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Epistatic interactions between neuraminidase mutations facilitated the emergence of the oseltamivir-resistant H1N1 influenza viruses.

Duan S, Govorkova EA, Bahl J, Zaraket H, Baranovich T, Seiler P, Prevost K, Webster RG, Webby RJ - Nat Commun (2014)

Bottom Line: Although several neuraminidase substitutions were found to be necessary to counteract the adverse effects of H275Y, the order and impact of evolutionary events involved remain elusive.Of the 12 neuraminidase substitutions that occurred during 1999-2009, 5 (chronologically, V234M, R222Q, K329E, D344N, H275Y and D354G) are necessary for maintaining full neuraminidase function in the presence of the H275Y mutation by altering protein accumulation or enzyme affinity/activity.The sequential emergence and cumulative effects of these mutations clearly illustrate a role for epistasis in shaping the emergence and subsequent evolution of a drug-resistant virus population, which can be useful in understanding emergence of novel viral phenotypes of influenza.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, St Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, Tennessee 38105, USA.

ABSTRACT
Oseltamivir-resistant H1N1 influenza viruses carrying the H275Y neuraminidase mutation predominated worldwide during the 2007-2009 seasons. Although several neuraminidase substitutions were found to be necessary to counteract the adverse effects of H275Y, the order and impact of evolutionary events involved remain elusive. Here we reconstruct H1N1 neuraminidase phylogeny during 1999-2009, estimate the timing and order of crucial amino acid changes and evaluate their impact on the biological outcome of the H275Y mutation. Of the 12 neuraminidase substitutions that occurred during 1999-2009, 5 (chronologically, V234M, R222Q, K329E, D344N, H275Y and D354G) are necessary for maintaining full neuraminidase function in the presence of the H275Y mutation by altering protein accumulation or enzyme affinity/activity. The sequential emergence and cumulative effects of these mutations clearly illustrate a role for epistasis in shaping the emergence and subsequent evolution of a drug-resistant virus population, which can be useful in understanding emergence of novel viral phenotypes of influenza.

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Plaque size, NA activity, NA accumulation, and substrate affinity of the BR/59/07 NA proteins with different amino acid substitutions(a) Plaque size of viruses carrying ten different NA amino acid substitutions, in the absence (white) or presence (gray) of the H275Y mutation. The ten amino acid residues were replaced with the corresponding NC99-like consensus residues. (N45H+E78K were substituted together, as both reside in the stalk region.) Data represent mean ± s.e.m. diameter of ~10 randomly selected plaques. Dashed line indicates mean plaque diameter of wild-type NABR/59. (b) Enzyme activity of total NABR07 and NABR07-H275Y variant proteins with 5 different amino acid substitutions, expressed by equal quantities (0.5 μg) of the respective plasmids. Dotted and dashed lines indicate mean activity of NABR/59and NABR/59-H275Y, respectively. (c) Representative western blot showing total accumulation of protein variants with substitutions; the variants were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA, respectively. Panels show detection by anti-HA-tag (upper), anti-β actin (center), and anti-GFP (lower). (d) NA Km values of rgBR/59/07 viruses carrying the respective NA amino acid substitutions in the absence and presence of the H275Y mutation. Dotted and dashed lines indicate the values of NABR/59and NABR/59-H275Y, respectively. All graphs represent mean ± s.e.m. of three to five times independent experiments. * p<0.05, two-tailed t test, versus NABR/59; † p<0.05, two-tailed t test, versus NABR/59-H275Y.
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Figure 4: Plaque size, NA activity, NA accumulation, and substrate affinity of the BR/59/07 NA proteins with different amino acid substitutions(a) Plaque size of viruses carrying ten different NA amino acid substitutions, in the absence (white) or presence (gray) of the H275Y mutation. The ten amino acid residues were replaced with the corresponding NC99-like consensus residues. (N45H+E78K were substituted together, as both reside in the stalk region.) Data represent mean ± s.e.m. diameter of ~10 randomly selected plaques. Dashed line indicates mean plaque diameter of wild-type NABR/59. (b) Enzyme activity of total NABR07 and NABR07-H275Y variant proteins with 5 different amino acid substitutions, expressed by equal quantities (0.5 μg) of the respective plasmids. Dotted and dashed lines indicate mean activity of NABR/59and NABR/59-H275Y, respectively. (c) Representative western blot showing total accumulation of protein variants with substitutions; the variants were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA, respectively. Panels show detection by anti-HA-tag (upper), anti-β actin (center), and anti-GFP (lower). (d) NA Km values of rgBR/59/07 viruses carrying the respective NA amino acid substitutions in the absence and presence of the H275Y mutation. Dotted and dashed lines indicate the values of NABR/59and NABR/59-H275Y, respectively. All graphs represent mean ± s.e.m. of three to five times independent experiments. * p<0.05, two-tailed t test, versus NABR/59; † p<0.05, two-tailed t test, versus NABR/59-H275Y.

