Rare variant MX1 alleles increase human susceptibility to zoonotic H7N9 influenza virus.

Zoonotic avian influenza A virus (IAV) infections are rare. Sustained transmission of these IAVs between humans has not been observed, suggesting a role for host genes. We used whole-genome sequencing to compare avian IAV H7N9 patients with healthy controls and observed a strong association between H7N9 infection and rare, heterozygous single-nucleotide variants in the MX1 gene. MX1 codes for myxovirus resistance protein A (MxA), an interferon-induced antiviral guanosine triphosphatase known to control IAV infections in transgenic mice. Most of the MxA variants identified lost the ability to inhibit avian IAVs, including H7N9, in transfected human cell lines. Nearly all of the inactive MxA variants exerted a dominant-negative effect on the antiviral function of wild-type MxA, suggesting an MxA null phenotype in heterozygous carriers. Our study provides genetic evidence for a crucial role of the MX1-based antiviral defense in controlling zoonotic IAV infections in humans.

Mx genes: host determinants controlling influenza virus infection and trans-species transmission.

The human MxA protein, encoded by the interferon-inducible MX1 gene, is an intracellular influenza A virus (IAV) restriction factor. It can protect transgenic mice from severe IAV-induced disease, indicating a key role of human MxA for host survival and suggesting that natural variations in MX1 may account for inter-individual differences in disease severity among humans. MxA also provides a robust barrier against zoonotic transmissions of avian and swine IAV strains. Therefore, zoonotic IAV must acquire MxA escape mutations to achieve sustained human-to-human transmission. Here, we discuss recent progress in the field.

Human MX2/MxB: a Potent Interferon-Induced Postentry Inhibitor of Herpesviruses and HIV-1.

Interferons limit viral replication by inducing intracellular restriction factors, such as the GTPase MxB (also designated MX2), which inhibits HIV-1 and, as recently shown, herpesviruses. Inhibition of these viruses occurs at ill-defined steps after viral entry and requires formation of MxB dimers or oligomers, but GTP hydrolysis is needed only for blocking herpesviruses. Together with previous findings on related MxA, the new research on MxB highlights the mechanistic diversity by which MX proteins interfere with viral replication.

The Discovery of the Antiviral Resistance Gene Mx: A Story of Great Ideas, Great Failures, and Some Success.

The discovery of the Mx gene-dependent, innate resistance of mice against influenza virus was a matter of pure chance. Although the subsequent analysis of this antiviral resistance was guided by straightforward logic, it nevertheless led us into many blind alleys and was full of surprising turns and twists. Unexpectedly, this research resulted in the identification of one of the first interferon-stimulated genes and provided a new view of interferon action. It also showed that in many species, MX proteins have activities against a broad range of viruses. To this day, Mx research continues to flourish and to provide insights into the never-ending battle between viruses and their hosts.

In vivo evasion of MxA by avian influenza viruses requires human signature in the viral nucleoprotein.

Zoonotic transmission of influenza A viruses can give rise to devastating pandemics, but currently it is impossible to predict the pandemic potential of circulating avian influenza viruses. Here, we describe a new mouse model suitable for such risk assessment, based on the observation that the innate restriction factor MxA represents an effective species barrier that must be overcome by zoonotic viruses. Our mouse lacks functional endogenous Mx genes but instead carries the human MX1 locus as a transgene. Such transgenic mice were largely resistant to highly pathogenic avian H5 and H7 influenza A viruses, but were almost as susceptible to infection with influenza viruses of human origin as nontransgenic littermates. Influenza A viruses that successfully established stable lineages in humans have acquired adaptive mutations which allow partial MxA escape. Accordingly, an engineered avian H7N7 influenza virus carrying a nucleoprotein with signature mutations typically found in human virus isolates was more virulent in transgenic mice than parental virus, demonstrating that a few amino acid changes in the viral target protein can mediate escape from MxA restriction in vivo. Similar mutations probably need to be acquired by emerging influenza A viruses before they can spread in the human population.

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.

A tribute to Jean Lindenmann, co-discoverer of interferon (1924-2015).

