Otto Haller

Mx protein: Constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.

cDNA structures and regulation of two interferon-induced human Mx proteins.

Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells.

Interferon-induced protein Mx accumulates in nuclei of mouse cells expressing resistance to influenza viruses

In mouse cells carrying the dominant influenza resistance allele Mx+ (but not in Mx- cells) interferon-alpha/beta (IFN) induces an efficient antiviral state against influenza viruses and, concomitantly, the synthesis of a 75,000-Da protein (protein Mx). Here, indirect immunofluorescence using monoclonal antibodies was used to demonstrate that protein Mx accumulates in the nucleus of IFN-treated Mx+ cells, suggesting a nuclear site of action. Protein Mx is present in the nucleus of untreated influenza virus-resistant macrophages freshly explanted from the peritoneal cavity of Mx+ mice but is lost with time in culture when peritoneal macrophages become permissive for influenza virus.

A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx.

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.

Inborn Resistance of Mice to Orthomyxoviruses

During evolution the vertebrate host has evolved a multitude of defense mechanisms to cope with virus infections. These are operative at several levels and range from simple physical barriers to complex structures such as the various parts of the immune system. Compared with acquired immunity, which has been so extensively studied, relatively little is known about resistance mechanisms in natural, genetically determined host defense against viruses.

Genetic control of interferon action: mouse strain distribution and inheritance of an induced protein with guanylate-binding property.

Interferons (IFNs) induce in responsive cells the synthesis of various proteins including a set with high binding affinities to guanylates. These guanylate-binding proteins (GBPs) were analyzed in cells from 46 inbred mouse strains using GMP-agarose affinity chromatography. In cells of 11 strains, including A/J, BALB/cJ, and C3H/HeJ, type I and II IFNs induced the synthesis of a major GBP of Mr 65,000, designated here GBP-1, and of at least three minor GBPs. In contrast, cells of the remaining 35 strains, including DBA/2J, C57BL/6J, and A2G, failed to synthesize GBP-1 in response to both types of IFNs. Induction of the minor GBPs was comparable in cells of both groups of mice, confirming that they were all responsive to IFNs. Analysis of F1, F2, and BC1 offspring of crosses between GBP-1 inducible (A/J) and noninducible (DBA/2J or A2G) strains showed that inducibility of GBP-1 was inherited as a single autosomal gene. The symbol Gbp-1 is proposed for this locus, designated Gbp-1a for the allele causing inducibility and Gbp-1b for the other allele.

Characterization and Expression of the Mx1 Gene in Wild Mouse Species

The mouse Mx1 gene encodes an interferon(IFN)-inducible nuclear protein and confers resistanceto influenza virus infection. The standard laboratorymouse strains all carry the Mx1- allele andare susceptible to influenza virus. In this study, severalmouse strains established from wild mice were tested todetermine their Mx1+ or Mx1-allele status with polymerase chain reaction-restrictionfragment length variation (PCR-RFLV), sequence analysis, reversetranscription (RT)-PCR, and immunofluorescence staining.All of the mouse strains originating from wild mice werefound uniformly to carry the Mx1+ allele.Therefore, it is conceivable that the Mx1+allele in wild populations serves a function againstsome pathogens related to orthomyxoviruses. The PCR-RFLVand sequence analysis allowed us to classify theMx1+ alleles of the laboratory and wild-origin mouse strainsinto distinct classes. RT-PCR and immunofluorescencestaining demonstrated that the Mx1 transcripts andproteins were induced by IFN-alpha/beta in macrophages from wild mouse species.

Mx genes show weaker primary response to virus than other interferon-regulated genes

Some interferon (IFN)-regulated genes are induced as a primary response to virus as well as secondarily through virus-induced IFN. Here we investigated whether this dual control mechanism would also regulate the activity of the human and mouse Mx genes that encode proteins with intrinsic antiviral potentials. To distinguish between a primary response to virus and a secondary response to virus-induced IFN, we studied virus-induced Mx gene expression in cell lines that lack a functional IFN system and in cells with blocked protein synthesis. In contrast to the two IFN-regulated human genes ISG56 and ISG15, the human MxA gene showed almost no primary response to Newcastle disease virus (NDV) or influenza virus. Similarly, direct activation of the mouse Mx1 gene by NDV or influenza virus was not significant in mouse embryo cells or explanted peritoneal macrophages. A moderate primary Mx1 response to NDV was observed in the permanent cell line L1210. Lack of a strong IFN-independent Mx response to virus indicates that this mode of gene regulation does not play a significant role in Mx-mediated resistance to viral disease.

Host gene influence on interferon action in adult mouse hepatocytes: specificity for influenza virus.

Abstract Primary monolayer cultures of hepatocytes isolated from adult mice either genetically resistant (bearing the autosomal dominant allele Mx ) or genetically susceptible toward influenza virus infection were tested for susceptibility to three viruses: a hepatotropic influenza A virus, vesicular stomatitis virus, and herpes simplex type I virus. Hepatocytes of both genotypes spontaneously released equal amounts of interferon during the first day of culture. Spontaneous interferon was sufficient to protect the cells markedly against vesicular stomatitis virus and marginally against herpesvirus infection. With respect to influenza virus, only Mx -bearing hepatocytes lost their initial susceptibility. Treatment with anti-interferon antibodies during the first 24 hr of culture rendered Mx -bearing cells as permissive for all three viruses as non- Mx -bearing cells. In such anti-interferon-treated cultures the antiviral resistance could be restored with highly purified mouse type I interferon. The antiviral state induced by exogenous interferon displayed the same characteristics as that induced by spontaneous interferon: it was highly efficient in inhibiting influenza A virus replication in Mx -bearing cells and inefficient in non- Mx -bearing cells. Inhibition of vesicular stomatitis virus or herpesvirus was independent of the Mx genotype.

Interferon-induced guanylate-binding proteins: Mapping murine Gbp-1 locus to chromosome 3

GBP-1 is the predominant species of a family of guanylate-binding proteins synthesized in mouse cells in response to interferons (IFNs) alpha, beta, or gamma. IFN inducibility of this 65,000-Da protein is controlled by alleles at a single autosomal locus, Gbp-1, with allele a encoding inducibility and allele b noninducibility. Here, we present evidence suggesting that both alleles occur in outbred populations of wild mice. Using recombinant inbred strains and classical linkage analysis of offspring of two-point and three-point backcrosses we demonstrate that Gbp-1 is linked to Adh-3 (encoding alcohol dehydrogenase C2) and VaJ (varitintwaddler-Jackson) located on the distal part of chromosome 3. The relevant recombination frequencies (RFs) (+/- SE) were 3.5 (+/- 1.1) and 11.7 (+/- 2.8)%, respectively. We further show that strain B6.C-H-23c/By(HW 53), congenic for a small segment of chromosome 3, carries the BALB/c alleles at both the Gbp-1 and the Adh-3 locus and not the alleles of the B6 background strain confirming the chromosomal location and close linkage of the two loci.