Dominik Dornfeld

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

The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored.

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.

Influenza A viruses escape from MxA restriction at the expense of efficient nuclear vRNP import

To establish a new lineage in the human population, avian influenza A viruses (AIV) must overcome the intracellular restriction factor MxA. Partial escape from MxA restriction can be achieved when the viral nucleoprotein (NP) acquires the critical human-adaptive amino acid residues 100I/V, 283P, and 313Y. Here, we show that introduction of these three residues into the NP of an avian H5N1 virus renders it genetically unstable, resulting in viruses harboring additional single mutations, including G16D. These substitutions restored genetic stability yet again yielded viruses with varying degrees of attenuation in mammalian and avian cells. Additionally, most of the mutant viruses lost the capacity to escape MxA restriction, with the exception of the G16D virus. We show that MxA escape is linked to attenuation by demonstrating that the three substitutions promoting MxA escape disturbed intracellular trafficking of incoming viral ribonucleoprotein complexes (vRNPs), thereby resulting in impaired nuclear import, and that the additional acquired mutations only partially compensate for this import block. We conclude that for adaptation to the human host, AIV must not only overcome MxA restriction but also an associated block in nuclear vRNP import. This inherent difficulty may partially explain the frequent failure of AIV to become pandemic.

SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

Corrigendum: Influenza A viruses escape from MxA restriction at the expense of efficient nuclear vRNP import.

Scientific Reports 6: Article number: 23138; Published online: 18 March 2016; Updated: 09 May 2016 The Acknowledgements section in this Article is incomplete. “We thank Richard Randall for providing A549 cells stably expressing MxA or shMxA, Georg Kochs for providing the NP-specific antibody and Geoffrey Chase, Georg Kochs and Peter Staeheli for critically reading of the manuscript.

Author Correction: SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

P107 Identification of adaptive mutations in the nucleoprotein of pandemic influenza A viruses required to evade restriction from the interferon induced MxA protein

Introduction The interferon induced human MxA protein is a potent restriction factor against avian influenza A virus infections. We therefore predicted that zoonotic transmission of these viruses into the human population is accompanied by the acquisition of adaptive mutations allowing evasion from MxA restriction. Methods The H5N1 polymerase reconstitution assay was utilized to identify adaptive mutations in the nucleoproteins (NP) of pandemic viruses required for resistance against human MxA. Highly pathogenic H5N1 viruses harboring the identified adaptive mutations in NP were generated and their growth properties were determined in cell culture. The evolutionary pressure to maintain the MxA resistance-contributing amino acids was assessed using the NCBI Influenza Virus Sequence Database. Results We identified the MxA-adaptive mutations in the nucleoprotein (NP) of the pandemic strains A/Brevig Mission/1/1918(H1N1) (1918) and A/Hamburg/4/2009(H1N1) (pH1N1). Intriguingly, the amino acids conferring resistance towards MxA differ in both strains, but cluster to the same area of the body domain of NP. Sequence analysis revealed that the amino acid cluster required for MxA resistance in the 1918 strain remained highly conserved in all descendant seasonal and pandemic strains. However, the amino acid cluster in NP of the pH1N1 strain contains three unprecedented adaptive mutations, indicating an independent evolution of MxA resistance in this porcine-derived virus. Introduction of either the 1918 or the pH1N1 amino acid cluster into NP of an H5N1 virus mediated resistance to MxA but also decreased viral fitness. Vice versa, mutation of the corresponding amino acid cluster in pH1N1 NP to avian signature impaired MxA resistance, while viral growth was increased. Conclusion Taken together, the acquisitions of NP mutations required for MxA resistance emerged in both the 1918 and the 2009 pandemic strain independently and were most likely accompanied by compensatory mutations to overcome the associated strong attenuation.