Mentions: We firstly adopted a strategy using plaque size to evaluate NA-mediated virus release during the replication cycle. A plaque is formed from a single virus particle via multiple cycles of replication, and release of the progeny viruses at each cycle is mediated by NA functionality. Thus NA mutation that could alter NA activity sufficiently would directly affect the speed of virus release and consequently determine the plaque size. We first validated the feasibility of this strategy by confirming that plaque size paralleled NA enzyme activity in two pairs of reverse-genetics viruses, a BR07-like pair (NABR/59 and NABR/59-H275Y) and an NC99-like pair (NAGA/17 and NAGA/20 (H275Y)) (each pair differs only at position 275) (Supplementary Fig. 4a, b). We next generated rgBR/59/07 viruses whose NA proteins carried the respective substitutions N45H+E78K, G214E, Q222R, M234V, K249G, I287T, E329K, N344D, and D354G (representing a change from BR07-like to NC99-like), in the presence and absence of H275Y. Four substitutions (Q222R, M234V, E329K, and N344D) in NABR/59 significantly reduced plaque size, with and/or without the H275Y (Fig. 4a, Supplementary Fig. 5a). These four positions and the final substitution site (354) were selected for further evaluation; the remaining five substitutions were unlikely to reduce virus growth appreciably and were not further evaluated.


Epistatic interactions between neuraminidase mutations facilitated the emergence of the oseltamivir-resistant H1N1 influenza viruses.

Duan S, Govorkova EA, Bahl J, Zaraket H, Baranovich T, Seiler P, Prevost K, Webster RG, Webby RJ - Nat Commun (2014)

