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J Virol. 2011 May; 85(9): 4596–4601.
PMCID: PMC3126221

Impact of Amino Acid Mutations in PB2, PB1-F2, and NS1 on the Replication and Pathogenicity of Pandemic (H1N1) 2009 Influenza Viruses[down-pointing small open triangle]


Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.

Although the pandemic (H1N1) 2009 influenza A viruses that emerged in the early spring of 2009 circulate widely (4), most infected individuals experience an uncomplicated upper respiratory disorder. These clinical manifestations indicate that the pathogenicity of the pandemic (H1N1) 2009 viruses in most humans is relatively mild, possibly due to preexisting cross-reactive immunity as exemplified by an appreciable immune response upon a single vaccination (7, 20, 28). Nevertheless, these viruses are clearly more pathogenic than seasonal influenza viruses in animal models (11, 16, 18) and in some humans (13). Moreover, their sustained transmission among humans may lead to the emergence of more pathogenic mutants, as has been suggested for the 1918 influenza pandemic virus (25).

PB1-F2, which is carried on a +1 reading frame of the gene segment for influenza viral RNA polymerase subunit PB1 (1), is a known virulence factor (3, 17, 27). Specifically, an asparagine-to-serine substitution at position 66 (F2-N66S), which is found in 1918 pandemic viruses, is partly responsible for the high pathogenicity of this virus (3). Most pandemic (H1N1) 2009 viruses, however, possess three stop codons in the region encoding amino acid positions 12, 58, and 88 in the PB1-F2 reading frame and, therefore, do not express full-length PB1-F2. Hai et al. (8) demonstrated that pandemic (H1N1) 2009 viruses possessing full-length PB1-F2 with or without F2-N66S do not exhibit increased in vitro and in vivo growth kinetics or mouse pathogenicity; however, the phenotype of viruses possessing shorter PB1-F2 proteins (i.e., of 57 or 87 amino acids) has not been studied. Such studies have become even more relevant with the recent isolation of three pandemic (H1N1) 2009 virus isolates (A/Wisconsin/629-D00485/2009 [GenBank accession number CY057428], A/Wisconsin/629-D00832/2009 [GenBank accession number CY057412], and A/Puerto Rico/51/2009 [GISAID {http://platform.gisaid.org/dante-cms/live/struktur.jdante?aid=1131} accession number EPI252209]) with a stop-to-leucine substitution at position 12 in the PB1-F2 (F2-stop12L) protein, hence creating a PB1-F2 protein of 57 amino acids in length.

NS1 is an influenza viral interferon antagonist (6). Its C-terminal four residues in most humans and several animal isolates possess an X-S/T-X-V-type PDZ ligand motif (21), which modulates protein-protein interactions in a sequence-specific fashion (22). These four residues are also thought to influence mouse pathogenicity (12). However, the pandemic (H1N1) 2009 viruses possess a truncated NS1 protein due to a stop codon in the region encoding amino acid position 220. Moreover, elimination of the stop codon would result in an NS1 protein carrying GTEI at positions 227 to 230, which deviates from the X-S/T-X-V-type PDZ ligand motifs. Hale et al. (9) characterized a pandemic (H1N1) 2009 virus that possesses the full-length NS1 protein carrying GTEI at positions 227 to 230 in vitro and in vivo: however, the effect of the PDZ ligand motifs on the pathogenicity of this virus remains unknown.

Here, we assessed the impact of PB1-F2 and NS1 mutations that are known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also examined the phenotype of viruses possessing the newly identified F2-stop12L mutants with and without mutations known to affect pathogenicity in the viral RNA polymerase subunit PB2 and in NS1.

