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Antimicrobial Agents and Chemotherapy, November 2009, p. 4640-4646, Vol. 53, No. 11
0066-4804/09/$08.00+0 doi:10.1128/AAC.00686-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Impact of Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutations P119S and T165A on 4'-Ethynylthymidine Analog Resistance Profile
Guangwei Yang,1,
Elijah Paintsil,1,2,
Ginger E. Dutschman,1
Susan P. Grill,1
Chuan-Jen Wang,1
Jimin Wang,3
Hiromichi Tanaka,4
Takayuki Hamasaki,5
Masanori Baba,5 and
Yung-Chi Cheng1*
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520,1 Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520,2 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520,3 School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan,4 Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890 8544, Japan5
Received 19 May 2009/ Returned for modification 27 July 2009/ Accepted 14 August 2009
2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4'-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4'-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4'-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4'-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4'-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4'-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4'-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4'-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4'-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4'-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.
* Corresponding author. Mailing address: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: (203) 785-7118. Fax: (203) 785-7129. E-mail: yccheng{at}yale.edu
Published ahead of print on 24 August 2009.
G.Y. and E.P. contributed equally to this work.
Antimicrobial Agents and Chemotherapy, November 2009, p. 4640-4646, Vol. 53, No. 11
0066-4804/09/$08.00+0 doi:10.1128/AAC.00686-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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