In 1957, the discovery of interferon was reported by Isaacs and Lindenmann in the prestigious journal Proceedings of the Royal Society (1957) [1,2]. This discovery was definitely one of the scientific landmarks in cell biology of the past century. It was the result of an initially unplanned and amazingly creative collaboration with Alick Isaacs that lasted from July 1956 to June 1957 at Mill Hill in London. Jean Lindenmann died in Zürich on January 15, 2015, at the age of 90, after having survived Alick Isaacs (1921-1967) for almost five decades.

Mx GTPases: dynamin-like antiviral machines of innate immunity.

The Mx dynamin-like GTPases are key antiviral effector proteins of the type I and type III interferon (IFN) systems. They inhibit several different viruses by blocking early steps of the viral replication cycle. We focus on new structural and functional insights and discuss recent data revealing that human MxA (MX1) provides a safeguard against introduction of avian influenza A viruses (FLUAV) into the human population. The related human MxB (MX2) serves as restriction factor for HIV-1 and other primate lentiviruses.

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

UNLABELLED: 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.

Structural requirements for the antiviral activity of the human MxA protein against Thogoto and influenza A virus.

The interferon-induced dynamin-like MxA protein has broad antiviral activity against many viruses, including orthomyxoviruses such as influenza A and Thogoto virus and bunyaviruses such as La Crosse virus. MxA consists of an N-terminal globular GTPase domain, a connecting bundle signaling element, and the C-terminal stalk that mediates oligomerization and antiviral specificity. We previously reported that the disordered loop L4 that protrudes from the compact stalk is a key determinant of antiviral specificity against influenza A and Thogoto virus. However, the role of individual amino acids for viral target recognition remained largely undefined. By mutational analyses, we identified two regions in the C-terminal part of L4 that contribute to an antiviral interface. Mutations in the proximal motif, at positions 561 and 562, abolished antiviral activity against orthomyxoviruses but not bunyaviruses. In contrast, mutations in the distal motif, around position 577, abolished antiviral activity against both viruses. These results indicate that at least two structural elements in L4 are responsible for antiviral activity and that the proximal motif determines specificity for orthomyxoviruses, whereas the distal sequence serves a conserved structural function.

Mx GTPases: efficient host defense against viral intruders.

Mx proteins are interferon-induced members of the dynamin superfamily of large GTPases. They inhibit a wide range of viruses by blocking early steps in the viral replication cycle. Recent evidence suggests that the human MxA (MX1) protein provides a barrier against zoonotic introduction of influenza A viruses into the human population, whereas the related human MxB (MX2) protein is an inhibitor of HIV-1 and other primate lentiviruses. Structural and functional data suggest that Mx proteins target the nucleocapsids of Mx-sensitive viruses and thereby inhibit their transcriptional and replicative function. Evolutionary studies revealed that Mx GTPases are subject to recurrent arms races with viral targets that shape their specificity determinants while the overall architecture is conserved. Here we briefly review the most salient features of Mx GTPases and their antiviral action as molecular machines.

Dynamins are forever: MxB inhibits HIV-1.

Human MxA (MX1) protein is an interferon-induced restriction factor for a diverse range of viruses, whereas the related MxB (MX2) protein was thought to lack such activity. Three recent papers, including one in this issue of Cell Host & Microbe, show that MxB inhibits human immunodeficiency virus type 1 (HIV-1) infection.

Emergence of a C-terminal seven-amino-acid elongation of NS1 in around 1950 conferred a minor growth advantage to former seasonal influenza A viruses.

Influenza A viruses circulating in humans from ∼1950 to ∼1987 featured a nonstructural (NS1) protein with a C-terminal extension of seven amino acids. The biological significance of this NS1 elongation remained elusive. We observed that replication kinetics of the wild-type virus A/Hong Kong/01/68 (H3N2) and a mutant encoding a truncated NS1 were indistinguishable in most experimental systems. However, wild-type virus outcompeted the mutant during mixed infections, suggesting that the NS1 extension conferred minor growth advantages.

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.

Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA.

