Bovine Mx1 Research Articles

Anti-Schmallenberg Virus Activities of Type I/III Interferons-Induced Mx1 GTPases from Different Mammalian Species.

Mx proteins are key factors of the innate intracellular defense mechanisms that act against viruses induced by type I/III interferons. The family Peribunyaviridae includes many viruses of veterinary importance, either because infection results in clinical disease or because animals serve as reservoirs for arthropod vectors. According to the evolutionary arms race hypothesis, evolutionary pressures should have led to the selection of the most appropriate Mx1 antiviral isoforms to resist these infections. Although human, mouse, bat, rat, and cotton rat Mx isoforms have been shown to inhibit different members of the Peribunyaviridae, the possible antiviral function of the Mx isoforms from domestic animals against bunyaviral infections has, to our knowledge, never been studied. Herein, we investigated the anti-Schmallenberg virus activity of bovine, canine, equine, and porcine Mx1 proteins. We concluded that Mx1 has a strong, dose-dependent anti-Schmallenberg activity in these four mammalian species.

The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region.

Mammalian myxovirus resistance (Mx) proteins are interferon-induced, large dynamin-like GTPases with a broad antiviral spectrum. Here, we analyzed the antiviral activity of selected mammalian Mx1 proteins against Thogoto virus (THOV). Of those, equine Mx1 (eqMx1) showed antiviral activity comparable to that of the human MX1 gene product, designated huMxA, whereas most Mx1 proteins were antivirally inactive. We previously demonstrated that the flexible loop L4 protruding from the stalk domain of huMxA, and especially the phenylalanine at position 561 (F561), determines its antiviral specificity against THOV (P. S. Mitchell, C. Patzina, M. Emerman, O. Haller, et al., Cell Host Microbe 12:598-604, 2012, https://doi.org/10.1016/j.chom.2012.09.005). However, despite the similar antiviral activity against THOV, the loop L4 sequence of eqMx1 substantially differs from the one of huMxA. Mutational analysis of eqMx1 L4 identified a tryptophan (W562) and the adjacent glycine (G563) as critical antiviral determinants against THOV, whereas the neighboring residues could be exchanged for nonpolar alanines without affecting the antiviral activity. Further mutational analyses revealed that a single bulky residue at position 562 and the adjacent tiny residue G563 were sufficient for antiviral activity. Moreover, this minimal set of L4 amino acids transferred anti-THOV activity to the otherwise inactive bovine Mx1 (boMx1) protein. Taken together, our data suggest a fairly simple architecture of the antiviral loop L4 that could serve as a mutational hot spot in an evolutionary arms race between Mx-escaping viral variants and their hosts. IMPORTANCE Most mammals encode two paralogs of the interferon-induced Mx proteins: Mx1, with antiviral activity largely against RNA viruses, like orthomyxoviruses and bunyaviruses; and Mx2, which is antivirally active against HIV-1 and herpesviruses. The human Mx1 protein, also called huMxA, is the best-characterized example of mammalian Mx1 proteins and was recently shown to prevent zoonotic virus transmissions. To evaluate the antiviral activity of other mammalian Mx1 proteins, we used Thogoto virus, a tick-transmitted orthomyxovirus, which is efficiently blocked by huMxA. Interestingly, we detected antiviral activity only with equine Mx1 (eqMx1) but not with other nonprimate Mx1 proteins. Detailed functional analysis of eqMx1 identified amino acid residues in the unstructured loop L4 of the stalk domain critical for antiviral activity. The structural insights of the present study explain the unique position of eqMx1 antiviral activity within the collection of nonhuman mammalian Mx1 proteins.