To assess the effect of PB1-F2 mutations on the replication and pathogenicity of pandemic (H1N1) 2009 viruses, we performed reverse genetics (19) to generate A/California/04/09 (H1N1; CA04) mutants possessing one of the following single or multiple mutations in the PB1-F2 reading frame: (i) a stop-to-serine substitution at position 12 (F2-stop12S) (note that the F2-stop12S mutants were generated and tested before natural isolates with a leucine at this position were reported; serine was chosen at that time because this mutation does not affect the PB1 amino acid composition); (ii) F2-stop12S and a stop-to-tryptophan substitution at position 58 (F2-stop58W); (iii) F2-stop12S, F2-stop58W, and a stop-to-tryptophan substitution at position 88 (F2-stop88W); (iv) F2-stop12S, F2-stop58W, and F2-N66S (F2-stop58W/N66S); and (v) F2-stop12S, F2-stop58W, F2-N66S, and F2-stop88W (F2-stop88W/N66S) (Fig. 1A). None of these PB1-F2 mutations changes the PB1 amino acid composition. The F2-stop88W and F2-stop88W/N66S mutants are identical to the mutants assessed by Hai et al. (8). To determine the in vitro growth kinetics of these PB1-F2 mutants, we infected MDCK cells with viruses at a multiplicity of infection (MOI) of 0.001. Supernatants of the infected cells were harvested at 12, 24, 48, and 72 h postinfection (p.i.) and subjected to plaque assays for virus titration. No substantial differences were observed in in vitro growth kinetics between wild-type and mutant viruses (Fig. 2A). Further, to assess the mouse pathogenicity of these CA04 PB1-F2 mutants, three mice (6-week-old female BALB/c mouse; Jackson Laboratory, Bar Harbor, ME) per group were intranasally inoculated with 105 PFU (50 μl) of virus and monitored daily for 14 days for changes in body weight. None of the virus-infected mice exhibited severe weight loss during the monitoring period (Fig. 3A). Hai et al. (8) also found that the F2-stop88W and F2-stop88W/N66S mutants were comparable to the wild-type virus in terms of replication in MDCK cells and mouse pathogenicity. These results indicate that the PB1-F2 mutations tested had limited effect on the in vitro growth kinetics and pathogenicity of pandemic (H1N1) 2009 viruses.

Fig. 1.
Schematic diagrams of the wild-type and mutant genes tested in this study. (A) PB1-F2 variants. Black bars represent stop codons in the regions encoding positions 12, 58, and 88. Red bars represent the stop12S, stop58W, and stop88W substitutions. Green ...
Fig. 2.
In vitro growth kinetics of CA04 mutants. MDCK cells were infected with wild-type CA04 (A to D), PB1-F2 mutants (A), NS1 mutants possessing stop220W (B), NS1 mutants possessing stop220R (C), or PB2, PB1-F2, and NS1 combination mutants (D) at an MOI of ...
Fig. 3.
Body weight changes in mice infected with CA04 mutants. BALB/c mice were infected with 105 PFU of wild-type CA04 (A to D), PB1-F2 mutants (A), NS1 mutants possessing stop220W (B), NS1 mutants possessing stop220R (C), or PB2 mutants (D). Body weights were ...

We also assessed the effect of NS1 mutations on virus replication and pathogenicity of pandemic (H1N1) 2009 viruses by using reverse genetics-derived CA04 mutants with one of the following NS1 mutations: (i) a stop-to-tryptophan substitution at position 220 (NS1-stop220W) (tryptophan is commonly observed at this position in the classical swine virus lineage whose NS gene is an ancestor of that of the pandemic [H1N1] 2009 virus [4]; note that this mutation results in a glutamic acid-to-glycine substitution at position 63 in another protein encoded by the NS gene, NS2); (ii) NS1-stop220W and a C-terminal GTEI-to-RSEV substitution (creating a seasonal H1N1 virus-type PDZ ligand motif) (NS1-W-RSEV); (iii) NS1-stop220W and a GTEI-to-RSKV substitution (creating a seasonal H3N2 virus-type PDZ ligand motif) (NS1-W-RSKV); (iv) NS1-stop220W and a C-terminal GTEI-to-KSEV substitution (creating a 1918 pandemic virus-type PDZ ligand motif) (NS1-W-KSEV); and (v) NS1-stop220W and a C-terminal GTEI-to-ESEV substitution (creating a highly pathogenic H5N1 virus-type PDZ ligand motif) (NS1-W-ESEV) (Fig. 1B). While the KSEV and ESEV mutations resulted in a glycine-to-serine substitution at position 70 in NS2 (as found in 1918 pandemic and H5N1 viruses), the remaining PDZ ligand motif mutations did not change the NS2 amino acid composition. Further, we prepared additional versions of these NS1 mutants with a stop-to-arginine substitution at position 220 (NS1-stop220R), which is found in most of human isolates and does not change the NS2 amino acid composition, i.e., NS1-stop220R, NS1-R-RSEV, NS1-R-RSKV, NS1-R-KSEV, and NS1-R-ESEV (Fig. 1B). The NS1-stop220R mutant is identical to one of the mutants assessed by Hale et al. (9).

