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Journal of Virology, June 2008, p. 5869-5878, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02325-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Ninety-Nine Is Not Enough: Molecular Characterization of Inhibitor-Resistant Human Immunodeficiency Virus Type 1 Protease Mutants with Insertions in the Flap Region{triangledown} ,{dagger}

Milan Kozísek,1,2 Klára Grantz Sasková,1,2 Pavlína Rezácová,1,3 Jirí Brynda,1,3 Noortje M. van Maarseveen,4 Dorien De Jong,4 Charles A. Boucher,4 Ron M. Kagan,5 Monique Nijhuis,4 and Jan Konvalinka1,2,3*

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 2, 166 10 Praha 6, Czech Republic,1 Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, Praha 2, Czech Republic,2 Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 140 00 Praha 4, Czech Republic,3 Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands,4 Department of Infectious Diseases, Quest Diagnostics Inc., San Juan Capistrano, California 926755

Received 26 October 2007/ Accepted 28 March 2008

While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

* Corresponding author. Mailing address: 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 2, 166 10 Praha 6, Czech Republic. Phone: 420 220183218. Fax: 420 220183578. E-mail: konval{at}uochb.cas.cz

{triangledown} Published ahead of print on 9 April 2008.

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

Journal of Virology, June 2008, p. 5869-5878, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02325-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Saskova, K. G., Kozisek, M., Rezacova, P., Brynda, J., Yashina, T., Kagan, R. M., Konvalinka, J. (2009). Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir. J. Virol. 83: 8810-8818 [Abstract] [Full Text]  


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