Development and assessment of a new bioassay for accurate quantification of Type I interferons induced by bovine respiratory viruses

Type I interferon (IFN-I) plays a major role in antiviral and inflammatory processes of the infected host. In the bovine industry, the bovine respiratory disease complex is a major cause of economic and health problems. This disease is caused by interactions of pathogens, together with environmental and host factors. Several pathogens have been identified as causal agents of respiratory diseases in cattle. To better understand how primary infections by viruses predispose animals to further infections by pathogenic bacteria, tools to accurately detect antiviral and immunoregulatory cytokines are needed. To facilitate the detection and quantification of bovine IFN-I, we have established a new specific and sensitive bioassay studies in the bovine host. This assay is based on a Madin–Darby Bovine Kidney (MDBK) cell line that carries a luciferase gene under the control of the IFN-I inducible bovine Mx1 promoter. Specific luciferase activity was measured after stimulation with serial dilutions of recombinant bovine alpha and beta IFNs and human IFN-α. With this novel bioassay we have successfully measured IFN-I production in supernatant from MDBK cells after stimulation of Toll-like receptors (TLR3, TLR7 and TLR8) and RIG-I-like receptors (RIG-I and MDA5), after viral infection with bovine respiratory pathogens, but also in samples from infected calves. Finally, this new bioassay is an easy-to-use and low cost tool to measure the production of bovine Type-I Interferon.

Computational analysis of non-synonymous SNPs in bovine Mx1 gene

The Mx gene encodes an antiviral protein and is induced by type 1 interferons (IFNs). In this study, bovine Mx1B gene was analysed using six online tools to evaluate the effect of single nucleotide polymorphisms (SNPs) that may cause some deleterious effect in the animal. Out of 115 nsSNPs evaluated, 23nsSNPs were classified as highly damaging. The results of computational analysis of the Mx1 gene further widens the scope of research on mutations that can be prioritized in future population and laboratory studies.

Genetic variations of the bovine MX1 and their association with mastitis

The primary agent of mastitis is a wide spectrum of bacterial strains; however, viral-related mastitis has also been reported. The MX dynamin-like GTPase 1 (MX1) gene has been demonstrated to confer positive antiviral responses to many viruses, and may be a suitable candidate gene for the study of disease resistance in dairy cattle. The present study was conducted to investigate the genetic diversity of the MX1 gene in Chinese cattle breeds and its effects on mastitis in Holstein cows. First, polymorphisms were identified in the complete coding region of the bovine MX1 gene in 14 Chinese cattle breeds. An association study was then carried out, utilizing polymorphisms detected in Holstein cows to determine the associations of these single nucleotide polymorphisms (SNPs) with mastitis. We identified 13 previously reported SNPs in Chinese domestic cattle and four of them in Holstein cattle. A novel 12 bp indel was also discovered in Holstein cattle. In addition, haplotype frequencies and linkage disequilibrium of four SNPs detected in Holstein cows were investigated. Analysis of these four SNPs in Chinese Holstein cows revealed two SNPs (g.143181370 T>C and g.143182088 C>T)<br /> significantly (P < 0.05) associated with somatic cell score (SCS). The results indicated that SNPs in the MX1 gene might contribute to the variations observed in the SCS of dairy cattle. Therefore, implementation of these two mutations in selection indexes of the dairy industry might be beneficial by favouring the selection individuals with lower SCS.

Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA.

Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy.

MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants—namely, MX1-a and MX1B. In ruminants, IFN-τ—a type I IFN—is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14–18, 25–40, 50–70, 80–100, and 130–150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14–18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.

Expression of bovine Mx1 protein inhibits the replication of foot-and-mouth disease virus in BHK-21 cells

Mx proteins belonging to the dynamin superfamily of large GTPases inhibit replication of a wide range of RNA viruses. In this study, we examined whether bovine Mx1 protein could interfere with the replication of foot-and-mouth disease virus (FMDV). For this purpose we established cloned BHK-21 cells expressing bovine Mx1 protein (BM1 cells) and infected them with FMDV serotype O. Cloned BHK-21 cells expressing neomycin resistance instead of Mx1 protein (BH1 cells) and original BHK-21 cells served as negative controls. The results showed that the expression of bovine Mx1 protein reduced viral yields by 90% and levels of viral VP1 mRNA by 60%. These findings correlated with a significant reduction of viral antigen detectable in infected cells by immunofluorescent assay. These results demonstrate that bovine Mx1 protein interferes with the replication of FMDV.

