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Journal of Virology, March 2004, p. 2790-2807, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2790-2807.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Genetic and Phenotypic Analyses of Human Immunodeficiency Virus Type 1 Escape from a Small-Molecule CCR5 Inhibitor{dagger}

Shawn E. Kuhmann,1 Pavel Pugach,1 Kevin J. Kunstman,2 Joann Taylor,2 Robyn L. Stanfield,3 Amy Snyder,1 Julie M. Strizki,4 Janice Riley,4 Bahige M. Baroudy,4 Ian A. Wilson,3 Bette T. Korber,5,6 Steven M. Wolinsky,2 and John P. Moore1*

Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021,1 Department of Medicine, The Feinberg Medical School, Northwestern University, Chicago, Illinois 60611,2 Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037,3 Schering Plough Research Institute, Kenilworth, New Jersey 07033,4 Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, New Mexico 87545,5 Santa Fe Institute, Santa Fe, New Mexico 875016

Received 21 August 2003/ Accepted 18 November 2003

We have described previously the generation of an escape variant of human immunodeficiency virus type 1 (HIV-1), under the selection pressure of AD101, a small molecule inhibitor that binds the CCR5 coreceptor (A. Trkola, S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan, P. Pugach, S. X. L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S. McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore, Proc. Natl. Acad. Sci. USA 99:395-400, 2002). The escape mutant, CC101.19, continued to use CCR5 for entry, but it was at least 20,000-fold more resistant to AD101 than the parental virus, CC1/85. We have now cloned the env genes from the the parental and escape mutant isolates and made chimeric infectious molecular clones that fully recapitulate the phenotypes of the corresponding isolates. Sequence analysis of the evolution of the escape mutants suggested that the most relevant changes were likely to be in the V3 loop of the gp120 glycoprotein. We therefore made a series of mutant viruses and found that full AD101 resistance was conferred by four amino acid changes in V3. Each change individually caused partial resistance when they were introduced into the V3 loop of a CC1/85 clone, but their impact was dependent on the gp120 context in which they were made. We assume that these amino acid changes alter how the HIV-1 Env complex interacts with CCR5. Perhaps unexpectedly, given the complete dependence of the escape mutant on CCR5 for entry, monomeric gp120 proteins expressed from clones of the fully resistant isolate failed to bind to CCR5 on the surface of L1.2-CCR5 cells under conditions where gp120 proteins from the parental virus and a partially AD101-resistant virus bound strongly. Hence, the full impact of the V3 substitutions may only be apparent at the level of the native Env complex.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Ave., W-805, New York, NY 10021. Phone: (212) 746-4462. Fax: (212) 746-8340. E-mail: jpm2003{at}med.cornell.edu.

{dagger} Contribution la-ur-03-5893 from the Los Alamos National Laboratory.

Journal of Virology, March 2004, p. 2790-2807, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2790-2807.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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