Giuseppe
Emeritus
[Source: PLoS Pathogens, full text: (LINK). Abstract, edited.]
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H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis
Erhard van der Vries<SUP>1</SUP>, Patrick J. Collins<SUP>2</SUP>, Sebastien G. Vachieri<SUP>2</SUP>, Xiaoli Xiong<SUP>2</SUP>, Junfeng Liu<SUP>2</SUP><SUP>,</SUP><SUP>3</SUP>, Philip A. Walker<SUP>2</SUP>, Lesley F. Haire<SUP>2</SUP>, Alan J. Hay<SUP>2</SUP>, Martin Schutten<SUP>1</SUP>, Albert D. M. E. Osterhaus<SUP>1</SUP>, Steve R. Martin<SUP>2</SUP>, Charles A. B. Boucher<SUP>1</SUP>, John J. Skehel<SUP>2</SUP><SUP>*</SUP>, Steve J. Gamblin<SUP>2</SUP>
<SUP></SUP>
1 Erasmus Medical Centre, Department of Virology, Rotterdam, The Netherlands, 2 Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom, 3 MOA Key Laboratory of Plant Pathology, China Agricultural University, Beijing, People's Republic of China
Abstract
Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K<SUB>M</SUB> values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K<SUB>I</SUB>) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.
Author Summary
Recently, a pandemic A/H1N1 influenza virus was isolated from an immune compromised patient with a novel antiviral resistance pattern to the neuraminidase inhibitor class of drugs. This virus had an amino acid change in the viral neuraminidase enzyme; an isoleucine at position 223 was substituted by an arginine (I223R). Patients infected with such a virus leave physicians with reduced antiviral treatment options, since pandemic viruses are naturally resistant to the other class of antivirals, the adamantanes. Previously, we have shown that this mutant virus retains its potential to cause disease and may still be able to spread in the human population. Here we used enzyme kinetic measurements and crystal structures of the I223R mutant neuraminidase to determine the resistance mechanism of this amino acid change. We found that the I223R change results in shrinkage of the active site of the enzyme. As a result, binding of the neuraminidase inhibitors is affected. In addition, we found that the active site of our pandemic neuraminidase structure, crystallized in the absence of inhibitor, has an extra cavity (150-cavity) adjacent to the active site. Our study could aid in the development of novel inhibitors designed to target the 150-cavity and active site of the enzyme.
Citation: van der Vries E, Collins PJ, Vachieri SG, Xiong X, Liu J, et al. (2012) H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis. PLoS Pathog 8(9): e1002914. doi:10.1371/journal.ppat.1002914
Editor: F?lix A. Rey, Institut Pasteur, France
Received: April 4, 2012; Accepted: August 5, 2012; Published: September 20, 2012
Copyright: ? 2012 van der Vries et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Part of this study is supported by the Influenza Resistance Information Study (IRIS-study). This study is sponsored by Hoffmann-La Roche, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and have the following conflicts: MS is consultant of ViroClinics Biosciences BV. MS is advisor for Hoffmann-La Roche, Inc. MS is advisor for Vertex Ltd. ADMEO is employe of ViroClinics Biosciences BV. ADMEO is chief scientific officer of ViroClinics Biosciences BV. ADMEO, MS, EVDV and CABB participate in the IRIS study sponsored by Hoffmann-La Roche, Inc. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.
* E-mail: skeheljj@nimr.mrc.ac.uk
-Erhard van der Vries<SUP>1</SUP>, Patrick J. Collins<SUP>2</SUP>, Sebastien G. Vachieri<SUP>2</SUP>, Xiaoli Xiong<SUP>2</SUP>, Junfeng Liu<SUP>2</SUP><SUP>,</SUP><SUP>3</SUP>, Philip A. Walker<SUP>2</SUP>, Lesley F. Haire<SUP>2</SUP>, Alan J. Hay<SUP>2</SUP>, Martin Schutten<SUP>1</SUP>, Albert D. M. E. Osterhaus<SUP>1</SUP>, Steve R. Martin<SUP>2</SUP>, Charles A. B. Boucher<SUP>1</SUP>, John J. Skehel<SUP>2</SUP><SUP>*</SUP>, Steve J. Gamblin<SUP>2</SUP>
<SUP></SUP>
1 Erasmus Medical Centre, Department of Virology, Rotterdam, The Netherlands, 2 Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom, 3 MOA Key Laboratory of Plant Pathology, China Agricultural University, Beijing, People's Republic of China
Abstract
Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K<SUB>M</SUB> values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K<SUB>I</SUB>) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.
Author Summary
Recently, a pandemic A/H1N1 influenza virus was isolated from an immune compromised patient with a novel antiviral resistance pattern to the neuraminidase inhibitor class of drugs. This virus had an amino acid change in the viral neuraminidase enzyme; an isoleucine at position 223 was substituted by an arginine (I223R). Patients infected with such a virus leave physicians with reduced antiviral treatment options, since pandemic viruses are naturally resistant to the other class of antivirals, the adamantanes. Previously, we have shown that this mutant virus retains its potential to cause disease and may still be able to spread in the human population. Here we used enzyme kinetic measurements and crystal structures of the I223R mutant neuraminidase to determine the resistance mechanism of this amino acid change. We found that the I223R change results in shrinkage of the active site of the enzyme. As a result, binding of the neuraminidase inhibitors is affected. In addition, we found that the active site of our pandemic neuraminidase structure, crystallized in the absence of inhibitor, has an extra cavity (150-cavity) adjacent to the active site. Our study could aid in the development of novel inhibitors designed to target the 150-cavity and active site of the enzyme.
Citation: van der Vries E, Collins PJ, Vachieri SG, Xiong X, Liu J, et al. (2012) H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis. PLoS Pathog 8(9): e1002914. doi:10.1371/journal.ppat.1002914
Editor: F?lix A. Rey, Institut Pasteur, France
Received: April 4, 2012; Accepted: August 5, 2012; Published: September 20, 2012
Copyright: ? 2012 van der Vries et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Part of this study is supported by the Influenza Resistance Information Study (IRIS-study). This study is sponsored by Hoffmann-La Roche, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and have the following conflicts: MS is consultant of ViroClinics Biosciences BV. MS is advisor for Hoffmann-La Roche, Inc. MS is advisor for Vertex Ltd. ADMEO is employe of ViroClinics Biosciences BV. ADMEO is chief scientific officer of ViroClinics Biosciences BV. ADMEO, MS, EVDV and CABB participate in the IRIS study sponsored by Hoffmann-La Roche, Inc. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.
* E-mail: skeheljj@nimr.mrc.ac.uk
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