Human/Bovine Chimeric MxA-Like GTPases Reveal a Contribution of N-Terminal Domains to the Magnitude of Anti-Influenza A Activity

Type I interferons (IFN-α/β) provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced by IFN-α/β, Mx proteins of some species appear as key components of defense against influenza A viruses. The body of work published to date suggests that to exert anti-influenza activity, an Mx protein should possess a GTP-binding site, structural bases allowing multimerisation, and a specific C-terminal GTPase effector domain (GED). Both the human MxA and bovine Mx1 proteins meet these minimal requirements, but the bovine protein is more active against influenza viruses. Here, we measured the anti-influenza activity exerted by 2 human/bovine chimeric Mx proteins. We show that substituting the bovine GED for the human one in human MxA does not affect the magnitude of anti-influenza activity. Strikingly, however, substituting the human GED for the bovine one in bovine Mx1 yields a chimeric protein with a much higher anti-influenza activity than the human protein. We conclude, in contradiction to the hypothesis currently in vogue in the literature, that the GED is not the sole determinant controlling the magnitude of the anti-influenza activity exercised by an Mx protein that can bind GTP and multimerise. Our results suggest that 1 or several motifs that remain to be discovered, located N-terminally with regard to the GED, may interact with a viral component or a cellular factor so as to alter the viral cycle. Identifying, in the N-terminal portion of bovine Mx1, the motif(s) responsible for its higher anti-influenza activity could contribute to the development of new anti-influenza molecules.

Specific Intracellular Localization and Antiviral Property of Genetic and Splicing Variants in Bovine Mx1

In bovine Mx1, only an amino acid substitution between Ile and Met at position 120 was detected by the nucleotide sequence and mismatched PCR-RFLP technique. The Ile variant was assumed to distribute mainly in the bovine population since the gene frequency was 79.3%. Furthermore, we cloned water buffalo Mx1 cDNA, which showed 51 nucleotide and 20 amino acid substitutions in comparison with that of the cow. Another kind of Mx1 cDNA, bovine Mx1B cDNA, was found and it was deduced to cause 27 amino acid substitutions at the N-terminus compared to the original Mx1 by alternative splicing. However, no variation was detected in 27 amino acids specific for Mx1B among 29 cows and a water buffalo. We established four kinds of mRNA-expressing 3T3 cells and Vero cells. When infection experiments were performed using recombinant vesicular stomatitis virus (VSVDeltaG*-G), bovine Ile and Met types and water buffalo Mx1 mRNA-expressing cell lines showed equally positive antiviral activities (p < 0.05). On the other hand, bovine Mx1B mRNA-expressing cell lines did not have activity against VSVDeltaG*-G. Intracellular localization of bovine Mx1 and Mx1B proteins was examined by a transiently GFP-fused expression system in 3T3 cells. Bovine Mx1 was localized in the cytoplasm, while bovine Mx1B was mainly localized in the nucleus. An arginine-rich nuclear localization signal was found in 27 amino acids specific for Mx1B. N-terminus-deleted Mx1B was only localized in the cytoplasm, and the deleted Mx1B-expressing cell lines showed significantly positive antiviral activities (p < 0.05) against VSVDeltaG*-G.

Structural and Functional Analysis of the BovineMx1Promoter

The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFκB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction. Several artificial deletion mutants were prepared to determine the important regulatory elements responsible for the promoter activity, and it was found that ISRE has a key function in IFN response. The proximal ISRE1 showed potential induction by IFN. Furthermore, the proximal GC boxes were found to be essential for IFN response in the bovine Mx1 promoter with the deletion mutants. In this case, the 2 GC boxes exhibited a synergistic activation in the IFN response. Mithramycin A, an agent that inhibits gene expression selectively by coating GC boxes, was used, and Mx mRNA expression in MDBK cells was suppressed by this chemical. Therefore, GC boxes were also shown to be essential for IFN response in the bovine Mx1 gene.