MxA is an interferon-induced dynamin-like GTPase with wide-ranging antiviral activity, which hinges upon detection of unique viral structures that differ across virus families. Despite elucidation of its structure, the basis of MxA antiviral specificity remains enigmatic. We used an evolution-guided approach to identify the loop L4 of MxA as a hotspot for recurrent positive selection in primates. Further, we show that single amino acid changes in L4 are necessary and sufficient to explain dramatic differences in species-specific antiviral activity of primate MxA proteins against the orthomyxoviruses Thogoto virus and influenza A virus. Taken together, our findings identify a genetic determinant of MxA target recognition and suggest a model by which MxA achieves antiviral breadth without compromising viral specificity.

Altered receptor specificity and fusion activity of the haemagglutinin contribute to high virulence of a mouse-adapted influenza A virus.

The viral haemagglutinin (HA) and the viral polymerase complex determine the replication fitness of a highly virulent variant of influenza A virus strain A/PR/8/34 (designated hvPR8) and its high pathogenicity in mice. We report here that the HA of the hvPR8 differs from the HA of a low virulent strain (lvPR8) by the efficiency of receptor binding and membrane fusion. hvPR8 bound to 2,6-linked as well as 2,3-linked sialic acid-containing receptors, whereas lvPR8 bound exclusively to 2,3-linked sialic acids with high avidity. Remarkably, hvPR8 infected its target cells faster than lvPR8 and tolerated an elevated pH for efficient membrane fusion. In spite of these differences, both viruses targeted type II but not type I pneumocytes in the lung of infected mice. The HA of hvPR8 differs from that of lvPR8 by 16 aa substitutions and one insertion. Mutational analyses revealed that amino acid at HA position 190 (H3 numbering) primarily determined the specificity of receptor binding, while the insertion at position 133 influenced the avidity of receptor binding. Both amino acid positions also strongly influenced viral virulence. Furthermore, leucine at position 78 and glutamine at position 354 were critical determinants of increased fusion activity and virulence of hvPR8. Our data suggest that the HA of hvPR8 enhances virulence by mediating optimal receptor binding and membrane fusion thereby promoting rapid and efficient viral entry into host cells.

Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function.

Human myxovirus resistance protein 1 (MxA) is an interferon-induced dynamin-like GTPase that acts as a cell-autonomous host restriction factor against many viral pathogens including influenza viruses. To study the molecular principles of its antiviral activity, we determined the crystal structure of nucleotide-free MxA, which showed an extended three-domain architecture. The central bundle signaling element (BSE) connected the amino-terminal GTPase domain with the stalk via two hinge regions. MxA oligomerized in the crystal via the stalk and the BSE, which in turn interacted with the stalk of the neighboring monomer. We demonstrated that the intra- and intermolecular domain interplay between the BSE and stalk was essential for oligomerization and the antiviral function of MxA. Based on these results, we propose a structural model for the mechano-chemical coupling in ring-like MxA oligomers as the principle mechanism for this unique antiviral effector protein.

Stalk domain of the dynamin-like MxA GTPase protein mediates membrane binding and liposome tubulation via the unstructured L4 loop.

The human MxA protein is an interferon-induced large GTPase with antiviral activity against a wide range of viruses, including influenza viruses. Recent structural data demonstrated that MxA oligomerizes into multimeric filamentous or ring-like structures by virtue of its stalk domain. Here, we show that negatively charged lipid membranes support MxA self-assembly. Like dynamin, MxA assembled around spherical liposomes inducing liposome tubulation. Cryo-transmission electron microscopy revealed that MxA oligomers around liposomes have a "T-bar" shape similar to dynamin. Moreover, biochemical assays indicated that the unstructured L4 loop of the MxA stalk serves as the lipid-binding moiety, and mutational analysis of L4 revealed that a stretch of four lysine residues is critical for binding. The orientation of the MxA molecule within the membrane-associated oligomer is in agreement with the proposed topology of MxA oligomers based on crystallographic data. Although oligomerization of wild-type MxA around liposomes led to the creation of helically decorated tubes similar to those formed by dynamin, this lipid interaction did not stimulate GTPase activity, in sharp contrast to the assembly-stimulated nucleotide hydrolysis observed with dynamin. Moreover, MxA readily self-assembles into rings at physiological conditions, as opposed to dynamin which self-assembles only at low salt conditions or onto lipids. Thus, the present results indicate that the oligomeric structures formed by MxA critically differ from those of dynamin.