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

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.

Figure 1. Temporally-structured maximum clade credibility phylogenetic tree of the NA gene of seasonal H1N1 viruses, showing the timeline of amino acid substitutions

Ancestral state reconstruction of tested amino acids is annotated on the tree backbone to indicate when specific mutations were fixed in the population. The trunk color change indicates a >95% posterior probability of amino acid fixation. Purple bars on tree nodes indicate the Bayesian credible intervals of ancestral divergence time estimates (>50% posterior probability). Isolates with H275Y mutation prior to H275Y fixation into the population are indicated with red branches.

Figure 2. NA protein phenotypes of seasonal H1N1 influenza viruses of the NC99 and BR07 lineages

(a) Neuraminidase enzyme activity of total NA proteins expressed by equal quantities (0.5 μg) of the respective plasmids. Rep: representative NA protein of the indicated lineage; Int: intermediate NA protein of the indicated lineage, fully or partially conforming to the 10 consensus lineage residues. The residue at the NA protein 275 position (wild-type [wt] or H275Y) is indicated in parentheses. NA-H275Y is shown when the H275Y was introduced by mutagenesis. Data were normalized to NA^BR07 protein. Dotted and dashed lines indicate the activity level of lineage-representative NA^GA/17 and NA^BR/59 proteins, respectively. (b) Representative western blot showing total accumulation of NA protein variants expressed by equal quantities (0.5 μg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA proteins, respectively. Panels show detection by anti-HA-tag (upper), anti-β actin (middle), and anti-GFP (lower). (c) Representative flow cytometric plots of cell surface accumulation of NA protein variants expressed by equal quantities (0.5 μg) of respective plasmids. The pairs differed only at position 275, with the exception of the last panel, the BR07-like NAs. (d) Km values of the NA proteins. The dotted and dashed lines indicate the value of lineage-representative NA^GA/17 and NA^BR/59, respectively. All graphs show mean ± s.e.m. from three to five times independent experiments. * p<0.05, two-tailed t test, versus NA^{BR/59; †} p<0.05, two-tailed t test, versus its counterpart wt NA at left side.

Figure 3. Transmissibility of seasonal H1N1 influenza viruses of the NC99 and BR07 lineages in ferrets

(a) Virus titers in nasal wash samples from individual donor ferrets (black), direct-contact (DC) ferrets (blue), and respiratory-droplet (RD) ferrets (red). Arrows indicate the day (day 1 p.i for donors) when recipient ferrets were housed with donor ferrets. (b) Virus titers in DC ferret nasal wash samples at day 1 post-contact. Each dot represents an individual ferret. * p<0.05, two-tailed t test, versus MEM/13. (c) Virus titers in RD ferret nasal wash samples on the first day of detection (the day post-contact), which is indicated above each column.

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 NA^BR/59. (b) Enzyme activity of total NA^BR07 and NA^BR07-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 NA^BR/59and NA^BR/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 NA^BR/59and NA^BR/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 NA^BR/59; † p<0.05, two-tailed t test, versus NA^BR/59-H275Y.

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 (NA^GA/17and NA^{GA/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 NA^GA/17 or NA^{GA/20 (H275Y)} with the indicated single substitutions. Dotted and dashed lines indicate Km values of NA^GA/17 and NA^{GA/20 (H275Y)}, respectively. (c) Representative western blot showing total accumulation of NA^GA/17 and NA^{GA/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 NA^GA/17 and NA^{GA/20 (H275Y)} with sequentially added substitutions. Dotted and dashed lines indicate Km values of NA^GA/17 and NA^{GA/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 NA^GA/17; † p<0.05, two-tailed t test, versus NA^{GA/20 (H275Y)}.

Figure 6. Defects in enzyme activity and protein accumulation caused by the H275Y mutation in other N1-subtype NA proteins

Each panel shows a pair of NA protein variants differing only in the absence or presence of the H275Y mutation, including two pairs of pdmH1N1 NA proteins (a, b) and two pairs of hpH5N1 NA proteins (c, d). Enzyme activity of total accumulated NA protein is shown at left in each panel; Representative flow cytometry plots showing surface accumulation are at right. NA proteins were expressed by equal quantities (0.5 μg) of respective plasmids. Data are the mean ± s.e.m. of at four determinations. * p<0.05, two-tailed t test, versus its counterpart wild-type NA.