Bovine and water buffalo Mx2 genes: polymorphism and antiviral activity

Millennia-long selective pressure of single-strand RNA viruses on the bovine Mx locus has increased the advantages of using the bovine Mx protein to evaluate the ultimate significance of the antiviral role of Mx proteins. The conclusions of research based only on the bovine Mx1 protein showed the need for comprehensive studies that demonstrate the role of all isoforms, individually or together, especially in the presence of a second isoform, the bovine Mx2 gene. This study provides information about bovine and water buffalo Mx2 genes, as well as their allelic polymorphism and basic antiviral potential. Observation of an Mx2 cDNA sequence (2,381 bp) obtained from 15 animals from 11 breeds using primers based on a previous sequence (NCBI accession no. AF335147) revealed several nucleotide substitutions, with eight different alleles and two amino acid exchanges: Gly to Ser at position 302 and Ile to Val at position 354, though the latter was found only in the NCBI database. A water buffalo Mx2 cDNA sequence was identified for the first time, revealing 46 nucleotide substitutions with 12 amino acid variations, in addition to a 9-bp insertion in the 5' untranslated region UTR, compared with the bovine Mx2 cDNA. Transfected 3T3 cells expressing bovine Mx2 mRNAs coding Gly or Ser at position 302, water buffalo Mx2 mRNA, positive control bovine Mx1 mRNA-expressing cells, and negative control parental 3T3 were subjected to infection with recombinant vesicular stomatitis virus (VSVDeltaG*-G), as were empty pCI-neo vector-transfected cells. The positive control and all cells expressing Mx2 mRNAs displayed significantly higher levels of antiviral activity against VSVDeltaG*-G (P < 0.01) than did the negative controls.

Expression of the interferon-alpha/beta-inducible bovine Mx1 dynamin interferes with replication of rabies virus

Rabies is a fatal anthropozoonotic viral infection of the central nervous system that remains a serious public health problem in many countries. As several animal cases of spontaneous survival to infection were reported and because type 1 interferons were shown to protect against the virus, it was suggested that innate resistance mechanisms exist. Among the antiviral proteins that are synthesized in response to interferon-alpha/beta stimulation, Mx proteins from several species are long known to block the replication of vesicular stomatitis virus (VSV). As both VSV and rabies virus belongs to the Rhabdoviridae family, this study was started with the aim to establish whether the anti-VSV activity of a mammalian Mx protein could be extended to rabies virus. This question was addressed by inoculating the virus onto a bovine Mx1 or human MxA-expressing Vero cell clone. Plaque formation was unambiguously blocked, and viral yields were reduced 100- to 1000-fold by bovine Mx1 expression for both SAG2 and SADB19 viral strains. In opposition, only SAG2 strain could be inhibited by the expression of human MxA protein. The effect of both proteins expression was then evaluated at the viral protein expression level. Again, boMx1 was able to repress protein expression in both strain, whereas only SAG2 proteins were inhibited in human MxA-expressing cells. These results suggest that protection conferred by interferon-alpha/beta against rabies could be, at least partially, attributable to the Mx pathway. Alternatively, bovine Mx1 could be unique in its ability to repress rabies virus which, if confirmed in vivo, would open an avenue for the development of new antirabies therapeutic strategies.

