Pandemic Influenza A Viruses Escape from Restriction by Human MxA through Adaptive Mutations in the Nucleoprotein

1Avian influenza A viruses sporadically transmit from waterfowl, their natural reservoir, into the human population –. These zoonotic viruses usually cannot propagate in the new human host, nor do they readily transmit between humans –. In rare cases, however, influenza A viruses of avian origin break the species barrier and establish new virus lineages in humans. In the last 100 years, the introduction of an influenza A virus with a novel nucleoprotein (NP) gene segment occurred only on two occasions, both of which led to pandemics: in 1918 (“Spanish” H1N1) an avian virus and in 2009 (pH1N1) a reassortant virus (comprising gene segments of two swine influenza viruses) established a stable lineage in humans,. In contrast, the 1957 (“Asian” H2N2) and the 1968 (“Hong-Kong” H3N2) pandemics were caused by genetic reassortment events, whereby the circulating human strains acquired some gene segments from avian sources but kept, among others, their 1918-derived NP.

2To overcome the species barrier, multiple adaptations to the new host are required. Theoretically, two categories of adaptive mechanisms can be envisaged. One comprises adaptations to cellular factors which promote viral infection yet differ between hosts. These include, for example, changes in the viral hemagglutinin during the adaptation of avian influenza A viruses to humans, or altered binding of viral proteins to different cellular importins,. The second category comprises adaptations to counteract cellular restriction factors that inhibit virus replication. These factors are part of the intrinsic and innate host defense mechanisms and may exert a strong selective pressure against newly invading viruses. Surprisingly little is known about adaptive mutations that overcome such host restriction factors and facilitate trans-species transmission of influenza viruses.

3The human interferon (IFN) system represents a major innate defense against zoonotic viruses. Among the many antiviral factors induced by IFNs, the MxA protein is one of the most potent characterized to date. It is a key effector molecule inhibiting influenza A virus as well as several other human RNA viruses,. MxA is a dynamin-like large GTPase which consists of an N-terminal globular GTPase domain, a bundle signaling element, and a C-terminal helical stalk. The recent atomic resolution of the MxA structure revealed that it forms stable tetramers and oligomers which assemble in a criss-cross manner via the stalk,. A current model proposes that, upon viral infection, MxA recognizes the incoming vRNPs and starts to self-assemble into rings, resulting in a higher-order oligomeric complex that blocks vRNP function,.

4In accordance with this model, recent findings suggest that NP determines the relative sensitivity of influenza A viruses toward the antiviral action of MxA. Avian influenza viruses were found to be generally more sensitive to MxA than human strains, which was subsequently shown through reassortant viruses to be dependent on the origin of NP. These findings suggest that human strains acquire adaptive mutations in NP to evade MxA restriction.

5Here, we identified the amino acids critical for MxA resistance in the two NP proteins introduced into the human population in 1918 (by the “Spanish” H1N1 influenza A virus) and in 2009 (by the pH1N1 strain). These residues clustered into two distinct but overlapping “patches” in the body domain of the protein. Introduction of these amino acids into an MxA-sensitive H5N1 NP was sufficient to render the avian polymerase resistant to MxA. Surprisingly, the resistance-associated substitutions resulted in impaired viral growth in both mammalian and avian cells when introduced into recombinant H5N1 virus A/Thailand/1(KAN-1)/04. The amino acid clusters identified here are highly conserved in circulating human isolates and virtually absent in NPs of avian influenza A viruses. Several of the amino acids that confer increased resistance to human MxA are also conserved in influenza A viruses of the classical swine lineage, correlating with resistance of these viruses to swine Mx1. These findings suggest that multiple adaptive amino acid changes would be required for H5N1 viruses to both escape from MxA restriction and maintain viral fitness. Partial adaptation in an intermediate host, such as the pig, might facilitate this demanding process.

