Asparagine 631 variants of the chicken Mx protein do not inhibit influenza virus replication in primary chicken embryo fibroblasts or in vitro surrogate assays

Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles.     

The Cytoplasmic Location of Chicken Mx Is Not the Determining Factor for Its Lack of Antiviral Activity

Background

Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.

Methodology/Principal Findings

Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.

Conclusions/Significance

The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.     

Resistance to influenza virus infection of Mx transgenic mice expressing Mx protein under the control of two constitutive promoters.

Transgenic mice constitutively expressing in the brain the influenza virus resistance protein Mx1 controlled by the HMG (3-hydroxy-3-methylglutaryl coenzyme A reductase) promoter showed specific resistance against the neurotropic influenza A virus strain NWS. Control mice of the A2G strain express Mx1 protein in all organs, but only after induction by interferon type I upon or without viral infection. The extent of specific resistance in transgenic mice of the best-expressing line reached about two-thirds that of controls, most likely because of considerably less total-body Mx protein activity in the transgenic mice. Thus, the theoretical advantage in these mice of the continuous presence of Mx protein with early inhibitory potential to viral replication was apparently offset by restricted organ expression. Strong evidence that the Mx1 protein on its own is a specific anti-influenza A virus agent and that its efficiency in the experimental setting is independent of interferon actions could be derived from the treatment of experimental and control mice with anti-interferon antibodies at the time of virus tests. Whereas in A2G mice, Mx1 mRNA and Mx1 protein synthesis were abolished and viral resistance was markedly reduced or abolished, resistance in the transgenic mice persisted to almost the same degree. Transgenic mice generated with a mouse albumin/Mx1 cDNA construct showed liver-specific expression. However, in two expressing transgenic lines, Mx1 protein synthesis was suppressed after a few months. The mechanism of suppression could not be elucidated, but increasing methylation of the transgene's coding region was not the cause. It is possible that continuous Mx1 protein expression in the liver is less well tolerated than that in the brain. Whether this partial suppression and, with the HMG promoter, restricted organ expression are the organism's responses to interference of Mx1 with normal cellular activities such as nucleocytoplasmic transport of RNA and proteins cannot be determined until the molecular mechanisms of antiviral activity of Mx1 protein are understood.     

Determination of influenza virus proteins required for genome replication.

An artificial vaccinia virus vector-driven replication system for influenza virus RNA has been developed. In this system, a synthetic NS-like gene is replicated and expressed by influenza virus proteins supplied through infection with vaccinia virus recombinant vectors. The minimum subset of influenza virus proteins needed for specific replication and expression of the viral ribonucleoprotein was found to be the three polymerase proteins (PB1, PB2, and PA) and the nucleoprotein.     

Transgenic mice with intracellular immunity to influenza virus

We have generated transgenic mice that express the intracellular anti-influenza virus protein Mx1 under control of an interferon-responsive regulatory element. Upon infection with influenza virus, mice of a high responder line produce Mx1 protein locally at the sites of initial viral replication, exhibit little viral spread, and survive infection. Mice of a low responder line show more extensive viral spread and survive infection only when virus is given at high doses. To survive low dose infections, these mice require injection of interferon along with virus. The results show that influenza viral pathogenesis is determined by a subtle balance between the dose of the infecting virus and the levels of the antiviral host factor Mx1 and that mice can be rendered resistant to a virulent infection by "intracellular immunization" achieved through germline transformation.     

A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.     

Switch in antiviral specificity of a GTPase upon translocation from the cytoplasm to the nucleus.

The Mx2 protein of rats is a cytoplasmic GTPase that protects cells against vesicular stomatitis virus but not against influenza virus. Since vesicular stomatitis virus replicates in the cytoplasm and influenza virus replicates in the nucleus, it was possible that the antiviral specificity of rat Mx2 protein was determined solely by the protein's subcellular localization. Here, we found that, indeed, rat Mx2 protein lost its anti-vesicular stomatitis virus activity and gained anti-influenza virus activity when it was directed to the nucleus by way of a foreign nuclear-transport signal appended to its amino terminus. These data show that rat Mx2 protein possesses an antiviral activity that is revealed only when the protein is shuttled to the nucleus.     

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.     

Peptide-mediated interference with influenza A virus polymerase

The assembly of the polymerase complex of influenza A virus from the three viral polymerase subunits PB1, PB2, and PA is required for viral RNA synthesis. We show that peptides which specifically bind to the protein-protein interaction domains in the subunits responsible for complex formation interfere with polymerase complex assembly and inhibit viral replication. Specifically, we provide evidence that a 25-amino-acid peptide corresponding to the PA-binding domain of PB1 blocks the polymerase activity of influenza A virus and inhibits viral spread. Targeting polymerase subunit interactions therefore provides a novel strategy to develop antiviral compounds against influenza A virus or other viruses.