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Growth of tumors induced by Rous sarcoma virus (RSV) is controlled by alleles at the major histocompatibility complex locus in chickens, indicating that immunological host defense mechanisms play a major role. We show here that the resistance phenotype of CB regressor chickens can be partially reverted by treating the animals with a monoclonal antibody that neutralizes the major serotype of chicken type I interferon, ChIFN-alpha. Injection of recombinant ChIFN-alpha into susceptible CC progressor chickens resulted in a dose-dependent inhibition of RSV-induced tumor development. This treatment was not effective, however, in CC chickens challenged with a DNA construct expressing the v-src oncogene, suggesting that the beneficial effect of type I interferon in this system resulted from its intrinsic antiviral activity and probably not from indirect immunmodulatory effects. By contrast, recombinant chicken interferon-gamma strongly inhibited tumor growth when given to CC chickens that were challenged with the v-src oncogene, indicating that the two cytokines target different steps of tumor development.

To counteract the host immune response, poxviruses have evolved secreted factors that bind cytokines and thereby neutralize their biological activities. The vaccinia virus B8R gene encodes a protein that neutralizes interferon-gamma (IFN-gamma) from several mammals including man, cow, rat, and rabbit but not mice. We now report that the activity of the B8R gene product is not restricted to cytokines of mammals: it also efficiently neutralized chicken IFN-gamma. B8R blocked chicken IFN-gamma-mediated induction of guanylate binding protein RNA in the chicken fibroblast cell line CEC-32 and secretion of nitric oxide in HD-11 cells. Radiolabeled baculovirus-expressed B8R efficiently bound to immobilized recombinant chicken IFN-gamma. Scatchard analysis revealed a binding constant of chicken IFN-gamma to B8R of approximately 0.5 nM. A mutant form of chicken IFN-gamma which lacks the 18 C-terminal amino acids and which has lost more than 99% of its biological activity was able to block the IFN-gamma-neutralizing effect of B8R. Binding studies showed that the mutant protein bound radiolabeled B8R only about threefold less well than wild-type chicken IFN-gamma but failed to compete with wild-type chicken IFN-gamma for binding to the cellular receptor. These results suggest that the extreme C terminus of chicken IFN-gamma is crucial for binding to its cellular receptor but less important for recognition by the viral cytokine receptor.

MxA is a GTPase encoded by an interferon-inducible human gene. Its constitutive expression renders transfected mammaliancells resistant to infections with several different RNA viruses, including vesicular stomatitis virus (VSV). Differences in viral RNA levels of VSV-infected cells either expressing or lacking MxA indicated that VSV mRNA synthesis is the principal target of MxA action. We now used purified histidine-tagged MxA (His-MxA) that we produced in Escherichia coli to successfully inhibit VSV in vitro transcription, a reaction catalyzed by VSV ribonucleoprotein complexes isolated from virus-infected cells or from purified virions. MxA was inactive when added to preformed VSV mRNAs, arguing against the possibility that it has a negative effect on viral RNA stability. MxA inhibited both leader RNA and mRNA synthesis of VSV, suggesting that it interfered with transcription initiation. The degree of VSV inhibition correlated directly with the specific GTPase activities of the various wild-type MxA preparations. No inhibition of viral mRNA synthesis was observed when a C-terminally truncated, GTPase-inactive variant of His-MxA was added to the transcription reactions. Purified His-MxA-E645R, a mutant of MxA with normal GTPase activity whose range of antiviral activity in vivo is altered so that it no longer inhibits VSV, showed no inhibitory effect on VSV in vitro transcription. Since MxA inhibited VSV RNA synthesis in the presence of GMP-PNP or GTPγS, GTP analogs that are readily accepted by the viral polymerase but cannot be hydrolyzed by MxA, the possibility was excluded that MxA acts by depleting the viral polymerase for its nucleotide substrates. Thus, binding of GTP rather than its hydrolysis seems of importance for the anti-VSV activity of MxA.

We used polymerase chain reaction techniques to isolate a highly conserved genomic fragment of the Mx gene of a wild duck. This probe recognized the transcripts of two distinct Mx alleles of virus-infected duck embryo cells, which we cloned and sequenced. Duck Mx mRNA was not detectable in uninduced cells, but it accumulated to a high level after infection with Newcastle disease virus or after treatment of cultured duck embryo cells with double-stranded RNA. Duck Mx protein exhibited the characteristic structural features of interferon-induced mammalian Mx proteins, including GTP-binding and leucine zipper motifs. Western blot analysis revealed multiple forms of duck Mx protein with molecular masses between 72 to 82 kDa. In induced duck embryo cells as well as in murine or avian cells permanently transfected with cloned duck Mx cDNAs, duck Mx protein accumulated predominantly in the cell nucleus. Its immuno-staining pattern differed markedly from that of the nuclear mouse and rat Mx1 proteins. A significant portion of duck Mx protein was also found in the cytoplasm where it formed large granules. Duck Mx proteins failed to render permanently transfected mouse or chick cells resistant to different strains of influenza virus. This unexpected result suggests that influenza virus resistance of ducks is not dependent on interferon-induced Mx proteins.