6We have previously shown that the polymerase activity of A/Thailand/1(KAN-1)/04 (H5N1) is highly sensitive to inhibition by murine Mx1, a close homolog of human MxA, in a polymerase reconstitution assay, and that this sensitivity is determined by the NP gene. The H5N1 NP is of typical avian origin and resembles the avian H5N1 amino acid consensus sequence. We therefore used this assay to identify the amino acids in NP of either the 1918 (A/Brevig Mission/1/1918) or the 2009 (A/Hamburg/4/2009) pandemic H1N1 strain (Figure 1A) critical for Mx1 resistance. In this assay, the polymerase activity was measured in the presence of overexpressed Mx1. In addition, we determined the polymerase activity in the presence of the inactive mutant Mx1-K49A. Mx1 resistance was defined as the relative activity of the viral polymerase in the presence of Mx1 divided by the activity obtained with Mx1-K49A. Substitution of the H5N1 NP with the NP of the pandemic 1918 strain rendered the H5N1 polymerase largely Mx1-resistant (Figure 1B). An alignment of the amino acid sequences of the 1918 NP with the NP of the Mx1-sensitive H5N1 strain revealed differences at 14 positions, including 4 positions in the C-terminal domain, namely amino acids 373, 377, 473 and 482 (Figure 1A). An artificial chimera (1918*-NP), consisting of the N-terminal 365 amino acids of the 1918 NP and the C-terminal domain (amino acids 366 to 498) of the H5N1 NP, behaved like the full-length 1918 NP, indicating that the 4 C-terminal differences in the 1918 protein do not contribute to the Mx1 resistance phenotype (Figure 1B). To investigate which of the 10 remaining 1918-specific amino acids in the chimeric protein contributed to Mx1 resistance, 1918*-NP mutants harboring single H5N1-derived substitutions were tested. A major decrease in Mx1 resistance was observed for the mutations P283L and Y313F, while a less pronounced phenotype was observed for I100R and several other mutants (Figure 1C). Various combinations of these putative adaptive mutations revealed that the triple mutant I100R, P283L, and Y313F led to a similar degree of Mx1 sensitivity as observed using the H5N1 NP (Figure 1D), whereas combinations of the remaining seven mutations failed to reduce Mx1 resistance (Figure 1D). Consistently, the 1918*-NP, carrying the mutations I100R, P283L and Y313F reduced Mx1 resistance by 50% (as compared to 1918*-NP) also in the context of the 1918 polymerase (Figure S1A–B).

7To test whether the amino acids apparently responsible for Mx1 resistance of 1918 NP could also confer Mx1 resistance to an Mx1-sensitive NP, we introduced the mutations R100I, L283P, and F313Y into H5N1 NP. We also tested the exchange R100V in NP, since screening of the NCBI influenza database revealed that valine rather than isoleucine is commonly found at this position in seasonal strains. Single amino acid exchanges slightly increased Mx1 resistance, while the combination of all three substitutions resulted in resistance comparable to the 1918 NP, irrespective of I or V at position 100 (Figure 1E). Importantly, this enhanced Mx1 resistance was not simply achieved by a higher polymerase activity, as it did not strictly correlate with increased activity in the presence of the antivirally inactive mutant Mx1-K49A. Nevertheless, some Mx1 resistance-enhancing amino acids appeared to improve the polymerase activity for unknown reasons in the absence of Mx1 or presence of Mx1-K49A protein (Figure S1C).

