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A computational-experimental approach identifies mutations that enhance surface expression of an oseltamivir-resistant influenza neuraminidase.

Bloom JD, Nayak JS, Baltimore D - PLoS ONE (2011)

Bottom Line: H274Y is currently rare in pandemic H1N1, and it remains unclear whether secondary mutations exist that might similarly counteract the decreased neuraminidase surface expression associated with this resistance mutation in pandemic H1N1.We then use the most successful computational approach to predict a set of candidate secondary mutations to the pandemic H1N1 neuraminidase.Two of the secondary mutations together restore surface-expressed neuraminidase activity to wildtype levels, and also eliminate the very slight decrease in viral growth in tissue-culture caused by H274Y.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT
The His274→Tyr (H274Y) oseltamivir (Tamiflu) resistance mutation causes a substantial decrease in the total levels of surface-expressed neuraminidase protein and activity in early isolates of human seasonal H1N1 influenza, and in the swine-origin pandemic H1N1. In seasonal H1N1, H274Y only became widespread after the occurrence of secondary mutations that counteracted this decrease. H274Y is currently rare in pandemic H1N1, and it remains unclear whether secondary mutations exist that might similarly counteract the decreased neuraminidase surface expression associated with this resistance mutation in pandemic H1N1. Here we investigate the possibility of predicting such secondary mutations. We first test the ability of several computational approaches to retrospectively identify the secondary mutations that enhanced levels of surface-expressed neuraminidase protein and activity in seasonal H1N1 shortly before the emergence of oseltamivir resistance. We then use the most successful computational approach to predict a set of candidate secondary mutations to the pandemic H1N1 neuraminidase. We experimentally screen these mutations, and find that several of them do indeed partially counteract the decrease in neuraminidase surface expression caused by H274Y. Two of the secondary mutations together restore surface-expressed neuraminidase activity to wildtype levels, and also eliminate the very slight decrease in viral growth in tissue-culture caused by H274Y. Our work therefore demonstrates a combined computational-experimental approach for identifying mutations that enhance neuraminidase surface expression, and describes several specific mutations with the potential to be of relevance to the spread of oseltamivir resistance in pandemic H1N1.

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Sites of the mutations mapped onto the neuraminidases protein structure.Shown in dark green is one monomer from an N1 neuraminidase crystal structure ([37], PDB code 3BEQ]. Residue 274 (N2 numbering) is shown in red, and the sites of the secondary mutations (N1 numbering) are shown in blue. Oseltamivir (yellow spheres) is modeled in its binding site based on a related crystal structure ([83], PDB code 2HU0). The other three monomers of the full neuraminidase tetramer are shown in light green, based on modeling from a related crystal structure ([83], PDB code 2HU0). The image was rendered with PyMOL.
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pone-0022201-g006: Sites of the mutations mapped onto the neuraminidases protein structure.Shown in dark green is one monomer from an N1 neuraminidase crystal structure ([37], PDB code 3BEQ]. Residue 274 (N2 numbering) is shown in red, and the sites of the secondary mutations (N1 numbering) are shown in blue. Oseltamivir (yellow spheres) is modeled in its binding site based on a related crystal structure ([83], PDB code 2HU0). The other three monomers of the full neuraminidase tetramer are shown in light green, based on modeling from a related crystal structure ([83], PDB code 2HU0). The image was rendered with PyMOL.

Mentions: Regardless of the eventual fate of H274Y in pandemic H1N1, our findings are relevant to broader issues in protein evolution. We began this paper by describing the burgeoning set of examples where a mutation causes a beneficial phenotypic alteration only when it is paired with a secondary mutation. We further noted that these secondary mutations often act in a general manner by bolstering a protein-level property such as folding, stability, or expression, thereby alleviating defects caused by a variety of other mutations [1], [5], [43]–[48]. The potential for this phenomenon appears to be pervasive in influenza neuraminidase, as evidenced by the existence of multiple secondary mutations that partially counteract the decreased surface expression caused by H274Y. The exact biophysical mechanism remains unclear, and is an important area for further research. However, it is interesting to note that the mutations are scattered about the neuraminidase protein structure (Figure 6), and so appear to be generally promoting surface expression rather than forming a specific structural interaction with H274Y.


