This Article Full Text Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Google Scholar Articles by Sasková, K. G. Articles by Konvalinka, J. PubMed PubMed Citation Articles by Sasková, K. G. Articles by Konvalinka, J.

 Previous Article  |  Next Article 

Journal of Virology, September 2009, p. 8810-8818, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00451-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir ^,

Klára Grantz aková,^1,2 Milan Koíek,^1,2 Pavlína ezáová,^1,3 Jií Brynda,^1,3 Tatyana Yashina,⁴ Ron M. Kagan,⁵ and Jan Konvalinka^1,2,3*

Gilead Sciences and IOCB Research Center, Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic, v.v.i., Flemingovo n. 2, 166 10 Prague 6, Czech Republic,¹ Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, Prague 2, Czech Republic,² Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 140 00 Prague 4, Czech Republic,³ Department of Infectious Diseases, Specialty Laboratories, Valencia, California 91355,⁴ Department of Infectious Diseases, Quest Diagnostics Inc., San Juan Capistrano, California 92675⁵

Received 4 March 2009/ Accepted 11 June 2009

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

---

* Corresponding author. Mailing address: Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic, v.v.i., Flemingovo n. 2, 166 10 Prague 6, Czech Republic. Phone: 420-220183218. Fax: 420-220183578. E-mail: konval{at}uochb.cas.cz

Published ahead of print on 17 June 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

---

Journal of Virology, September 2009, p. 8810-8818, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00451-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.