8Next, we evaluated the capacity of NP from the 2009 pandemic H1N1 influenza A virus (pH1N1) to confer Mx1 resistance in the context of the H5N1 polymerase. Figure 2A shows that pH1N1 NP rendered the H5N1 polymerase activity relatively resistant to Mx1 inhibition, as previously reported. Sequence comparisons between the NP of pH1N1 and H5N1 origin revealed that pH1N1 NP carried only one (V100) out of the three Mx1 resistance determinants identified in 1918 NP (Figure 1A). We therefore assumed that different amino acids contribute to Mx1 resistance in pH1N1 NP than in 1918 NP. Since the pH1N1 NP differs by 32 amino acids from the H5N1 sequence (Figure 1A), we did not assay all individual amino acid positions, but rather focused on discordant and surface exposed amino acids in close proximity (27 Å) to the resistance cluster identified in the 1918 NP, utilizing the published NP crystal structures,. Five of the resulting 10 pH1N1-specific amino acids, which were closest to the 1918 resistance cluster, were analyzed in the H5N1 polymerase reconstitution assay using pH1N1 NP mutants harboring single H5N1-derived substitutions (Figure 2B). A significant decrease in Mx1 resistance was observed for the mutations D53E, H289Y and V313F, while a less pronounced phenotype was observed for V100R and K305R (Figure 2B). Next, pH1N1-specific amino acids were introduced into H5N1 NP and tested for their contributions to Mx1 resistance in the H5N1 polymerase reconstitution assay (Figure 2C). While no individual amino acid substitution had a major effect, the combination of mutations at 4 positions (E53D, R100V, Y289H, and F313V) enhanced Mx1 resistance to a similar extent as 1918 NP. The additional mutations R305K, I316M, T350K, R351K, V353I, and Q357K together further increased Mx1 resistance to the degree of pH1N1 NP (Figure 2C). Although we observed variations in NP expression levels (Figure 2C), these differences did not correlate with Mx sensitivity. Together, these results demonstrate that the cluster of amino acids conferring Mx1 resistance differs between the NP of the 2009 and 1918 pandemic strains, although the crucial residues in both cases are located in the surface-exposed body domain.

9Next, we investigated whether the identified amino acid clusters in NP of the 1918 and the pH1N1 strains also confer resistance to human MxA. Consistent with the findings observed with Mx1, both 1918 and pH1N1 NP increased resistance in the H5N1 polymerase reconstitution assay (Figure 3A), however, the 1918 NP confers greater resistance to human MxA than to murine Mx1, whereas the opposite is true for the pH1N1 NP (Figure 2C and 3A). Importantly, the mutant H5N1 NP containing the 1918-derived Mx1 resistance determinants R100V, L283P, and F313Y exhibited an MxA resistance approximately 85% of that by 1918 NP itself (Figure 3A). To identify additional amino acid residues that contribute to the increased MxA resistance of the 1918 NP, we changed single amino acid positions in the 1918*-NP to avian residues. This revealed that D16, in addition to V100, P283, and Y313, contributed to MxA resistance (Figure S2). To confirm the relevance of this finding, we tested H5N1 NP harboring all four mutations (G16D, R100V, L283P and F313Y). This mutant NP displayed an MxA resistance comparable to 1918 NP (Figure 3A). Next, the pH1N1-specific adaptive mutations were tested in the context of H5N1 NP. In particular E53D, R100V and F313V increased resistance to MxA, while introduction of the additional mutations Y289H, R305K, I316M, T350K, R351K V353I and Q357K was required to achieve a resistance comparable to pH1N1-NP (Figure 3A). These results indicate that the adaptive mutations in 1918 or pH1N1 NP lead to increased resistance for both murine Mx1 and human MxA. Again, the observed resistance towards MxA did not strictly correlate with polymerase activity (Figure S3).

10The atomic crystal structure of the H5N1 NP revealed that the 1918-specific amino acids 100, 283 and 313 form a surface exposed cluster in the body domain of the viral NP (Figure 3B). Amino acid 16 is located in the N-terminal region of NP that is predicted to form a flexible loop adjacent to the 1918 cluster (Figure S4). The amino acids forming the pH1N1 cluster are located in the same area of the NP body domain as the 1918 cluster (Figure 3C).

11V100, P283, and V/Y313 mainly responsible for Mx1 or MxA resistance (Figure 3B and C), we analyzed the NP sequences of various isolates deposited in the NCBI Influenza Virus Sequence Database. In avian isolates, each resistance-conferring amino acid could be identified in only ≤1% of the sequences (n = 5350) investigated (Table 1). In contrast, in classical seasonal human isolates representing H1N1, H1N2, H2N2, and H3N2 subtypes (n = 4969), the resistance-associated amino acids D16, I/V100, P283, and Y313 were each found at very high frequencies (>98%). Similarly, analyses of the human-derived pH1N1 NP sequences (n = 4104) revealed a high conservation of amino acids D53, I/V100 and V313 (>99%). Intriguingly, a chronological sequence comparison revealed that additional Mx resistance-enhancing mutations occurred in the NP of strains which are classified as descendents of the 1918 virus, namely R305K and R351K (Figure 4, Figure S5). These mutations emerged in early seasonal H1N1 viruses and were maintained in subsequent H2N2 and H3N2 strains (Figure 4). Taken together, these findings suggest a continuous selection pressure for increased Mx resistance in seasonal influenza viruses.

