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.

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.

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 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 basis of oligomerization in the stalk region of dynamin-like MxA.

The interferon-inducible dynamin-like myxovirus resistance protein 1 (MxA; also called MX1) GTPase is a key mediator of cell-autonomous innate immunity against pathogens such as influenza viruses. MxA partially localizes to COPI-positive membranes of the smooth endoplasmic reticulum-Golgi intermediate compartment. At the point of infection, it redistributes to sites of viral replication and promotes missorting of essential viral constituents. It has been proposed that the middle domain and the GTPase effector domain of dynamin-like GTPases constitute a stalk that mediates oligomerization and transmits conformational changes from the G domain to the target structure; however, the molecular architecture of this stalk has remained elusive. Here we report the crystal structure of the stalk of human MxA, which folds into a four-helical bundle. This structure tightly oligomerizes in the crystal in a criss-cross pattern involving three distinct interfaces and one loop. Mutations in each of these interaction sites interfere with native assembly, oligomerization, membrane binding and antiviral activity of MxA. On the basis of these results, we propose a structural model for dynamin oligomerization and stimulated GTP hydrolysis that is consistent with previous structural predictions and has functional implications for all members of the dynamin family.

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.

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.

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.

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.

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.

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.

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.

The viral nucleoprotein determines Mx sensitivity of influenza A viruses.

Host restriction factors play a crucial role in preventing trans-species transmission of viral pathogens. In mammals, the interferon-induced Mx GTPases are powerful antiviral proteins restricting orthomyxoviruses. Hence, the human MxA GTPase may function as an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Successful viruses are likely to acquire adaptive mutations allowing them to evade MxA restriction. We compared the 2009 pandemic influenza A virus [strain A/Hamburg/4/09 (pH1N1)] with a highly pathogenic avian H5N1 isolate [strain A/Thailand/1(KAN-1)/04] for their relative sensitivities to human MxA and murine Mx1. The H5N1 virus was highly sensitive to both Mx GTPases, whereas the pandemic H1N1 virus was almost insensitive. Substitutions of the viral polymerase subunits or the nucleoprotein (NP) in a polymerase reconstitution assay demonstrated that NP was the main determinant of Mx sensitivity. The NP of H5N1 conferred Mx sensitivity to the pandemic H1N1 polymerase, whereas the NP of pandemic H1N1 rendered the H5N1 polymerase insensitive. Reassortant viruses which expressed the NP of H5N1 in a pH1N1 genetic background and vice versa were generated. Congenic Mx1-positive mice survived intranasal infection with these reassortants if the challenge virus contained the avian NP. In contrast, they succumbed to infection if the NP of pH1N1 origin was present. These findings clearly indicate that the origin of NP determines Mx sensitivity and that human influenza viruses acquired adaptive mutations to evade MxA restriction. This also explains our previous observations that human and avian influenza A viruses differ in their sensitivities to Mx.

Dynamin-like MxA GTPase: structural insights into oligomerization and implications for antiviral activity.

The interferon-inducible MxA GTPase is a key mediator of cell-autonomous innate immunity against a broad range of viruses such as influenza and bunyaviruses. MxA shares a similar domain structure with the dynamin superfamily of mechanochemical enzymes, including an N-terminal GTPase domain, a central middle domain, and a C-terminal GTPase effector domain. Recently, crystal structures of a GTPase domain dimer of dynamin 1 and of the oligomerized stalk of MxA (built by the middle and GTPase effector domains) were determined. These data provide exciting insights into the architecture and antiviral function of the MxA oligomer. Moreover, the structural knowledge paves the way for the development of novel antiviral drugs against influenza and other highly pathogenic viruses.

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.

Adaptive mutations resulting in enhanced polymerase activity contribute to high virulence of influenza A virus in mice.

High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.

Inhibition of a large double-stranded DNA virus by MxA protein.