Consistent with the earlier report that the NS1-stop220W mutant replicates comparably to wild-type virus in primary differentiated human tracheobronchial epithelial cells and a swine cell line (9), its growth kinetics in MDCK cells was also comparable to that of wild-type virus; however, different PDZ ligand motifs at the C terminus of NS1 affected virus replication to some extent (Fig. 2B). Interestingly, the three mutants that exhibited slightly increased titers compared to wild-type virus at 48 and 72 h p.i., NS1-W-RSEV, -KSEV, and -ESEV, share the amino acid sequence SEV at the last three residues of the PDZ ligand motif. In contrast, the SEV motif at the three C-terminal amino acids of NS1 did not increase viral replication in the context of NS1-stop220R (Fig. 2C). These results indicate that the X-S-E-V PDZ ligand motif in NS1 contributes to the efficient replication of pandemic (H1N1) 2009 viruses in cell culture in concert with a tryptophan, but not an arginine, at position 220.

In mice intranasally inoculated with 105 PFU (50 μl) of virus, the NS1-W-RSEV, -RSKV, and -ESEV mutants exhibited higher pathogenicity, as measured by body weight loss, than the wild-type virus (Fig. 3B). The mouse pathogenicity of the three mutants, NS1-W-RSEV, -RSKV, and -ESEV, was tested further by infecting animals with different doses of virus (104, 105, or 106 PFU) (Fig. 4A to C and data not shown). In animals infected with 104 and 105 PFU of virus, all three mutants caused slightly more weight loss than was caused by the wild-type virus. More importantly, all mice infected with 106 PFU of NS1-W-RSKV died by day 5 p.i. (Fig. 4C), in contrast to animals infected with the wild type and the other two mutant viruses; these data confirm the substantially increased pathogenicity of the NS1-W-RSKV mutant. In contrast, NS1-R-RSEV and -RSKV were attenuated in mice (Fig. 3C). The remaining NS1 mutants were comparable to wild-type virus in their pathogenicity in mice (Fig. 3B and C). The pathogenicity in mice of NS1-stop220R as reported by Hale et al. (9) was comparable to that shown in Fig. 3C.

Fig. 4.
Body weight changes in mice infected with CA04 NS1 and PB2, PB1, and NS1 combination mutants. BALB/c mice were infected with 104 (A, D), 105 (B, E), or 106 (C, F) PFU of wild-type CA04 (A to F), the NS1-W-RSKV mutant (A to C), the PB2-E627K+F2-stop12L+NS1-W-RSKV ...

To determine the in vivo virus replication levels of NS1 mutants, six mice per group were intranasally infected with 105 PFU of each mutant and three mice per group were euthanized on days 3 and 6 p.i. NS1 mutants possessing NS1-stop220W were comparable to wild-type virus in their replicative ability in mouse organs (Table 1). In contrast, the NS1 mutants possessing NS1-stop220R replicated poorly in mice compared to wild-type virus (Table 1), consistent with our earlier finding that these mutants cause less severe weight loss in mice than did wild-type virus. Hale et al. (9) also reported that the replication of NS1-stop220R was slightly impaired in mouse lungs. Collectively, these results indicate that the PDZ ligand motifs RSEV, RSKV, and ESEV, in combination with a tryptophan at position 220 in NS1, increase the mouse pathogenicity of a pandemic (H1N1) 2009 virus, although they do not substantially increase in vitro and in vivo virus replication.

Table 1.
Virus titers in organs of mice infected with CA04 mutantsa

In the spring of 2010, three pandemic (H1N1) 2009 viruses possessing F2-stop12L, which does not change the PB1 amino acid composition, were isolated. We evaluated the effect of this mutation with or without an NS1 mutation that exhibits increased pathogenicity in mice (NS1-W-RSKV) and one of the following two PB2 mutations: a glutamic acid-to-lysine change at position 627 (PB2-E627K) or an aspartic acid-to-asparagine substitution at position 701 (PB2-D701N). These two mutations are involved in adaptation (2, 5, 14, 24) and increased pathogenicity (10, 15, 23) of avian-origin influenza viruses to mammalian hosts. To this end, we generated CA04 mutants possessing the following mutations: (i) F2-stop12L (Fig. 1A); (ii) PB2-E627K, F2-stop12L, and NS1-W-RSKV (PB2-E627K+F2-stop12L+NS1-W-RSKV); and (iii) PB2-D701N, F2-stop12L, and NS1-W-RSKV (PB2-D701N+F2-stop12L+NS1-W-RSKV). We found that all three mutants possessing the F2-stop12L mutation replicated more efficiently in MDCK cells than did wild-type virus (Fig. 2D), in contrast to the F2-stop12S mutant, which was comparable in its replicative ability to wild-type virus (Fig. 2A). These findings indicate that F2-stop12L promotes the in vitro replication of pandemic (H1N1) 2009 viruses. However, we did not observe any synergistic effect of NS1-W-RSKV and PB2-E627K or PB2-D701N on in vitro replication.

