Michel A. Horisberger

Interferon-induced proteins: Identification of Mx proteins in various mammalian species

Mx protein controls influenza virus pathogenicity in mice in vivo. It is an abundant protein synthesized in response to interferon-alpha/beta. Searches for homolog proteins in various animal species have been conducted using several methods: (1) radioactive labeling of proteins induced by interferon and analysis by 2-D gel electrophoresis, (2) immunoprecipitation, (3) protoblot-ELISA analysis of cell-protein extracts, and (4) immunostaining of fixed cells. All mammalian species tested so far (including human, horse, and pig, which are prone to influenza virus infection) responded to interferon-alpha by synthesizing one or two Mx proteins differing in relative molecular weight and pl. The expression of Mx proteins within one species differed sometimes between primary cultures of diploid cells and established cell lines. Mx proteins were detected in all species in the cytoplasm and, in some rodents only, also in the nucleus. This study indicates that the site of action of the Mx gene family might be cytoplasmic. The conservation and ubiquity of this family of proteins reflects a more general and essential cellular function than was initially considered.

A Recombinant Human Interferon-α B/D Hybrid with a Broad Host-range

Summary A recombinant interferon (IFN) hybrid has been found to have a broad host-range of activity in an antiviral assay (plaque reduction of vesicular stomatitis virus) and also high efficacy as an antiviral agent in at least 12 different animal cell species. The IFN hybrid consists of amino acids 1 to 60 from HuIFN-αB and amino acids 61 to 166 from HuIFN-αD. The profile of cross-species activity of the IFN-αB/D hybrid has been compared with that of HuIFN-αF, and of the parents HuIFN-αB and -αD. When both IFN-αB and -αD were active in a cell species, the hybrid IFN had comparable or better activity than the more active parental IFN. The hybrid shared a broad cross-species activity with IFN-αD. However, the IFN-αB/D hybrid was 10-fold more active on human cells, 30-fold more active on rabbit cells, and 50-fold more active on mouse cells than IFN-αD.

Mx-dependent resistance to influenza viruses is induced by mouse interferons α and β but not γ

Abstract In cells derived from congenic BALB.A2G- Mx mice carrying the resistance gene Mx , but not in cells from BALB/c mice lacking Mx , mouse interferons α and β induced the synthesis of a unique cellular protein that was associated with an efficient antiviral state with selectivity for influenza viruses. In contrast, native or recombinant mouse interferon γ failed to efficiently protect Mx -bearing cells against influenza viruses and did not noticeably induce the synthesis of the Mx -associated protein, although interferon γ was as effective as interferons α and β in protecting BALB.A2G- Mx and BALB/c cells against the rhabdovirus VSV. These results demonstrate that different types of interferons differentially regulate the expression of the Mx gene and thereby induce distinct antiviral states.

cDNA cloning and assignment to chromosome 21 ofIFI-78K gene, the human equivalent of murineMx gene

Recently we have purified to homogeneity and characterized an interferon-induced human protein (p78 protein) which is the equivalent of the interferon-induced murine Mx protein responsible for a specific antiviral state against influenza virus infection. A cDNA library was constructed using mRNAs from interferon-induced human diploid fibroblasts. cDNA clones coding for the human p78 protein were identified and used to determine the chromosomal location of the corresponding gene (termedIFI-78K gene) by hybridization to DNA from a panel of human x rodent somatic cell hybrids. The newly identified gene is located on chromosome 21. This has been confirmed by the observation of a gene dosage effect using chromosome 21 trisomic cells (fibroblasts derived from Downs syndrome patients). Among all interferon-inducible genes mapped so far, theIFI-78K gene is the only one located on chromosome 21, together with the gene for the receptor of type I interferon. Our results also provide further evidence for homology between human chromosome 21 and mouse chromosome 16, since the gene encoding the mouse Mx protein (the presumed mouse homolog protein of human p78 protein) has been assigned to chromosome 16.

The large P proteins of influenza A viruses are composed of one acidic and two basic polypeptides

Abstract Six strains of influenza A virus, grown and labeled with [35S]methionine in tissue culture, were purified and their proteins were analyzed in a two-dimensional system combining non-equilibrium pH gradient electrophoresis (NEPHGE) with NaDodSO4-polyacrylamide gel electrophoresis. The P polypeptides separated as one acidic polypeptide (pI 6) and two basic polypeptides (pI above 8). This method facilitated the identification of P polypeptides, especially of P2 and P3 whose separation was difficult in a one-dimensional system.

Cloning and chromosomal location of human genes inducible by type I interferon.

