The nucleoprotein of newly emerged H7N9 influenza A virus harbors a unique motif conferring resistance to antiviral human MxA

Abstract

Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans.

Importance: The natural host of influenza A viruses (IAVs) are aquatic birds. Occasionally, these viruses cross the species barrier, as in early 2013 when an avian H7N9 virus infected humans in China. Since then, multiple transmissions of H7N9 viruses to humans have occurred, leaving experts puzzled about molecular causes for such efficient crossing of the species barrier compared to other avian influenza viruses. Mx proteins are known restriction factors preventing influenza virus replication. Unfortunately, some viruses (e.g., human IAV) have developed some resistance, which is associated with specific amino acids in their nucleoproteins, the target of Mx function. Here, we demonstrate that the novel H7N9 bird IAV already carries a nucleoprotein that overcomes the inhibition of viral replication by human MxA. This is the first example of an avian IAV that is naturally less sensitive to Mx-mediated inhibition and might explain why H7N9 viruses transmitted efficiently to humans.

FIG 1

Mx sensitivity of PR/8 NP mutants at positions 100, 283, and 313. (A and B) Viral polymerase reconstitution assay. HEK 293T cells were transfected with expression plasmids coding for the polymerase subunits PB2, PB1, PA, and the indicated NP protein of hvPR/8, a firefly luciferase minigenome under the control of the RNA polymerase I promoter and the Mx1-encoding plasmid. In addition, a plasmid encoding Renilla luciferase under the control of the SV40 promoter was cotransfected to normalize variations in transfection efficiency. Mx1(K49A) is an inactive mutant that was used as a control to normalize the data obtained by coexpression of wild-type Mx1. Relative polymerase activity was calculated as the ratio of luciferase activity in the presence of wild-type Mx1 compared to the luciferase activity in the presence of antiviral inactive Mx1(K49A). The polymerase activity with wild-type NP was set to 100%. Bars represent mean values with standard deviations of two independent experiments performed in triplicates. Two-way analysis of variance was performed to calculate P values. ns, not significant; *, P < 0,01; ***, P < 0,001. A representative Western blot analysis shows the expression levels of wild-type Mx1 (72 kDa), NP (56 kDa), and β-actin (42 kDa) in the cell lysates. (A) Inhibition of the viral polymerase activity by Mx1. Relative luciferase activity was determined in the presence of Mx1 or Mx1(K49A). (B) Point mutations in NP influence Mx1 sensitivity of the viral polymerase. Wild-type NP or NP encoding single or combinations of mutations were used in the replicon assay. Luciferase activity obtained in the presence of wild-type Mx1 was normalized to the activity in the presence of Mx1(K49A). (C) Growth kinetics of hvPR/8 encoding either wild-type NP or NP with the indicated mutations. Calu-3 cells were infected with an MOI of 0.001 in the presence of 0.5 μg/ml trypsin and were incubated at 37°C. At the indicated time points, virus titers in the cell supernatants were determined by plaque assay. Error bars indicate the standard deviation of one experiment performed in duplicates. (D and E) Lung titers of hvPR/8 encoding wild-type NP compared to the viruses encoding the indicated NP mutants in C57BL/6 (D) or B6.A2G-Mx1 (E) mice. Mice were infected intranasally with 200 PFU for 48 h. Viral titers of the lung homogenates were determined by plaque assay. (F) MLD₅₀ values of hvPR/8 encoding wild-type NP or the indicated NP mutants determined in C57BL/6 and B6.A2G-Mx1 mice (n = 5/group). α, anti.

FIG 2

Passaging Mx-sensitive hvPR/8-NP(P283L/Y313F) in B6.A2G-Mx1 mice. Two independent lines of Mx1-positive mice were infected intranasally with 100 PFU of hvPR/8-NP(P283L/Y313F) for 48 h. Then lungs were harvested, and virus titers were determined. The lung homogenates were diluted appropriately with PBS and directly used for the next passage. (A) Lung virus titers of each step in the passage lines A and B. The first appearance of NP mutations in codon 52 in line A and codon 309 in line B is indicated. (B) RNA was isolated from the lung homogenates and used for segment 5-specific RT-PCR and sequencing.

FIG 3

Mx sensitivity of NP mutants at positions 52 and 309. (A) A polymerase reconstitution assay was performed as described in the legend of Fig. 1B, including the compensatory NP mutants. Relative polymerase activity was calculated as the ratio of luciferase activity in the presence of wild-type Mx1 compared to the luciferase activity in the presence of inactive Mx1(K49A). The activity with wild-type NP was set to 100%. Bars represent mean values with standard deviations of two independent experiments performed in triplicates. Two-way analysis of variance was performed to calculate P values. ns, not significant; ***, P < 0.001. Western blot analysis shows the expression levels of Mx1, NP, and β-actin in the cell lysates. (B) Growth kinetics of the recombinant hvPR/8 viruses on Calu-3 cells infected at an MOI of 0.001 in the presence of 0.5 μg/ml trypsin. Error bars indicate the standard deviations of one experiment performed in duplicates. (C and D) Lung titers of hvPR/8 viruses in C57BL/6 (C) and B6.A2G-Mx1 (D) mice upon intranasal infection with 200 PFU. At 48 h postinfection virus titers were determined in lung homogenates. Student's t test was performed to calculate P values. (E) MLD₅₀ values of hvPR/8 encoding wild-type NP or the indicated NP mutants determined in B6.A2G-Mx1 mice (n = 5/group). (F) Detection of Mx1 expression by quantitative RT-PCR. Lung homogenates of B6.A2G-Mx1 mice (n = 5) infected with 200 PFU for 48 h were used to extract RNA and determine the expression of Mx1 and GAPDH by qRT-PCR. The Mx1 signals were normalized to the GAPDH levels. qRT-PCR results were calculated as Mx1-to-GAPDH ratio by the 2^ΔCT method (59). The relative induction of Mx1 in mock-infected animals was set to 1. Bars represent mean values with standard deviations using data from five animals each (three animals in the mock control). Two-way analysis of variance was performed to calculate P values. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. α, anti.

