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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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The three secondary mutations V234M, R222Q, and D344N largely explain the differences in total surface-expressed activity and protein between 1999 and 2007 seasonal H1N1 neuraminidases.Shown are wildtype (WT) and indicated mutants of the A/New Caledonia/20/1999 neuraminidase, in addition to WT and H274Y neuraminidases from the A/Brisbane/59/2007 (BR07) strain. All neuraminidases contain C-terminal epitope tags, except for the untagged WT and H274Y A/New Caledonia/20/1999 variants. For the measurements, 293T cells were transfected with plasmids encoding the neuraminidase proteins. After 20 hours, the cells were assayed for the total surface-expressed neuraminidase activity (top panel) or protein using an antibody against the epitope tag (bottom panel). Bars show the mean and standard error for at least six replicates.
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pone-0022201-g001: The three secondary mutations V234M, R222Q, and D344N largely explain the differences in total surface-expressed activity and protein between 1999 and 2007 seasonal H1N1 neuraminidases.Shown are wildtype (WT) and indicated mutants of the A/New Caledonia/20/1999 neuraminidase, in addition to WT and H274Y neuraminidases from the A/Brisbane/59/2007 (BR07) strain. All neuraminidases contain C-terminal epitope tags, except for the untagged WT and H274Y A/New Caledonia/20/1999 variants. For the measurements, 293T cells were transfected with plasmids encoding the neuraminidase proteins. After 20 hours, the cells were assayed for the total surface-expressed neuraminidase activity (top panel) or protein using an antibody against the epitope tag (bottom panel). Bars show the mean and standard error for at least six replicates.

Mentions: The A/New Caledonia/20/1999 seasonal H1N1 strain is attenuated by H274Y [13], while the A/Brisbane/59/2007 strain is not attenuated by this mutation [14] and is an immediate ancestor of the lineage of oseltamivir-resistant viruses that went to fixation beginning in 2007. We performed assays to measure both the total surface-expressed neuraminidase activity and protein levels in mammalian cells transfected with plasmids encoding wildtype and H274Y neuraminidase proteins from these two strains. As described previously [8], H274Y caused an approximately two-fold decrease in surface-expressed neuraminidase protein and activity for the 1999 strain (Figure 1). In comparison, the wildtype 2007 neuraminidase was expressed on the cell surface at over 1.5-fold higher levels than its 1999 counterpart, and the relative magnitude of the decrease caused by H274Y was substantially smaller (Figure 1).


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)

The three secondary mutations V234M, R222Q, and D344N largely explain the differences in total surface-expressed activity and protein between 1999 and 2007 seasonal H1N1 neuraminidases.Shown are wildtype (WT) and indicated mutants of the A/New Caledonia/20/1999 neuraminidase, in addition to WT and H274Y neuraminidases from the A/Brisbane/59/2007 (BR07) strain. All neuraminidases contain C-terminal epitope tags, except for the untagged WT and H274Y A/New Caledonia/20/1999 variants. For the measurements, 293T cells were transfected with plasmids encoding the neuraminidase proteins. After 20 hours, the cells were assayed for the total surface-expressed neuraminidase activity (top panel) or protein using an antibody against the epitope tag (bottom panel). Bars show the mean and standard error for at least six replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022201-g001: The three secondary mutations V234M, R222Q, and D344N largely explain the differences in total surface-expressed activity and protein between 1999 and 2007 seasonal H1N1 neuraminidases.Shown are wildtype (WT) and indicated mutants of the A/New Caledonia/20/1999 neuraminidase, in addition to WT and H274Y neuraminidases from the A/Brisbane/59/2007 (BR07) strain. All neuraminidases contain C-terminal epitope tags, except for the untagged WT and H274Y A/New Caledonia/20/1999 variants. For the measurements, 293T cells were transfected with plasmids encoding the neuraminidase proteins. After 20 hours, the cells were assayed for the total surface-expressed neuraminidase activity (top panel) or protein using an antibody against the epitope tag (bottom panel). Bars show the mean and standard error for at least six replicates.
Mentions: The A/New Caledonia/20/1999 seasonal H1N1 strain is attenuated by H274Y [13], while the A/Brisbane/59/2007 strain is not attenuated by this mutation [14] and is an immediate ancestor of the lineage of oseltamivir-resistant viruses that went to fixation beginning in 2007. We performed assays to measure both the total surface-expressed neuraminidase activity and protein levels in mammalian cells transfected with plasmids encoding wildtype and H274Y neuraminidase proteins from these two strains. As described previously [8], H274Y caused an approximately two-fold decrease in surface-expressed neuraminidase protein and activity for the 1999 strain (Figure 1). In comparison, the wildtype 2007 neuraminidase was expressed on the cell surface at over 1.5-fold higher levels than its 1999 counterpart, and the relative magnitude of the decrease caused by H274Y was substantially smaller (Figure 1).

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