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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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Total protein accumulation and Km values of the NC99-like NA proteins with different amino acid substitutions(a) Representative western blot showing total accumulation of the NC99-like NA proteins (NAGA/17and NAGA/20 (H275Y)) with the indicated single substitutions; the variant NAs were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA proteins, respectively. (b) NA Km values of rgBR/59/07 viruses carrying NAGA/17 or NAGA/20 (H275Y) with the indicated single substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. (c) Representative western blot showing total accumulation of NAGA/17 and NAGA/20 (H275Y) proteins with sequentially added substitutions (sub), the order is the same in next panel; NAs were expressed by equal quantities (0.5 μg) of respective plasmids. (d) NA Km values of rgBR/59/07 viruses carrying the NAGA/17 and NAGA/20 (H275Y) with sequentially added substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. All graphs show mean ± s.e.m. of three to five times independent experiments. *p<0.05, two-tailed t test, versus NAGA/17; † p<0.05, two-tailed t test, versus NAGA/20 (H275Y).
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Figure 5: Total protein accumulation and Km values of the NC99-like NA proteins with different amino acid substitutions(a) Representative western blot showing total accumulation of the NC99-like NA proteins (NAGA/17and NAGA/20 (H275Y)) with the indicated single substitutions; the variant NAs were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA proteins, respectively. (b) NA Km values of rgBR/59/07 viruses carrying NAGA/17 or NAGA/20 (H275Y) with the indicated single substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. (c) Representative western blot showing total accumulation of NAGA/17 and NAGA/20 (H275Y) proteins with sequentially added substitutions (sub), the order is the same in next panel; NAs were expressed by equal quantities (0.5 μg) of respective plasmids. (d) NA Km values of rgBR/59/07 viruses carrying the NAGA/17 and NAGA/20 (H275Y) with sequentially added substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. All graphs show mean ± s.e.m. of three to five times independent experiments. *p<0.05, two-tailed t test, versus NAGA/17; † p<0.05, two-tailed t test, versus NAGA/20 (H275Y).

Mentions: We next tested whether the molecular determinants identified above were sufficient to explain the NA phenotypic transformations that accompanied the NC99-to-BR07 transition. The V234M, R222Q, K329E and D344N mutations (representing a change from NC99-like to BR07- like) were introduced into the NAGA/17 and NAGA/20 (H275Y) (already possessing the D354G mutation) NC99-like proteins, which differed only at position 275. All substitutions, except D344N, significantly increased the functional activity of NAGA/17 and, to a lesser extent, of NAGA/20 (H275Y) (Supplementary Fig. 6a). Enzyme activity paralleled the plaque size of the mutant viruses (Supplementary Fig. 6b). In NAGA/17, two substitutions (R222Q and V234M)greatly increased NA protein accumulation (Fig. 5a), and three (R222Q, K329E and D344N) variably reduced the Km value (Fig. 5b); all of these changes occurred to a lesser extent in NAGA/20 (H275Y). These findings confirmed that these four substitutions play a role in increasing overall NA function and in reducing the H275Y-generated defects, although no single substitution could alone fully transform the NA phenotype and fully offset the H275Y-generated defects. Thus, the intermediate level of NA accumulation observed in NAMS/03, NAMS/03 (H275Y) (which had acquired R222Q and D344N), and unusually high Km value in NANY/3467 (H275Y) (which had not acquire the D344N) (Fig. 2) can be explained by partial acquisition of these phenotypic determinants.


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)

Total protein accumulation and Km values of the NC99-like NA proteins with different amino acid substitutions(a) Representative western blot showing total accumulation of the NC99-like NA proteins (NAGA/17and NAGA/20 (H275Y)) with the indicated single substitutions; the variant NAs were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA proteins, respectively. (b) NA Km values of rgBR/59/07 viruses carrying NAGA/17 or NAGA/20 (H275Y) with the indicated single substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. (c) Representative western blot showing total accumulation of NAGA/17 and NAGA/20 (H275Y) proteins with sequentially added substitutions (sub), the order is the same in next panel; NAs were expressed by equal quantities (0.5 μg) of respective plasmids. (d) NA Km values of rgBR/59/07 viruses carrying the NAGA/17 and NAGA/20 (H275Y) with sequentially added substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. All graphs show mean ± s.e.m. of three to five times independent experiments. *p<0.05, two-tailed t test, versus NAGA/17; † p<0.05, two-tailed t test, versus NAGA/20 (H275Y).
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Figure 5: Total protein accumulation and Km values of the NC99-like NA proteins with different amino acid substitutions(a) Representative western blot showing total accumulation of the NC99-like NA proteins (NAGA/17and NAGA/20 (H275Y)) with the indicated single substitutions; the variant NAs were expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA proteins, respectively. (b) NA Km values of rgBR/59/07 viruses carrying NAGA/17 or NAGA/20 (H275Y) with the indicated single substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. (c) Representative western blot showing total accumulation of NAGA/17 and NAGA/20 (H275Y) proteins with sequentially added substitutions (sub), the order is the same in next panel; NAs were expressed by equal quantities (0.5 μg) of respective plasmids. (d) NA Km values of rgBR/59/07 viruses carrying the NAGA/17 and NAGA/20 (H275Y) with sequentially added substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. All graphs show mean ± s.e.m. of three to five times independent experiments. *p<0.05, two-tailed t test, versus NAGA/17; † p<0.05, two-tailed t test, versus NAGA/20 (H275Y).
Mentions: We next tested whether the molecular determinants identified above were sufficient to explain the NA phenotypic transformations that accompanied the NC99-to-BR07 transition. The V234M, R222Q, K329E and D344N mutations (representing a change from NC99-like to BR07- like) were introduced into the NAGA/17 and NAGA/20 (H275Y) (already possessing the D354G mutation) NC99-like proteins, which differed only at position 275. All substitutions, except D344N, significantly increased the functional activity of NAGA/17 and, to a lesser extent, of NAGA/20 (H275Y) (Supplementary Fig. 6a). Enzyme activity paralleled the plaque size of the mutant viruses (Supplementary Fig. 6b). In NAGA/17, two substitutions (R222Q and V234M)greatly increased NA protein accumulation (Fig. 5a), and three (R222Q, K329E and D344N) variably reduced the Km value (Fig. 5b); all of these changes occurred to a lesser extent in NAGA/20 (H275Y). These findings confirmed that these four substitutions play a role in increasing overall NA function and in reducing the H275Y-generated defects, although no single substitution could alone fully transform the NA phenotype and fully offset the H275Y-generated defects. Thus, the intermediate level of NA accumulation observed in NAMS/03, NAMS/03 (H275Y) (which had acquired R222Q and D344N), and unusually high Km value in NANY/3467 (H275Y) (which had not acquire the D344N) (Fig. 2) can be explained by partial acquisition of these phenotypic determinants.

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