Plaque size, NA activity, NA accumulation, and substrate affinity of the BR/59/07 NA proteins with different amino acid substitutions(a) Plaque size of viruses carrying ten different NA amino acid substitutions, in the absence (white) or presence (gray) of the H275Y mutation. The ten amino acid residues were replaced with the corresponding NC99-like consensus residues. (N45H+E78K were substituted together, as both reside in the stalk region.) Data represent mean ± s.e.m. diameter of ~10 randomly selected plaques. Dashed line indicates mean plaque diameter of wild-type NABR/59. (b) Enzyme activity of total NABR07 and NABR07-H275Y variant proteins with 5 different amino acid substitutions, expressed by equal quantities (0.5 μg) of the respective plasmids. Dotted and dashed lines indicate mean activity of NABR/59and NABR/59-H275Y, respectively. (c) Representative western blot showing total accumulation of protein variants with substitutions; the variants were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA, respectively. Panels show detection by anti-HA-tag (upper), anti-β actin (center), and anti-GFP (lower). (d) NA Km values of rgBR/59/07 viruses carrying the respective NA amino acid substitutions in the absence and presence of the H275Y mutation. Dotted and dashed lines indicate the values of NABR/59and NABR/59-H275Y, respectively. All graphs represent mean ± s.e.m. of three to five times independent experiments. * p<0.05, two-tailed t test, versus NABR/59; † p<0.05, two-tailed t test, versus NABR/59-H275Y.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Plaque size, NA activity, NA accumulation, and substrate affinity of the BR/59/07 NA proteins with different amino acid substitutions(a) Plaque size of viruses carrying ten different NA amino acid substitutions, in the absence (white) or presence (gray) of the H275Y mutation. The ten amino acid residues were replaced with the corresponding NC99-like consensus residues. (N45H+E78K were substituted together, as both reside in the stalk region.) Data represent mean ± s.e.m. diameter of ~10 randomly selected plaques. Dashed line indicates mean plaque diameter of wild-type NABR/59. (b) Enzyme activity of total NABR07 and NABR07-H275Y variant proteins with 5 different amino acid substitutions, expressed by equal quantities (0.5 μg) of the respective plasmids. Dotted and dashed lines indicate mean activity of NABR/59and NABR/59-H275Y, respectively. (c) Representative western blot showing total accumulation of protein variants with substitutions; the variants were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA, respectively. Panels show detection by anti-HA-tag (upper), anti-β actin (center), and anti-GFP (lower). (d) NA Km values of rgBR/59/07 viruses carrying the respective NA amino acid substitutions in the absence and presence of the H275Y mutation. Dotted and dashed lines indicate the values of NABR/59and NABR/59-H275Y, respectively. All graphs represent mean ± s.e.m. of three to five times independent experiments. * p<0.05, two-tailed t test, versus NABR/59; † p<0.05, two-tailed t test, versus NABR/59-H275Y.
Mentions: We firstly adopted a strategy using plaque size to evaluate NA-mediated virus release during the replication cycle. A plaque is formed from a single virus particle via multiple cycles of replication, and release of the progeny viruses at each cycle is mediated by NA functionality. Thus NA mutation that could alter NA activity sufficiently would directly affect the speed of virus release and consequently determine the plaque size. We first validated the feasibility of this strategy by confirming that plaque size paralleled NA enzyme activity in two pairs of reverse-genetics viruses, a BR07-like pair (NABR/59 and NABR/59-H275Y) and an NC99-like pair (NAGA/17 and NAGA/20 (H275Y)) (each pair differs only at position 275) (Supplementary Fig. 4a, b). We next generated rgBR/59/07 viruses whose NA proteins carried the respective substitutions N45H+E78K, G214E, Q222R, M234V, K249G, I287T, E329K, N344D, and D354G (representing a change from BR07-like to NC99-like), in the presence and absence of H275Y. Four substitutions (Q222R, M234V, E329K, and N344D) in NABR/59 significantly reduced plaque size, with and/or without the H275Y (Fig. 4a, Supplementary Fig. 5a). These four positions and the final substitution site (354) were selected for further evaluation; the remaining five substitutions were unlikely to reduce virus growth appreciably and were not further evaluated.

Bottom Line: Although several neuraminidase substitutions were found to be necessary to counteract the adverse effects of H275Y, the order and impact of evolutionary events involved remain elusive.Of the 12 neuraminidase substitutions that occurred during 1999-2009, 5 (chronologically, V234M, R222Q, K329E, D344N, H275Y and D354G) are necessary for maintaining full neuraminidase function in the presence of the H275Y mutation by altering protein accumulation or enzyme affinity/activity.The sequential emergence and cumulative effects of these mutations clearly illustrate a role for epistasis in shaping the emergence and subsequent evolution of a drug-resistant virus population, which can be useful in understanding emergence of novel viral phenotypes of influenza.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, St Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, Tennessee 38105, USA.

ABSTRACT
Oseltamivir-resistant H1N1 influenza viruses carrying the H275Y neuraminidase mutation predominated worldwide during the 2007-2009 seasons. Although several neuraminidase substitutions were found to be necessary to counteract the adverse effects of H275Y, the order and impact of evolutionary events involved remain elusive. Here we reconstruct H1N1 neuraminidase phylogeny during 1999-2009, estimate the timing and order of crucial amino acid changes and evaluate their impact on the biological outcome of the H275Y mutation. Of the 12 neuraminidase substitutions that occurred during 1999-2009, 5 (chronologically, V234M, R222Q, K329E, D344N, H275Y and D354G) are necessary for maintaining full neuraminidase function in the presence of the H275Y mutation by altering protein accumulation or enzyme affinity/activity. The sequential emergence and cumulative effects of these mutations clearly illustrate a role for epistasis in shaping the emergence and subsequent evolution of a drug-resistant virus population, which can be useful in understanding emergence of novel viral phenotypes of influenza.

Show MeSH
Related in: MedlinePlus