12Since pH1N1 NP is derived from an influenza A virus of the classical swine lineage,, we analyzed the NP sequences of a number (n = 393) of corresponding swine isolates obtained between 1930 and 2012. Amino acids I/V100 were highly conserved (>99%), but D53 and V313 were not present in any of the NP sequences, which instead harbored the avian consensus amino acids at these positions (Table 1). We therefore anticipated that NPs of the classical swine influenza strains would confer less MxA resistance than pH1N1 NP. Indeed, NP of one of the first swine isolates such as A/swine/Iowa/1976/1931 displayed comparatively poor MxA resistance in the H5N1 polymerase reconstitution assay (Figure 5A). Importantly, NP of the classical swine influenza A virus lineage acquired the additional mutations 305K, 351K, 353I and 357K over time (Figure 4), resulting in a gradual increase in MxA resistance (Figure 5A, pH1N1-NP-D53E, V313F, M316I, third column from the left and Figure S6A). These changes, however, were not sufficient to confer MxA resistance comparable to that of pH1N1 NP. Interestingly, NP of the recent triple reassortant swine isolate A/swine/Ohio/02026/2008 (H1N1) and a hypothetical NP precursor of pH1N1 (pH1N1-NP-D53E, V313F, M316I) share the adaptive mutations 100I/V, 289H, 305K, 350K, 351K, 353I and 357K. The hypothetical NP precursor of pH1N1 was created by altering the human pH1N1 specific positions D53, V313 and M316 to the consensus found in classical swine H1N1 strains (D53E, V313F, M316I). These mutations are found at the branching point of classical swine influenza viruses and pH1N1 viruses (Figure 4). The hypothetical NP precursor conferred only partial MxA resistance in the H5N1 (Figure 5A and Figure S6A) as well as in the pH1N1 background (Figure S6C). These findings suggested that further adaptive mutations are needed which may affect MxA recognition or otherwise improve NP functions such as binding to viral (e.g PB2 or cellular components (importins, helicases, ). Indeed, acquisition of E53D, F313V, and I316M was required to gain the full resistance of pH1N1 NP (Figure 5A and Figure S6C).

13The antiviral potency of Mx proteins of pigs is still insufficiently characterized –. In the present reconstitution assay using HEK293T cells, porcine Mx1 (Sus scrofa domestica) decreased the H5N1 polymerase activity to 50% (Figure 5B), whereas human MxA reduced the activity to approximately 10% (Figure 5A), despite similar expression levels of both Mx proteins (Figure 5C). Likewise, the H5N1 polymerase activity in the presence of different NPs from several distinct swine isolates was not significantly affected by porcine Mx1 (Figure 5B, Figure S6B). To further test the antiviral strength of porcine Mx, we used porcine cells for the polymerase reconstitution assay. We found essentially the same extent of inhibition by the porcine Mx1 as in human 293T cells. As shown in Figure S7A, porcine Mx1 reduced the H5N1 polymerase activity to ca. 50% in swine NPTr or NSK cells. In contrast, human MxA reduced the activity to 10% in porcine cells (Figure S7A). We conclude that the antiviral effect of porcine Mx1 is weak both in porcine and human cells. Together, these data suggest that while there is a clear selection pressure for swine influenza A viruses to acquire Mx resistance, the selection pressure in the porcine host is comparatively weak. Clearly, additional adaptive mutations are required to escape MxA restriction in humans.

14Re-transmission of pH1N1 from human to swine resulted in 17% of the documented cases in a substitution of aspartic acid at position 53 to the avian consensus glutamic acid (D53E) (swine pH1N1 in Table 1 and Figure 4), a mutation that confers loss of resistance to human MxA (Figure 5A), but not to porcine Mx1 (Figure 5B). This might suggest that MxA resistance-enhancing mutations are not necessarily favorable for NP function and might therefore cause impaired viral fitness.