Next, we tested the weight loss of mice infected with the above-described mutants. While the weight loss of mice infected with the F2-stop12L mutant was comparable to that of mice infected with wild-type virus, the two combination mutants PB2-E627K+F2-stop12L+NS1-W-RSKV and PB2-D701N+F2-stop12L+NS1-W-RSKV caused increased pathogenicity in mice infected with 106 PFU (Fig. 4D to F), as has been reported with mutant viruses possessing either PB2-E627K or PB2-D701N alone (Fig. 2D) (26). These combination mutants, however, did not kill mice even at 106 PFU, although the mutant virus possessing only NS1-W-RSKV exhibited mouse lethality (Fig. 4C); this finding suggests potential attenuating effects of the additional mutations in the viral background tested here. No significant differences in virus titers in mouse organs were observed between wild-type virus and the mutants tested (Table 1). Thus, F2-stop12L increases the replicative ability of pandemic (H1N1) 2009 viruses in cell culture, although an increase in mouse pathogenicity was not detectable.

In this study, we assessed the effects of previously identified pathogenic mutations in PB1-F2 and NS1 and of a recently identified PB1-F2 mutation and the synergistic effects of some of these mutations on the replication and mouse pathogenicity of pandemic (H1N1) 2009 influenza viruses.

As previously reported (8, 9), F2-stop88W, F2-stop88W/N66S, and NS1-stop220R mutations did not increase the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses (Fig. 2A and C and 3A and C). In contrast, our results show that NS1 mutants possessing stop220W in combination with an XSEV PDZ ligand motif (Fig. 2B), and the F2-stop12L mutant (Fig. 2D), replicated in MDCK cells more efficiently than did the wild-type virus. Further, NS1-W-RSEV, -RSKV, and -ESEV mutants exhibited increased pathogenicity in mice (Fig. 3B and 4A to C), while the virus titers in respiratory organs were comparable to those of the wild-type virus (Table 1). Interestingly, the PDZ ligand motif RSKV of seasonal H3N2 viruses, which are less pathogenic than the 1918 pandemic virus or highly pathogenic H5N1 viruses, conferred the highest pathogenicity in mice among the PDZ motives tested (Fig. 4A to C). These results suggest that the effect of the NS1 PDZ ligand motif on pathogenicity may differ depending upon the context of the viral gene backbone. In fact, the introduction of ESEV into the C terminus of the NS1 protein of the highly pathogenic A/Vietnam/1203/04 (H5N1) virus, whose NS1 protein does not possess the PDZ ligand motif, decreases mouse pathogenicity (29). No synergistic effects of F2-stop12L, NS1-W-RSKV, and PB2-E627K or PB2-D701N were observed in cell culture (Fig. 2D) or in mice (Fig. 4D to F and Table 1). The mouse pathogenicity of the two combination mutants (PB2-E627K+ F2-stop12L+NS1-W-RSKV and PB2-D701N+F2-stop12L+NS1-W-RSKV) may have been impaired compared to that of the NS1-W-RSKV mutant (Fig. 3B and 4A to C) because the F2-stop12L mutation may decrease the mouse pathogenicity of pandemic (H1N1) 2009 influenza viruses. In fact, the pathogenicity of the F2-stop12L mutant was slightly decreased compared to that of the wild-type virus in mice infected with 106 PFU of virus (Fig. 4D to F). Although some mutations affected replication but not pathogenicity or vice versa, these results show that certain mutations in NS1 and PB1-F2 can impact the pathogenicity of pandemic (H1N1) 2009 influenza viruses.


We thank Susan Watson for editing the manuscript.

This work was supported by ERATO (Japan Science and Technology Agency), by a grant-in-aid for Specially Promoted Research from the Ministries of Education, Culture, Sports, Science, and Technology, by grants-in-aid from Health, Labor, and Welfare of Japan, by a Contract Research Fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, by National Institute of Allergy and Infectious Disease Public Health Service research grants, and by an NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN266200700010C).


[down-pointing small open triangle]Published ahead of print on 16 February 2011.


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