Eurasian Avian-Like Swine Influenza A Viruses Escape Human MxA Restriction through Distinct Mutations in Their Nucleoprotein

Abstract

To cross the human species barrier, influenza A viruses (IAV) of avian origin have to overcome the interferon-induced host restriction factor MxA by acquiring distinct mutations in their nucleoprotein (NP). We recently demonstrated that North American classical swine IAV are able to partially escape MxA restriction. Here we investigated whether the Eurasian avian-like swine IAV lineage currently circulating in European swine would likewise evade restriction by human MxA. We found that the NP of the influenza virus isolate A/Swine/Belzig/2/2001 (Belzig-NP) exhibits increased MxA escape, similar in extent to that with human IAV NPs. Mutational analysis revealed that the MxA escape mutations in Belzig-NP differ from the known MxA resistance cluster of the North American classical swine lineage and human-derived IAV NPs. A mouse-adapted avian IAV of the H7N7 subtype encoding Belzig-NP showed significantly greater viral growth in both MxA-expressing cells and MxA-transgenic mice than control viruses lacking the MxA escape mutations. Similarly, the growth of the recombinant Belzig virus was only marginally affected in MxA-expressing cells and MxA-transgenic mice, in contrast to that of Belzig mutant viruses lacking MxA escape mutations in the NP. Phylogenetic analysis of the Eurasian avian-like swine IAV revealed that the NP amino acids required for MxA escape were acquired successively and were maintained after their introduction. Our results suggest that the circulation of IAV in the swine population can result in the selection of NP variants with a high degree of MxA resistance, thereby increasing the zoonotic potential of these viruses. IMPORTANCE The human MxA protein efficiently blocks the replication of IAV from nonhuman species. In rare cases, however, these IAV overcome the species barrier and become pandemic. All known pandemic viruses have acquired and maintained MxA escape mutations in the viral NP and thus are not efficiently controlled by MxA. Intriguingly, partial MxA resistance can also be acquired in other hosts that express antivirally active Mx proteins, such as swine. To perform a risk assessment of IAV circulating in the European swine population, we analyzed the degree of MxA resistance of Eurasian avian-like swine IAV. Our data demonstrate that these viruses carry formerly undescribed Mx resistance mutations in the NP that mediate efficient escape from human MxA. We conclude that Eurasian avian-like swine IAV possess substantial zoonotic potential.

FIG 1

The nucleoprotein of Eurasian avian-like swine influenza viruses confers MxA resistance in polymerase reconstitution assays. (A) Amino acid differences between the NPs of H5N1, H7N7 (dark gray), pH1N1 (red), and Belzig (green) strains (for a complete alignment, see Fig. S1A in the supplemental material). Underlined positions have been shown previously to influence the MxA sensitivity phenotype. (B) (Top) The ratio of the activity of MxA to the activity of antivirally inactive MxA-T103A was normalized to that for pH1N1-NP (set at 1) and is shown as relative activity (shaded bars). (Center) Viral polymerase activity in the presence of MxA (filled bars) or MxA-T103A (open bars). HEK293T cells were transfected with expression plasmids encoding H5N1-PB2, -PB1, and -PA (10 ng), the indicated NP (100 ng), MxA (50 ng), and an artificial minigenome encoding firefly luciferase under the control of the polI promoter (100 ng). Additionally, a plasmid encoding Renilla luciferase (30 ng) was transfected as a control for transfection efficiency. Error bars indicate the standard errors of the means from at least four independent experiments. Student’s t test was performed to determine the P value for the difference from the respective wild-type NP. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (Bottom) Expression levels of MxA and NP were detected via Western blotting. Actin was used as a loading control. (C) MxA escape mutations previously identified in pH1N1-NP and the newly identified mutations in Belzig-NP are shown in red and green, respectively, in the structural model of influenza virus A/HK/483/97 (H5N1) NP (PDB code 2Q06).

FIG 2

Influenza A viruses encoding Belzig-NP escape MxA restriction in MxA-overexpressing cells. (A and B) MDCKII cells were infected at an MOI of 0.001 with wild-type or mutant H7N7 (A) or rBelzig (B) viruses and were incubated at 37°C. Viral titers were determined at 12, 24, 36, and 48 h postinfection via plaque assay. Error bars indicate the standard errors of the means from at least three independent experiments. (C and D) MDCK-SIAT1 cells stably expressing either MxA (filled bars) or the antivirally inactive MxA-T103A mutant (open bars) were infected at an MOI of 0.001 with wild-type or mutant H7N7 (C) or rBelzig (D) viruses and were incubated at 37°C. The H5N1 and pH1N1 viruses were used as controls for MxA-sensitive and -resistant viruses, respectively. Viral titers were determined 24 h postinfection via plaque assay. Error bars indicate the standard errors of the means from at least three independent experiments. Student’s t test was performed to determine the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Influenza A viruses encoding Belzig-NP escape restriction in MxA-transgenic mice. (A and B) C57BL/6N (B6) mice lacking functional Mx proteins and B6 mice homozygous for the human MxA transgene (hMxtg^+/+) were challenged intranasally with 10⁴ PFU of the indicated viruses with the H7N7 (A) or rBelzig (B) backbone. Viral titers in the lungs at 3 days postinfection were determined via plaque assay. Error bars indicate the standard errors of the means for at least four mice. The fold decrease in the titer in hMxtg^+/+ mice from that in B6 mice is shown at the top. Student’s t test was performed to determine the P value for the difference between B6 and hMxtg^+/+ mice. *, P < 0.05; ***, P < 0.001.

FIG 4

Phylogenetic analysis of representative NP sequences and the presence of MxA resistance-enhancing mutations. Shown is a maximum-likelihood tree of 140 aligned representative NP sequences. Nucleotide sequences were retrieved from GenBank, manually aligned, and used for Bayesian tree inference with MrBayes, v3.2. The GTR+G+I substitution model was used. Convergence was reached after 3 million generations. The bar indicates substitutions per site. Avian sequences are printed in gray, human seasonal sequences (H1N1, H3N2) in blue, Eurasian porcine sequences in green, and the classical swine-derived H1N1 sequences in red. Amino acid substitutions resulting in MxA resistance are indicated. The complete phylogenetic tree is shown in Fig. S2 in the supplemental material. Zoonotic events are highlighted with asterisks.

FIG 5

Temporal appearance of MxA resistance-enhancing amino acids in NPs of Eurasian avian-like swine IAV. Shown is the consecutive acquisition of MxA resistance-enhancing NP mutations in the 1918, “classical” North American swine, and Eurasian avian-like swine lineages. For more-detailed information, see reference , Fig. 4, and Fig. S2 in the supplemental material.