15To compare the replication fitness of viruses containing MxA-sensitive or MxA-resistant NPs, we infected MDCKII cells (which do not express antivirally active Mx proteins ) with pH1N1 or mutant viruses with enhanced MxA sensitivity. A recombinant pH1N1 virus with the single D53E reversion (pH1N1-NP-D53E) grew equally well as the parental pH1N1 virus (Figure 5D). In contrast, the pH1N1 precursor virus lacking three MxA resistance-enhancing mutations (pH1N1-NP-D53E,V313F,M316I) grew to approximately one log10 higher infectious titers throughout the course of infection (Figure 5D). These results demonstrate that reversions to the original amino acids of the putative swine precursor virus (Figure 4) provided a strong growth advantage in the absence of an antivirally active Mx. Thus, the acquisition of MxA resistance appears to cause some growth disadvantage.

16To confirm this hypothesis, we tested the human H5N1 strain KAN-1 containing MxA resistance-enhancing mutations in MDCKII cells. Consistent with previous observations, the polymerase activity of H5N1 was not affected in the presence of canine Mx1 or Mx2 (Figure S7B). The triple mutant H5N1-NP-R100V,L283P,F313Y achieved reduced viral titers in the order of 1–2 log10 (Figure 5E). The recombinant H5N1 virus with the single MxA resistance mutation L283P (H5N1-NP-L283P) grew slightly less well than the parental H5N1 strain, while the double mutant virus H5N1-NP-R100I,F313Y showed comparable growth (Figure 5E). Intriguingly, the latter virus showed severely impaired replication efficiency in avian LMH cells which lack antiviral Mx proteins – (Figure S8). We conclude that MxA resistance is linked to impaired viral growth and may be easily lost in the absence of selective pressure.

17We argued that the acquisition of Mx resistance should also increase the pathogenicity of H5N1 in Mx1-positive mice. To test this hypothesis we selected the double mutant virus H5N1-NP-R100I,F313Y which had almost wild-type growth characteristics in tissue culture (Figure 5E) and exhibited comparable polymerase activity (Figure S8A), in spite of its Mx resistance-enhancing mutations (Figure 1E). We could not include the triple mutant virus H5N1-NP-R100V,L283P,F313Y in our studies due to the emergence of escape mutants (data not shown) and its strong attenuation. First, we compared the growth of wild-type and mutant H5N1 viruses in Mx1-negative BALB/c mice. Infection with 10 PFU of wild-type H5N1 virus lead to pronounced weight loss and death of all animals, as expected. In contrast, infection with the same challenge dose (10 PFU) of mutant H5N1-NP-R100I,F313Y virus resulted in survival of all BALB/c mice without significant weight loss (Figure 6A and B), indicating that the two amino acid substitutions associated with Mx resistance caused impaired viral growth. Indeed, challenge of BALB/c mice with 1000 PFU of the mutant virus resulted in viral lung titers that were 15-fold reduced as compared to wild-type virus at 48 h after infection (Figure 6C). Next, the growth properties of the two viruses were studied in congenic Mx1-positive mice. In these animals, infection with wild-type H5N1 virus produced no pronounced pathological effects, even at high doses of 106 PFU (Figure 6D and E). In contrast, Mx1-positive mice showed significant weight loss and mortality when challenged with 106 PFU of mutant H5N1-NP-R100I,F313Y virus (Figure 6D and E). To assess viral growth in Mx1-positive mice, viral lung titers were determined at various time points after intranasal infection with 104 PFU (Figure 6F). Two days after infection, the titers in mice infected with the mutant virus were approximately 28-fold lower than those in mice infected with the wild-type virus, demonstrating the attenuating effect of the Mx resistance-enhancing mutations in NP. Four days after infection, a 5-fold difference was observed, and 6 days after infection the mutant virus was still present in 5 out of 9 Mx1-positive mice with titers up to 2×105 PFU. In contrast, only low titers of wild-type virus were detected at the same time in 2 out of 9 animals. We conclude from these experiments that H5N1 viruses harboring MxA resistance-enhancing mutations partially overcome the antiviral effect mediated by Mx1.

