Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection.

An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.     

Capsid protein of eastern equine encephalitis virus inhibits host cell gene expression

Eastern equine encephalitis virus (EEEV) causes sporadic but often severe cases of human and equine neurological disease in North America. To determine how EEEV may evade innate immune responses, we screened individual EEEV proteins for the ability to rescue the growth of a Newcastle disease virus expressing green fluorescent protein (NDV-GFP) from the antiviral effects of interferon (IFN). Only expression of the EEEV capsid facilitated NDV-GFP replication. Inhibition of the antiviral effects of IFN by the capsid appears to occur through a general inhibition of cellular gene expression. For example, the capsid inhibited the expression of several reporter genes under the control of RNA polymerase II promoters. In contrast, capsid did not inhibit expression from a T7 RNA polymerase promoter construct, suggesting that the inhibition of gene expression is specific and is not a simple manifestation of toxicity. The inhibition correlated both with capsid-induced phosphorylation of eukaryotic initiation factor 2 alpha and with capsid-mediated inhibition of cellular mRNA accumulation. Mapping analysis identified the N terminus as the region important for the inhibition of host gene expression, suggesting that this inhibition is independent of capsid protease activity. Finally, when cell lines containing EEEV replicons encoding capsid were selected, replicons consistently acquired mutations that deleted all or part of the capsid, for example, amino acids 18 to 135. Given that the amino terminus of the capsid is required to inhibit host cell gene expression, these data suggest that capsid expression from the replicons is ultimately toxic to host cells, presumably because of its ability to inhibit gene expression.     

Interferon structural genes do not participate in quantitative regulation of interferon production by If loci as shown in C57BL/6 mice that are congenic with BALB/c mice at the alpha interferon gene cluster.

Previous studies have shown that serum interferon (IFN) production in mice is quantitatively influenced by If loci, whose alleles determine high or low production. Although different loci influence IFN production in response to different inducers, such as Newcastle disease virus, Sendai virus, herpes simplex virus type 1, and polyriboinosinic-polyribocytidylic acid, BALB/c mice are in every instance low producers. It was therefore possible that, in addition to If loci, some feature of the BALB/c structural IFN genes contributed to low production. This was examined in the present work, in which IFN production was measured in two strains of C57BL/6 mice congenic with BALB/c at the murine alpha IFN (IFN-alpha) gene cluster on chromosome 4. One line, HW13 (B6.C-H-15c-H-16c-H-20c-H-21c/By) has a BALB/c fragment on chromosome 4 of at least 35 centimorgans which includes the BALB/c IFN-alpha gene cluster and four loci of the brown histocompatibility complex; the other line, HW13J (B6.C-H-15c/By), has a much shorter fragment (about 15 centimorgans), but it also comprises the BALB/c IFN-alpha gene cluster. We show that these mice, carrying the BALB/c IFN-alpha structural genes on a C57BL/6 background, are high IFN producers when stimulated by Newcastle disease virus, Sendai virus, herpes simplex virus type 1, or polyriboinosinic-polyribocytidylic acid. Thus, the low IFN production of BALB/c mice is not directly due to some feature of the IFN-alpha structural genes but is mainly the result of different alleles at If loci.     

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.     

Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V,W, and C proteins

We have generated a recombinant Newcastle disease virus (NDV) that expresses the green fluorescence protein (GFP) in infected chicken embryo fibroblasts (CEFs). This virus is interferon (IFN) sensitive, and pretreatment of cells with chicken alpha/beta IFN (IFN-alpha/beta) completely blocks viral GFP expression. Prior transfection of plasmid DNA induces an IFN response in CEFs and blocks NDV-GFP replication. However, transfection of known inhibitors of the IFN-alpha/beta system, including the influenza A virus NS1 protein and the Ebola virus VP35 protein, restores NDV-GFP replication. We therefore conclude that the NDV-GFP virus could be used to screen proteins expressed from plasmids for the ability to counteract the host cell IFN response. Using this system, we show that expression of the NDV V protein or the Nipah virus V, W, or C proteins rescues NDV-GFP replication in the face of the transfection-induced IFN response. The V and W proteins of Nipah virus, a highly lethal pathogen in humans, also block activation of an IFN-inducible promoter in primate cells. Interestingly, the amino-terminal region of the Nipah virus V protein, which is identical to the amino terminus of Nipah virus W, is sufficient to exert the IFN-antagonist activity. In contrast, the anti-IFN activity of the NDV V protein appears to be located in the carboxy-terminal region of the protein, a region implicated in the IFN-antagonist activity exhibited by the V proteins of mumps virus and human parainfluenza virus type 2.     