A computational-experimental approach identifies mutations that enhance surface expression of an oseltamivir-resistant influenza neuraminidase.

Bloom JD, Nayak JS, Baltimore D - PLoS ONE (2011)

Sites of the mutations mapped onto the neuraminidases protein structure.Shown in dark green is one monomer from an N1 neuraminidase crystal structure ([37], PDB code 3BEQ]. Residue 274 (N2 numbering) is shown in red, and the sites of the secondary mutations (N1 numbering) are shown in blue. Oseltamivir (yellow spheres) is modeled in its binding site based on a related crystal structure ([83], PDB code 2HU0). The other three monomers of the full neuraminidase tetramer are shown in light green, based on modeling from a related crystal structure ([83], PDB code 2HU0). The image was rendered with PyMOL.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140507&req=5

pone-0022201-g006: Sites of the mutations mapped onto the neuraminidases protein structure.Shown in dark green is one monomer from an N1 neuraminidase crystal structure ([37], PDB code 3BEQ]. Residue 274 (N2 numbering) is shown in red, and the sites of the secondary mutations (N1 numbering) are shown in blue. Oseltamivir (yellow spheres) is modeled in its binding site based on a related crystal structure ([83], PDB code 2HU0). The other three monomers of the full neuraminidase tetramer are shown in light green, based on modeling from a related crystal structure ([83], PDB code 2HU0). The image was rendered with PyMOL.
Mentions: Regardless of the eventual fate of H274Y in pandemic H1N1, our findings are relevant to broader issues in protein evolution. We began this paper by describing the burgeoning set of examples where a mutation causes a beneficial phenotypic alteration only when it is paired with a secondary mutation. We further noted that these secondary mutations often act in a general manner by bolstering a protein-level property such as folding, stability, or expression, thereby alleviating defects caused by a variety of other mutations [1], [5], [43]–[48]. The potential for this phenomenon appears to be pervasive in influenza neuraminidase, as evidenced by the existence of multiple secondary mutations that partially counteract the decreased surface expression caused by H274Y. The exact biophysical mechanism remains unclear, and is an important area for further research. However, it is interesting to note that the mutations are scattered about the neuraminidase protein structure (Figure 6), and so appear to be generally promoting surface expression rather than forming a specific structural interaction with H274Y.

Bottom Line: H274Y is currently rare in pandemic H1N1, and it remains unclear whether secondary mutations exist that might similarly counteract the decreased neuraminidase surface expression associated with this resistance mutation in pandemic H1N1.We then use the most successful computational approach to predict a set of candidate secondary mutations to the pandemic H1N1 neuraminidase.Two of the secondary mutations together restore surface-expressed neuraminidase activity to wildtype levels, and also eliminate the very slight decrease in viral growth in tissue-culture caused by H274Y.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT
The His274→Tyr (H274Y) oseltamivir (Tamiflu) resistance mutation causes a substantial decrease in the total levels of surface-expressed neuraminidase protein and activity in early isolates of human seasonal H1N1 influenza, and in the swine-origin pandemic H1N1. In seasonal H1N1, H274Y only became widespread after the occurrence of secondary mutations that counteracted this decrease. H274Y is currently rare in pandemic H1N1, and it remains unclear whether secondary mutations exist that might similarly counteract the decreased neuraminidase surface expression associated with this resistance mutation in pandemic H1N1. Here we investigate the possibility of predicting such secondary mutations. We first test the ability of several computational approaches to retrospectively identify the secondary mutations that enhanced levels of surface-expressed neuraminidase protein and activity in seasonal H1N1 shortly before the emergence of oseltamivir resistance. We then use the most successful computational approach to predict a set of candidate secondary mutations to the pandemic H1N1 neuraminidase. We experimentally screen these mutations, and find that several of them do indeed partially counteract the decrease in neuraminidase surface expression caused by H274Y. Two of the secondary mutations together restore surface-expressed neuraminidase activity to wildtype levels, and also eliminate the very slight decrease in viral growth in tissue-culture caused by H274Y. Our work therefore demonstrates a combined computational-experimental approach for identifying mutations that enhance neuraminidase surface expression, and describes several specific mutations with the potential to be of relevance to the spread of oseltamivir resistance in pandemic H1N1.

Show MeSH
Related in: MedlinePlus