18Influenza A viruses sporadically transmit from the avian reservoir into the human population. Here we describe specific mutations found in the NP of the 1918 and 2009 pandemic viruses that confer resistance to the IFN-induced human MxA GTPase, a major restriction factor for influenza and other orthomyxoviruses. As MxA strongly inhibits transcription and replication of the viral genome early in infection, its antiviral activity can be readily analyzed in polymerase reconstitution (minireplicon) assays,. Using this assay, we identified a cluster of surface-exposed amino acids in the body domain of NP crucial for Mx resistance. Interestingly, different amino acid positions were identified in 1918 and pH1N1 NP, yet all were located in the same domain. All resistance-associated amino acids are conserved in previous and current human influenza A viruses (Table 1), and the continuing acquisition of resistance-enhancing mutations (Figure 7) suggests strong positive selection pressure by MxA. Of note, mutations conferring MxA resistance are absent in avian influenza A viruses, although we did observe the emergence of adaptive NP mutations in avian-derived viruses circulating in swine (Figure 4 and Figure 7). These substitutions in NP not only increased resistance to swine Mx1 but also to human MxA, supporting the theory that swine are an excellent intermediate host for the generation of viruses with pandemic potential.

19Influenza A viruses carrying a novel NP gene were introduced into the human population in 1918 and 2009,, but the distinct MxA resistance clusters in the NP genes of these pandemic viruses suggest independent evolution (Figure 7). In case of the 2009 pH1N1 virus, the NP gene originated from the classical swine lineage, which itself is of avian origin. Our data suggest that the swine precursor virus of pH1N1 acquired additional amino acid changes, which together increased the ability to counteract human MxA. The evolution of the 1918 NP is comparatively less clear. Recent data suggest that both the 1918 and the classical swine virus lineage share a common avian ancestor. It is unresolved whether the avian precursor virus was first transmitted to humans and then onto swine or vice versa. In the latter case, the precursor virus may have first adapted to swine Mx1 through the mutation R100I in NP which is found in early swine isolates. In the first case, mutations at position 283 and 313 might have been lost after transmission from humans to swine, as observed with the pH1N1 virus. However, analyses of host specificity markers which discriminate human from avian influenza viruses indicate that four adaptive mutations in NP (16D, 283P, 313Y and 357K) were likely required for transmission of the 1918 precursor virus to humans. Remarkably, all of these mutations contribute to MxA resistance (Figure 3) and may have evolved in a pre-pandemic phase in the human population. Since circulating human influenza A virus strains maintain these adaptive mutations (Table 1), it is conceivable that viruses are under constant selection pressure mediated by MxA.

20We observed that the acquisition of Mx resistance had a negative effect on viral growth in the absence of MxA. When MxA resistance-enhancing mutations were introduced into highly pathogenic avian H5N1 viruses, the recombinant viruses grew less well than the wild-type H5N1 virus in MxA-negative MDCKII cells (Figure 5E), in BALB/c mice (Figure 6C) and even in avian cells (Figure S8). These mutations have no major effect in the viral polymerase reconstitution assay (Figure S1C and S3), and it is unclear which step of the viral replication cycle is attenuated in infected cells. If MxA resistance-associated amino acids are also counter-selected in circulating avian influenza strains, then the emergence of MxA resistance in the avian reservoir is expected to be an extremely rare event (Table 1). Of note, human H5N1 isolates have developed few if any of the identified MxA resistance-enhancing mutations (Table 1), most likely due to the associated strong attenuation (Figure 5E, 6A–C). Perhaps for this reason, the 1957 and 1968 pandemic viruses retained the well-adapted 1918-origin NP, despite acquiring other avian genome segments by reassortment. Thus, more than 90 years passed before a new NP lineage was established 2009 in humans, a process aided by gradual adaptation of the new NP in swine.

21Correspondingly, we observed an increase in viral growth of pH1N1 lacking the MxA resistance-enhancing mutations D53, V313 and M316 in MDCKII cells (Figure 5D) which do not express antivirally active Mx proteins. We therefore propose that passage of viruses in hosts with weak or inactive Mx proteins (such as swine or laboratory mouse strains, respectively) would result in a loss of MxA resistance. In fact, experimental adaptation of the pandemic 2009 virus to Mx1-negative mice led to the acquisition of mutations in NP which strongly diminished Mx resistance, such as D101G (Figure S9). Similarly, mutations conferring MxA resistance were lost (D53E, D101G, H289L) following re-transmission of pH1N1 from human to swine (Figure 4).