FIG 4

Sensitivity of A/SH/01/13 to murine and human Mx. (A) Amino acid differences in NP of A/BM/1/18 (BM/18), A/PR/8/34 (PR/8), A/WSN/33, A/AH/1/13 (AH/1), A/SH/01/13 (SH/1), and A/Vietnam/1203/04 (VN/04). Blue boxes highlight positions increasing Mx resistance; red boxes indicate differences between AH/1 and SH/1 relative to VN/04. (B) Structural models of NP with the positions critical for Mx resistance highlighted in blue for BM/18 and PR/8 and the positions of SH/1 that differ from VN/04 highlighted in red. (C and D) Polymerase reconstitution assay with the polymerase subunits of VN/04 combined with expression plasmids encoding NP (100 ng) of SH/1, VN/04, or WSN/33. The assays were performed as described in the legend of Fig. 1B with increasing amounts of cotransfected expression plasmids for mouse Mx1 (C) or human MxA (D). Relative polymerase activity was calculated as the ratio of wild-type Mx1 or MxA to the inactive Mx1(K49A) or MxA(T103A), respectively. The activity with wild-type NP in the absence of Mx was set to 100%. Error bars indicate standard deviations of three independent experiments. Student's t test was performed to determine the P values. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Western blot analysis shows the expression levels of Mx proteins, viral PB2 and beta-actin in the cell lysates.

FIG 5

Amino acid 52N in H7N9 NP contributes to reduced sensitivity to murine Mx1. (A) Direct comparison of the Mx1 sensitivity of SH/1-NP(wt), SH/1-NP(N52Y), and VN/04 in the VN/04 polymerase reconstitution system and increasing amounts of Mx1 as described in the legend of Fig. 4C. Expression of Mx1 and viral PB2 was monitored by Western blotting. (B) Introduction of SH/1-NP(N52Y) or VN/04-NP does not alter virus growth of SH/1 in MDCKII cells. Cells were infected at an MOI of 0.01 with the indicated viruses in the presence of 0.5 μg/ml trypsin. At the indicated time points postinfection (p.i.), virus titers were determined by plaque assay. Error bars indicate the standard deviations of one experiment performed in duplicates. (C and D) Lung titers of H7N9 SH/1 viruses encoding an N52Y exchange in NP or the NP of VN/04. C57BL/6 (C) and B6.A2G-Mx1 (D) mice (n = 3) were infected intranasally with 100 PFU. At 3 and 6 days postinfection, virus titers were determined in lung homogenates by plaque assay. (E and F) Lung titers of H7N9 in IFN-pretreated animals. C57BL/6 (E) and B6.A2G-Mx1 (F) mice (n = 3) were intranasally pretreated with 20,000 units of IFN-α or PBS (ctrl). After 24 h the animals were infected intranasally with 200 PFU of the indicated viruses for 2 days, and virus titers were determined in lung homogenates by plaque assay. Student's t test was performed to determine the P values. *, P < 0.05; **P < 0.01; ***, P < 0.001; ns, not significant. D, day.

FIG 6

Amino acid 52N in H7N9 NP contributes to resistance against human MxA. (A) MxA sensitivity of SH/1-NP(wt), SH1-NP(N52Y), and VN/04 was determined in the VN/04 polymerase reconstitution system with increasing amounts of MxA expression plasmid as described in the legend of Fig. 4D. Relative polymerase activity was calculated as the ratio of luciferase activity of wild-type MxA and MxA(T103A). The activity in the absence of MxA was set to 100%. Error bars indicate standard deviations of three independent experiments. Student's t test was performed to determine the P values. **, P < 0.01; ***, P < 0.001. Western blot analysis shows the expression levels of MxA and PB2 in the cell lysates. (B) Expression of MxA limits replication of H7N9 NP(N52Y). Control A549 cells and A549 cells constitutively expressing MxA were infected at an MOI of 2 with the indicated viruses. At 16 h p.i. cells were lysed and analyzed for viral NS1, MxA, and β-actin by Western blotting. The second panel shows a longer exposure of the anti-NS1 blot. The Western blot signals of NS1 were quantified using ImageJ program and normalized to the signals for actin in the cell lysates (graphs). (C) Viral titers in A549 cells stably expressing MxA and control cells. Cells were infected for 16 h at an MOI of 2 with indicated viruses in the absence of TPCK-trypsin. Viral titers are depicted as PFU/ml from three independent samples; error bars represent standard deviations.