Although interferon is widely used to treat chronic hepatitis B virus infections, its mode of action against hepadnaviruses is largely unknown, This deficit is due mainly to the lack of suitable model systems. The duck system could not be used because purified duck interferon was not available in sufficient quantities. We have now cloned a DNA fragment that contains an intronless gene for duck interferon. The primary translation product consists of 191 amino acids, the N-terminal 30 residues of which constitute a signal peptide. Mature duck interferon is 50% identical to the recently cloned chicken interferon. Sequence hornology to mammalian interferons is marginal, but conservation of four cysteine residues and inducibility by virus indicate a distant relationship between duck interferon and mammalian type I interferons. Purified recombinant duck interferon from Escherichia coli is biologically active: it activates the interferon-inducible Mx gene, prevents cell destruction by cytolytic RNA viruses, and has a strong inhibitory effect on duck hepatitis B virus in cultured primary duck hepatocytes. This new reagent should help to define the interferon-sensitive step of the hepadnavirus life cycle. Furthermore, the duck system can now be used for systematic studies of the in vivo effectiveness of interferon in chronic hepatitis B virus infections.

Genomic DNA containing the first four exons and upstream sequences of the interferon (IFN)-inducible chicken Mx gene was cloned and sequenced. The exon/intron structure of the chicken Mx gene resembles that of the mouse Mx1 gene: exon 1 is very small, the first intron contains a noncoding exon 1′ that is alternatively spliced in different chicken breeds and exon 2 harbors the ATG initiation codon. Exons 3 and 4 of the chicken and mouse Mx genes have identical lengths and the encoded polypeptides show a high degree of homology. Sequences upstream of the transcription start site functioned as an IFN-inducible promoter in chick and monkey cells when cloned in front of a promoterless luciferase reporter gene. Deletion analysis suggested that the sequence 5′AGTTTCGTTTCT3′ is of critical importance for inducibility. It conforms to the consensus sequence of the IFN stimulated response element (ISRE) present in Mx and other IFN-α/β-inducible promoters of mammals These results suggest that the ISRE motif has been conserved and that it regulates expression of IFN-inducible genes in both mammals and birds.

The interferon-induced murine Mx1 protein possesses an intrinsic antiviral activity with selectivity for influenza viruses. Mx1 accumulates in the nucleus and inhibits the replication of influenza virus at the level of primary transcription. Simultaneous overexpression of the three influenza virus polymerase subunits via recombinant vaccinia virus vectors can titrate out the inhibitory action of Mx1 as determined by the amplification of a transfected recombinant viral reporter gene. A low degree of neutralization was also observed, when PB2 was overexpressed alone [Huang, T., Pavlovic, J., Staeheli, P., and Krystal, M. (1992) J. Virol. 66, 4154-4160]. We now employed a much simpler experimental setting which allowed us to directly measure the effect of PB2 on the antiviral activity of Mx1. We stably transfected a cell line derived from an A2G mouse (homozygous for a functional Mx1 gene) with expression vectors coding for cDNAs of the influenza virus polymerase subunits PB1 and PB2 or of the nucleoprotein (NP). Cells coexpressing Mx1 and PB1 or NP remained resistant to influenza virus infection whereas cells coexpressing Mx1 and PB2 became sensitive to influenza virus infection. The degree of neutralization of Mx1 activity by PB2 was dependent on the Mx1 concentration in the cell. Immunofluorescence analysis revealed that the nuclear localization of Mx1 and PB2 overlapped to a great extent. These findings support the view that Mx1 exerts its antiviral activity by interfering with the function of the influenza virus polymerase subunit PB2.

Some but not all known Mx proteins possess intrinsic antiviral activity. The mouse genome contains two related interferon-regulated genes, designated Mx1 and Mx2. Mx1 codes for a nuclear 72-kDa protein which selectively interferes with the multiplication of influenza viruses. The Mx2 gene is crippled by a mutation in commonly used laboratory mouse strains and, hence, the antiviral potential of the Mx2 protein was unknown. We have corrected the frameshift mutation in a cloned Mx2 cDNA by site-directed mutagenesis. Expression of the repaired Mx2 cDNA in Swiss mouse 3T3 cells gave rise to an 80-kDa cytoplasmic protein that cross-reacted with antibodies to other Mx proteins. In contrast to the cases of mouse Mx1 and human Mx proteins, permanent cell lines were extremely unstable with respect to Mx2 expression. Analysis at the single-cell level revealed that mouse Mx2 conferred to the transfected cells a high degree of resistance to vesicular stomatitis virus, but had no inhibitory effect on influenza virus. The antiviral potential of mouse Mx2 is thus similar to that of rat Mx2 protein.

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.

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.

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.

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.

In mouse cells carrying the dominant influenza resistance allele Mx+ (but not in Mx− cells) interferon-α/β (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.