22The mechanism by which MxA exerts its antiviral function during infection or in the polymerase reconstitution assays is presently not known. MxA may block the viral life cycle at several early steps by interfering with the processes of vRNP entry and intracellular transport, as well as primary and secondary transcription. We proposed a model in which MxA recognizes vRNPs and begins to self-assemble into rings, thereby sterically inhibiting vRNP function,,. This model has recently been suggested also for mouse Mx1, but in a modified version involving in addition also the polymerase subunit PB2, in agreement with previous functional work implicating PB2 as putative target of mouse Mx1,. Modeling the present MxA resistance clusters into the available vRNP structure revealed that the sites on NP are most likely solvent-exposed, and thus accessible to cellular factors. Initial contact of MxA to single binding sites on NP might be weak but reinforced by oligomerization, involving multiple repetitive contacts exposed on the many NP molecules that form the vRNP. Weak but extensive contacts to repetitive viral target motifs have been demonstrated for other intracellular restriction factors. For example, TRIM5α specifically binds to several surface-exposed amino acids of the capsid protein of HIV-1, thereby forming an array or lattice on top of the viral capsid,. To date, physical interaction between influenza A virus NP and MxA could be demonstrated after covalent protein crosslinking and thus MxA might also bind to free NP in the cytoplasm thereby blocking polymerase activity indirectly. However, it is also likely that further cellular proteins modulate MxA activity and its interaction with viral proteins. Previous work identified a number of potential MxA co-factors, but their contribution is still unclear –. One promising candidate is the helicase UAP56, a DEAD box RNA helicase which was shown to prevent double-strand RNA formation and subsequent innate immune activation in influenza virus-infected cells. UAP56 binds both MxA and NP,, in the latter case via the N-terminal domain of NP which contains the MxA resistance-enhancing mutation G16D. Nonetheless, the significance of this observation and the role of UAP56 for antiviral activity remain to be demonstrated.

23In summary, we have found functional and evolutionary evidence that the human MxA GTPase provides an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Thus, the human MxA is a significant driving force in influenza A virus nucleoprotein evolution. We therefore propose that amino acids known to contribute to MxA resistance should be monitored as a strong indicator for the pandemic potential of newly emerging influenza A viruses.

24All animal experiments were performed in compliance with the German animal protection law (TierSchG). The mice were housed and handled in accordance with good animal practice as defined by FELASA (www.felasa.eu/guidelines.php) and the national animal welfare body GV-SOLAS (www.gv-solas.de/index.html). The animal welfare committees of the university of Freiburg, as well as the local authorities (Regierungspräsidium Freiburg) approved all animal experiments.

25Canine MDCKII, porcine NSK and NPTr cells, and human HEK 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 1% penicillin-streptomycin. Chicken hepatocellular epithelial cell line (LMH) was grown in DMEM supplemented with 8% fetal calf serum 2% chicken serum, 2 mM L-glutamine and 1% penicillin/streptomycin.

26The pHW2000 rescue plasmids and pCAGGS expression plasmids coding for NP were used for site directed mutagenesis. The coding region of MxA was cloned into pCAGGS, whereas murine Mx1 was expressed using pcDNA 3.1. The cDNA of porcine Mx1 (poMx1) corresponding to the full length 1992 nt long open reading frame as described in, encoding a Flag tag at its 5′-end was cloned into pCAGGS using KpnI and XhoI. poMx1 cDNA was generated from mRNA isolated from IFNα-2a-treated cell cultures from domestic pig (Sus scrofa domestica).

27The recombinant viruses A/Hamburg/4/09 (pH1N1) and A/Thailand/1(KAN-1)/04 (H5N1), and the NP-mutant viruses were generated by the eight-plasmid reverse-genetics system as described previously. All recombinant viruses were plaque purified on MDCKII cells. Virus stocks were prepared on MDCKII cells and titers were determined by plaque assay.