Resistance of Paramyxoviridae to Type I Interferon-InducedBos taurusMx1 Dynamin

Typical targets of type I interferon (IFN)-induced antiviral Mx proteins known to date have been shown to share a common profile: single-stranded negative-sense RNA viruses. Among them, human MxA is known to interfere with the replication of measles, human, and bovine parainfluenza-3 viruses (BoPi3V), that is, three members of the Paramyxoviridae family. Recently, bovine Mx1 protein (BoMx1) was included in the group of Mx proteins with authenticated antiviral potential, as it dramatically represses the replication of vesicular stomatitis virus (VSV). As replication in bovine cells of Pi3, respiratory syncytial (RS), and Sendai (Se) viruses, all members of the same family, is known to be reduced on IFN-alpha incorporation into the culture medium, it was hypothesized that the BoMx1 pathway possibly was involved, its antiviral spectrum thus probably extending to Paramyxoviridae. In this study, probing of BoMx1-inhibiting effects was carried out by infecting a transgenic Vero cell line that allows tightly regulated conditional expression of BoMx1 after doxycycline treatment with a wide array of Paramyxoviridae. Expressing and nonexpressing cells displayed similar viability, cytopathic effects (CPEs), and amounts of infectious virus yields, whatever the infecting virus or the multiplicity of infection (moi) imposed. It is, therefore, concluded that BoMx1 does not interfere with Paramyxoviridae.

Conditional expression of type I interferon-induced bovine Mx1 GTPase in a stable transgenic vero cell line interferes with replication of vesicular stomatitis virus.

In some vertebrate species, type I interferon(IFN)-induced Mx gene expression has been shown to confer resistance to some single-stranded RNA (ssRNA) viruses in vitro. Because the bovine species is subject to an exceptionally wide array of infections caused by such viruses, it is anticipated that an antiviral allele should have been retained by evolution at the bovine Mx locus. The identification of such allele may help in evaluating the real significance of the Mx genotype for disease resistance in vivo, in deciphering host-virus molecular interactions involved, or in improving innate disease resistance of livestock through marker-assisted selection. We validated a double transgenic Vero cell clone in which the bovine Mx1 reference allele is placed under control of the human cytomegalovirus (CMV) enhancer-promoter sequence containing elements from the bacterial tetracycline resistance operon to regulate transcription. In the selected clone, transgene repression was very tight, and derepression by doxycycline led to homogeneous 48-h duration expression of physiologic levels of bovine Mx1. Expression of the transgene caused a dramatic decrease in cytopathic efficiency and a 500-5000-fold yield reduction of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV). To our knowledge, the transgenic clone developed here is the first ever reported that allows conditional expression of an Mx protein, thus providing a valuable tool for studying functions of Mx proteins in general and that of bovine Mx1 in particular. This latter may henceforward be included in the group of Mx proteins with authenticated anti-VSV activity, which offers new research avenues into the field of host-virus interactions.

Conditional Expression of Type I Interferon-Induced Bovine Mx1 GTPase in a Stable Transgenic Vero Cell Line Interferes with Replication of Vesicular Stomatitis Virus

In some vertebrate species, type I interferon(IFN)-induced Mx gene expression has been shown to confer resistance to some single-stranded RNA (ssRNA) viruses in vitro. Because the bovine species is subject to an exceptionally wide array of infections caused by such viruses, it is anticipated that an antiviral allele should have been retained by evolution at the bovine Mx locus. The identification of such allele may help in evaluating the real significance of the Mx genotype for disease resistance in vivo, in deciphering host-virus molecular interactions involved, or in improving innate disease resistance of livestock through marker-assisted selection. We validated a double transgenic Vero cell clone in which the bovine Mx1 reference allele is placed under control of the human cytomegalovirus (CMV) enhancer-promoter sequence containing elements from the bacterial tetracycline resistance operon to regulate transcription. In the selected clone, transgene repression was very tight, and derepression by doxycycline led to homogeneous 48-h duration expression of physiologic levels of bovine Mx1. Expression of the transgene caused a dramatic decrease in cytopathic efficiency and a 500–5000-fold yield reduction of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV). To our knowledge, the transgenic clone developed here is the first ever reported that allows conditional expression of an Mx protein, thus providing a valuable tool for studying functions of Mx proteins in general and that of bovine Mx1 in particular. This latter may henceforward be included in the group of Mx proteins with authenticated anti-VSV activity, which offers new research avenues into the field of host-virus interactions.

Genetic polymorphisms and antiviral activity in the bovine MX1 gene

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.

Genomic structure, organisation, and promoter analysis of the bovine (Bos taurus) Mx1 gene.