When cells are treated with interferon several new proteins are induced. We have isolated by differential screening two cDNA clones corresponding to human genes inducible by IFN-α, termed IFI-4 and IFI-54K. The accumulation of the corresponding mRNA was followed as a function of either IFN dose or of time. The IFI-4 and IFI-54K genes, as well as two previously isolated IFN-inducible genes, namely the IFI-56K and low-molecular-weight 2–5A synthetase, were localized on the human chromosomes. Using cloned probes on Southern blots of DNA from a panel of rodent-human somatic cell hybrids, we have assigned the IFI-4 gene to chromosome 1 and the gene coding for the low-molecular-weight 2–5A synthetase to chromosome 12. We also showed that the IFI-54K and IFI-56K genes, unlike most of the IFN-inducible genes, are syntenic. They are both located on chromosome 10. In addition, evidence is given for the presence of a pseudogene homologous to IFI-56K on chromosome 13.

The interferon-induced Mx-homologous protein in people with symptomatic Hiv-1 infection

Twenty-six people with symptomatic HIV-1 infection were screened for the presence of interferon (IFN) alpha and IFN alpha antibodies in their sera and the presence of the IFN-induced intracellular Mx-homologous protein in their peripheral blood leukocytes. Eleven people had measurable IFN alpha levels ranging from 1 to 40 IU/ml. None of the sera tested was positive for IFN alpha binding or IFN alpha neutralizing antibodies in the assays employed. Twenty-five of the 26 people had significant levels of the Mx-homologous protein in their peripheral mononuclear cells. The Mx concentrations varied from 0.3 to 6 U/ml in the people studied. IFN alpha-positive people had significantly higher levels of the Mx homolog than IFN alpha-negative people (P less than 0.03). Furthermore, the Mx homolog content in Walter-Reed class 2 people was significantly lower than in Walter-Reed class 5/6 people (P less than 0.01). Our results suggest that the IFN system is activated in more than 90% of the people with lymphadenopathy-associated syndrome, AIDS-related complex and AIDS. Since acid-labile IFN alpha can induce the Mx homolog in vitro endogenously produced IFN alpha seems likely to be responsible for the high Mx homolog levels detected.

Quantitation of ineterferon-induced Mx protein in whole blood lysates by an immunochemiluminsescent assay: elimination of protease activity of cell lysates in toto

Due to the rapidly expanding usage of interferons and its costliness of therapy, it is important to evaluate the clinical efficacy of the various interferons. Directly assaying circulating interferon is technically quite difficult. Here, we present an alternate method to evaluate interferon therapy by assaying a unique protein, called Mx protein, which is a 78 kDa cytoplasmic protein selectively induced by type-1 interferon in human leukocytes. The current assay is a two-site chemiluminescent immunoassay, designed to detect Mx protein in whole blood lysates. Since the Mx protein once solubilized, is highly susceptible to proteolysis in whole blood lysates, we have devised a new procedure both to maximize its solubility and virtually eliminate its proteolytic degradation. A mouse monoclonal antibody conjugated to the derivatized-paramagnetic particles and an acridinium ester-labeled antibody serve as the solid phase capture and detector antibodies, respectively. This assay is applicable to both manual and automated modes with a detection limit of Mx protein at 20 ng/ml whole blood. Availability of a reliable assay for Mx protein should facilitate the clinical evaluation of many of the newly constructed type-1 interferons.

The action of recombinant bovine interferons on influenza virus replication correlates with the induction of two Mx-related proteins in bovine cells☆

Recombinant bovine interferon-alpha and -gamma differ in their action against influenza virus on bovine cells. Bovine IFN-alpha severely impairs early protein synthesis and replication of influenza virus in bovine cells in contrast to bovine IFN-gamma which fails to induce an antiviral state against influenza virus. Otherwise the IFN system seems to function normally in bovine cells since both bovine IFN-alpha and -gamma induce an antiviral state against vesicular stomatitis virus. The establishment of the specific antiviral state against influenza virus correlates with the induction by bovine IFN-alpha, but not -gamma, of two cytoplasmic proteins related to the IFN-induced mouse protein Mx involved in the mechanism of resistance of mice to influenza virus infection. This study suggests that bovines possess a system for resistance to influenza virus similar to the mouse Mx system.

Interferon-dependent genetic resistance to influenza virus in mice: virus replication in macrophages is inhibited at an early step.

Inborn resistance to orthomyxoviruses due to the allele Mx was investigated at the molecular level in macrophages from A2G mice. In the absence of interferon, Mx-bearing and control cells were equally permissive for M-TUR, a macrophage-adapted strain of influenza A virus. After treatment with various doses of mouse interferon type I, Mx-bearing cells were fully resistant to this virus whereas cells lacking Mx were only marginally protected. Virus attachment and penetration were equally efficient in Mx-carrying and control cells and were not influenced by interferon. However, virus protein synthesis was blocked in Mx-bearing macrophages expressing resistance. These findings indicate that the resistance mechanism specific for influenza viruses realized by Mx in cooperation with interferon inhibits influenza virus replication in macrophages at an early stage following attachment and penetration of the virus.