28HEK 293T cells seeded in 12-well plates were transfected using the Nanofectin transfection reagent (PAA Laboratories) according to the manufacturer's protocol. 10 ng of pCAGGS plasmids encoding PB2, PB1, and PA and 100 ng of NP-encoding plasmid were cotransfected with 100 ng of the firefly luciferase- encoding viral minigenome construct pPolI-FFLuc-RT, which is flanked by the noncoding regions of segment 8 of influenza A virus. The transfection mixture also contained 30 ng of pRL-SV40, a plasmid constitutively expressing Renilla luciferase under the control of the simian virus 40 promoter to normalize variations in transfection efficiency. To evaluate the antiviral potential of Mx1 and MxA, we cotransfected Mx1- or MxA- encoding plasmid. A simultaneous experiment with cotransfection of the antivirally inactive mutants Mx1-K49A or MxA-T103A, respectively, was used as a control. To achieve equal amounts of transfected DNA, an empty vector plasmid was added. Twenty-four hours post transfection, cells were lysed and firefly and Renilla luciferase activities were measured using the dual luciferase reporter assay (Promega) according to the manufacturer's protocol. Reconstitution of the viral polymerase complex in avian and porcine cells was performed as above with the exception that the minigenome RNA was expressed under the control of a chicken or porcine Pol I promoter (pSPOM2).

29MDCKII cells seeded in 6-well plates were incubated with virus at a multiplicity of infection (MOI) of 0.001 in PBS+/+ containing 0.2% BSA for 1 h at 37°C. The inoculum was removed and 3 ml infection medium (DMEM supplemented with 0.2% BSA), additionally containing 1 µg/ml TPCK-treated trypsin for pH1N1 viruses, was added. Virus titers in cell culture supernatants were determined at the indicated time points by plaque assay and are expressed as PFU per ml.

30For determination of viral transcript levels in virus-infected MDCKII cells, cells were seeded in 6-well plates and infection was carried out with infection media. After the indicated time point post infection, cells were harvested in Trizol™ and RNA was purified according to the manufacturer's protocol (Invitrogen). Primer extension analysis was performed as described using specific primers for the NA segment (mRNA, cRNA and vRNA) and cellular 5sRNA.

31BALB/c mice were obtained from Janvier (Straßburg) and congenic BALB.A2G-Mx1 mice (designated BALB-Mx1) carrying the functional Mx1 allele were bred locally. Six- to eight-week-old mice were anesthetized with a mixture of ketamine (100 µg per gram body weight) and xylazine (5 µg per gram) administered intraperitoneally (i.p.) and inoculated intranasally (i.n.) with the indicated doses of viruses in 50 µl phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA). Mice were monitored daily for weight loss until 14 days postinfection (p.i.). Animals with severe symptoms or more than 25% weight loss were euthanized. Lung homogenates were prepared using the FastPrep24 system (MP Biomedicals). Briefly, after addition of 800 µl of PBS containing 0.2% BSA, lungs were subjected to two rounds of mechanical treatment for 10 s each at 6.5 m/s. Tissue debris was removed by low-speed centrifugation. The LD50 values were calculated based on the infectious dose (PFU). All animal work was conducted under BSL 3 conditions in accordance with the guidelines of the local animal care committee.

32The program PyMOL (www.pymol.org) was used to assign the indicated positions in the structural model of the NP of A/HK/483/97(H5N1) (PDB code:2Q06). The program I-TASSER (zhanglab.ccmb.med.umich.edu/I-TASSER) was used to generate a full length NP model of A/Thailand/1(KAN-1)/04 (H5N1), including amino acids 1–20.

33Alignments and phylogenetic analyses were conducted with MEGA5. For maximum likelihood (ML) tree inference, the GTR substitution model assuming gamma distribution (four gamma categories) and invariant sites was selected, and the initial tree was made automatically. Bootstrap analysis was performed with 1,000 replications. The optimal substitution model was selected on the basis of the Bayesian information criterion (BIC) and the corrected Akaike information criterion (AICc) using a model test implemented in MEGA5.