A family of interferon-induced Mx-related mRNAs encodes cytoplasmic and nuclear proteins in rat cells.

Mouse Mx protein, an interferon (IFN)-induced nuclear protein, confers selective resistance to influenza virus. We show here that, as with influenza virus-resistant Mx+ mouse embryo cells, influenza virus mRNA accumulation and protein synthesis are strongly inhibited in rat embryo cells treated with IFN-alpha/beta. IFN-alpha/beta induced in rat cells the synthesis of Mx-related mRNAs migrating on Northern (RNA) gels as two bands of about 3.5 and 2.5 kilobases which directed the synthesis of three electrophoretically distinct proteins called rat Mx proteins 1, 2, and 3. The three rat proteins were antigenically related to the mouse Mx protein but differed in molecular weight and intracellular location. Rat Mx protein 1 was found predominantly in the nucleus and, on the basis of several criteria, resembled the nuclear mouse Mx protein. It was induced by IFN-alpha/beta in all 28 inbred rat strains tested. Rat Mx proteins 2 and 3 differed from protein 1 at the carboxy terminus and were predominantly cytoplasmic like the human Mx homolog. Sequence data of partial cDNA clones indicate that three Mx-related genes, rather than one, exist in the rat.     

First report of invertebrate Mx: cloning, characterization and expression analysis of Mx cDNA in disk abalone (Haliotis discus discus)

Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN alpha/beta) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (Haliotis discus discus) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664 bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L(468), L(475), L(489) and L(510), suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48 h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.     

Upstream regulatory elements of murine alpha 4-interferon gene confer inducibility and cell type-restricted expression

We have identified and functionally characterized DNA sequences that are required for the inducible and cell-restricted expression of the murine alpha 4-interferon gene. Hybrid plasmids in which the alpha 4 promoter region or its 5' deletions were inserted upstream of the CAT gene were constructed, and the expression of these hybrid genes was studied in mouse L-cells both in permanent and transient assays with comparable results. Inducible expression was not affected by deletions up to -109; however, when the deletion was extended to -96, inducibility by Newcastle disease virus was abolished; however, this hybrid plasmid was expressed constitutively. Further deletion to -88 did not permit either constitutive or inducible expression. Insertion of the 35-base pair-long sequence (-109 to -75 base pairs) from the alpha 4 promoter region 5' of the minimal alpha 4 or human immunodeficiency virus promoter region, conferred inducibility to these two inactive promoters. The 5' deleted hybrids or plasmids containing the inducible element were induced only at low levels in transfected NIH/3T3 cells that do not express endogenous alpha 4 gene efficiently, indicating that the inducible region also determines the cell-specific expression. A tandem repeat of AGTGAA, which is present in the -109 to -88 region of alpha 4 in two copies, showed both basal levels of expression and inducibility in L-cells, while its analogue AATGAA was highly inducible but was not expressed constitutively. The inducibility of the synthetic hexamer repeats did not show cell type-restricted expression, suggesting that their response does not fully reflect the range of expression observed for the inducible region and the endogenous alpha genes.     

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.     

Protective role of beta interferon in host defense against influenza A virus

Type I interferon (IFN), which includes the IFN-alpha and -beta subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-beta remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-beta-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-beta-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-beta and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-beta plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-alpha.     

Influenza virus evades innate and adaptive immunity via the NS1 protein

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.     

Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-alpha, IFN-beta and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-lambda uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1(0/0)) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-lambda might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-lambda readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1(0/0) mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-lambda failed to induce Mx1 in the liver of IFNAR1(0/0) mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-lambda receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-alpha/beta and IFN-lambda were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1(0/0) mice. From these results we conclude that IFN-lambda contributes to inborn resistance against viral pathogens infecting the lung but not the liver.