Increasing evidence points to the importance of the interferon (IFN) response in determining the host range and virulence of African swine fever virus (ASFV). Infection with attenuated strains of ASFV leads to the upregulation of genes controlled by IFN pathways, including myxovirus resistance (Mx) genes that are potent effectors of the antiviral state. Mx gene products are known to inhibit the replication of many negative-sense single-stranded RNA viruses, as well as double-stranded RNA viruses, positive-sense single-stranded RNA viruses, and the reverse-transcribing DNA virus hepatitis B virus. Here, we provide data that extend the known range of viruses inhibited by Mx to include the large double-stranded DNA viruses. Stably transfected Vero cells expressing human MxA protein did not support ASFV plaque formation, and virus replication in these cells was reduced 100-fold compared with that in control cells. In contrast, ASFV replication in cells expressing MxB protein or a mutant MxA protein was similar to that in control Vero cells. There was a drastic reduction in ASFV late protein synthesis in MxA-expressing cells, correlating with the results of previous work on the effect of IFN on viral replication. Strikingly, the inhibition of ASFV replication was linked to the recruitment of MxA protein to perinuclear viral assembly sites, where the protein surrounded the virus factories. Interactions between ASFV and MxA were similar to those seen between MxA and different RNA viruses, suggesting a common inhibitory mechanism.

Intranasal administration of alpha interferon reduces seasonal influenza A virus morbidity in ferrets.

The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, we assessed the protective potential of exogenous IFN-alpha against seasonal and highly pathogenic influenza viruses in ferrets. Intranasal treatment with IFN-alpha several hours before infection with the H1N1 influenza A virus strain A/USSR/90/77 reduced viral titers in nasal washes at least 100-fold compared to mock-treated controls. IFN-treated animals developed only mild and transient respiratory symptoms, and the characteristic fever peak seen in mock-treated ferrets 2 days after infection was not observed. Repeated application of IFN-alpha substantially increased the protective effect of the cytokine treatment. IFN-alpha did not increase survival after infection with the highly pathogenic H5N1 avian influenza A virus strain A/Vietnam/1203/2004. However, viral titers in nasal washes were significantly reduced at days 1 and 3 postinfection. Our study shows that intranasal application of IFN-alpha can protect ferrets from seasonal influenza viruses, which replicate mainly in the upper respiratory tract, but not from highly pathogenic influenza viruses, which also disseminate to the lung. Based on these results, a more intensive evaluation of IFN-alpha as an emergency drug against pandemic influenza A is warranted.

Interferon, Mx, and viral countermeasures.

The interferon system provides a powerful and universal intracellular defense mechanism against viruses. Knockout mice defective in IFN signaling quickly succumb to all kinds of viral infections. Likewise, humans with genetic defects in interferon signaling die of viral disease at an early age. Among the known interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins belong to the dynamin superfamily of large GTPases and have direct antiviral activity. They inhibit a wide range of viruses by blocking an early stage of the viral replication cycle. Likewise, the protein kinase R (PKR), and the 2-5 OAS/RNaseL system represent major antiviral pathways and have been extensively studied. Viruses, in turn, have evolved multiple strategies to escape the IFN system. They try to go undetected, suppress IFN synthesis, bind and neutralize secreted IFN molecules, block IFN signaling, or inhibit the action of IFN-induced antiviral proteins. Here, we summarize recent findings about the astonishing interplay of viruses with the IFN response pathway.

Influenza A virus strains differ in sensitivity to the antiviral action of Mx-GTPase.

Interferon-mediated host responses are of great importance for controlling influenza A virus infections. It is well established that the interferon-induced Mx proteins possess powerful antiviral activities toward most influenza viruses. Here we analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and human MxA proteins and found remarkable differences. Virus strains of avian origin were highly sensitive to Mx1, whereas strains of human origin showed much weaker responses. Artificial reassortments of the viral components in a minireplicon system identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the recently reconstructed 1918 H1N1 "Spanish flu" virus was much less sensitive than the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a minireplicon system. Importantly, the human 1918 virus-based minireplicon system was almost insensitive to inhibition by human MxA, whereas the avian influenza A virus H5N1-derived system was well controlled by MxA. These findings suggest that Mx proteins provide a formidable hurdle that hinders influenza A viruses of avian origin from crossing the species barrier to humans. They further imply that the observed insensitivity of the 1918 virus-based replicon to the antiviral activity of human MxA is a hitherto unrecognized characteristic of the "Spanish flu" virus that may contribute to the high virulence of this unusual pandemic strain.