RT. P236L demonstrated slowing of both DNA 3'-end- and RNA 5'-end-directed RNase H cleavage, consistent with its reduced replication efficiency relative to K103N. These data suggest that NNRTI resistance mutations can lead to reductions in the efficiency of RNase H cleavage, which may contribute to a reduction in the replication fitness of HIV-1.

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

dagger Present address: Amersham Pharmacia Biotech, Inc., Cleveland, OH 44128.

Journal of Virology, July 1999, p. 5803-5813, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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