Some MX proteins are known to confer a specific resistance against a panel of single-stranded RNA viruses. Many diseases due to such viruses are known to affect cattle worldwide, raising the possibility that the identification of an antiviral isoform of a bovine MX protein would allow the implementation of genetic selection programs aimed at improving innate resistance of cattle. With this potential application in mind, the present study was designed to isolate the bovine Mx1 gene including its promoter region and to investigate its genomic organisation and promoter reactivity. The bovine Mx1 gene is made up of 15 exons. All exon-intron boundaries conformed to the consensus sequences. A PCR product that contained a approximately 1-kb, 5'-flanking region upstream from the putative transcription start site was sequenced. Unexpectedly, this DNA region did not contain TATA or CCAAT motifs. A computer scan of the region disclosed a series of putative binding sites for known cytokines and transcription factors. There was a GAAAN(1-2)GAAA(C/G) motif, typical of an interferon-sensitive responsive element, between -118 and -107 from the putative transcription start site. There were also a NF-kappaB, two interleukin-6 binding sites, two Sp1 sites and five GC-rich boxes. The region also contained 12 stretches of the GAAA type, as described in all IFN-inducible genes. Bovine Mx1 expression was assessed by Northern blotting and immunofluorescence in the Madin Darby bovine kidney cells (MDBK) cell line treated with several stimuli. In conclusion, the bovine Mx1 gene and promoter region share the major structural and functional characteristics displayed by their homologs described in the rainbow trout, chicken, mouse and man.

The bovine Mx1 gene: characterization of the gene structure, alternative splicing, and promoter region.

The Mx gene encodes an antiviral protein and is induced by type 1 interferons (IFNs). In this study, a new bovine Mx gene (designated Mx1B) was isolated from the endometrial cDNA library of the early pregnant cow. Although the Mx1B cDNA contained a single open reading frame (ORF) the same as the known Mx1, the 5' untranslated region (UTR) and 5' coding region of Mx1B were rather different from the corresponding regions of Mx1. Genomic structure analysis revealed that bovine Mx1B was an alternative splicing variant of Mx1 and had transcription regulatory sequences in the upstream region. RT-PCR and its sequencing identified another Mx1 splicing variant and demonstrated that these bovine Mx1 splicing variants were ubiquitously expressed in various tissues. Furthermore, it was found that all the bovine breeds investigated had identical splice sites of Mx1 and Mx1B. It is speculated that cattle have at least two functional Mx isoforms that might provide strong natural resistance to specific viruses.

In vitro and in vivo detection of Mx gene products in bovine cells following stimulation with alpha/beta interferon and viruses.

This study focused on products of the bovine Mx1 gene as specific markers for acute viral infections. The rationale for this is the fact that viral infections are commonly paralleled by the synthesis, release, and remote action of alpha/beta interferons (IFN-alpha/beta). Released IFN-alpha/beta act through specific receptors present on nucleated cells to transduce signals for the transcription of numerous IFN-regulated genes, such as the ones for double-stranded-RNA-dependent protein kinase, 2'-5'-oligoadenylate synthetase, or the Mx proteins. In this study, cultured MDBK cells and bovine white blood cells (WBC) were treated with recombinant IFN-alpha or infected with either bovine herpesvirus 1 (BHV-1) or bovine rotavirus (BRV). Treatment of cultured cells with IFN-alpha was followed within 4 h by a time- and dose-dependent accumulation of intracytoplasmic Mx protein as revealed by immunostaining and Western blot immunoassay. This was preceded by a distinct rise of Mx mRNA in similarly treated cells, as revealed by a newly established quantitative TaqMan PCR technique. The two viruses displayed a cell-dependent in vitro ability to induce Mx proteins, which was limited to bovine WBC with BHV-1 and to MDBK cells with BRV. The established methods were successfully used to show that infection of calves with a noncytopathic strain of bovine viral diarrhea virus, a pestivirus, was followed within 2 days postinfection by strong expression of both Mx mRNA and Mx proteins in WBC.