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See detailModulating mouse innate immunity to RNA viruses by expressing the Bos taurus Mx system.
Garigliany, Mutien-Marie ULg; Cloquette, Karine ULg; Leroy, Michael et al

in Transgenic Research (2009), 18(5), 719-32

Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted much attention because some display antiviral activity against ... [more ▼]

Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted much attention because some display antiviral activity against pathogenic RNA viruses, such as members of the orthomyxoviridae, bunyaviridae, and rhabdoviridae families. Among the diverse mammalian Mx proteins examined so far, we have recently demonstrated in vitro that the Bos taurus isoform 1 (boMx1) is endowed with exceptional anti-rabies-virus activity. This finding has prompted us to seek an appropriate in vivo model for confirming and evaluating gene therapy strategies. Using a BAC transgene, we have generated transgenic mouse lines expressing the antiviral boMx1 protein and boMx2 proteins under the control of their natural promoter and short- and long-range regulatory elements. Expressed boMx1 and boMx2 are correctly assembled, as deduced from mRNA sequencing and western blotting. Poly-I/C-subordinated expression of boMx1 was detected in various organs by immunohistochemistry, and transgenic lines were readily classified as high- or low-expression lines on the basis of tissue boMx1 concentrations measured by ELISA. Poly-I/C-induced Madin-Darby bovine kidney cells, bovine turbinate cells, and cultured cells from high-expression line of transgenic mice were found to contain about the same concentration of boMx1, suggesting that this protein is produced at near-physiological levels. Furthermore, insertion of the bovine Mx system rendered transgenic mice resistant to vesicular-stomatitis-virus-associated morbidity and mortality, and embryonic fibroblasts derived from high-expression transgenic mice were far less permissive to the virus. These results demonstrate that the Bos taurus Mx system is a powerful anti-VSV agent in vivo and suggest that the transgenic mouse lines generated here constitute a good model for studying in vivo the various antiviral functions-known and yet to be discovered-exerted by bovine Mx proteins, with priority emphasis on the antirabic function of boMx1. [less ▲]

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See detailPathology of influenza virus H5N1 infection in resistant and susceptible laboratory mice.
Garigliany, Mutien-Marie ULg; Cloquette, Karine; Leroy, Michaël et al

Conference (2008, September)

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See detailLes dynamines MX et leur activité anti-influenza.
Garigliany, Mutien-Marie ULg; Zecchinon, Laurent; Cloquette, Karine et al

Poster (2008, September)

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See detailMX dynamins and the innate resistance opposed to influenza virus.
Garigliany, Mutien-Marie ULg; Baise, Etienne; Cloquette, Karine et al

Conference (2008, March)

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See detailDifferential anti-influenza activity among allelic variants at the Sus scrofa Mx1 locus
Palm, Mélanie; Leroy, Michael; Thomas, Anne et al

in Journal of Interferon & Cytokine Research (2007), 27(2), 147-155

A promising way to oppose infectious challenges would be to improve the resistance of the target species through genetic selection. Theoretically, a candidate gene is available against influenza viruses ... [more ▼]

A promising way to oppose infectious challenges would be to improve the resistance of the target species through genetic selection. Theoretically, a candidate gene is available against influenza viruses since a resistance trait was fortuitously discovered in the A2G mouse strain. This trait was demonstrated to be correlated with the expression of a specific isoform of the type I interferon (IFN)-dependent protein MX, an isoform coded by a specific allele at the mouse Mx1 locus. Two allelic polymorphisms were described recently in the Sus scrofa homologous gene. In this study, the frequencies and distribution of both alleles were evaluated among European domestic pig and wild boar populations by PCR-RFLP, and the anti-influenza activity conferred by both MX1 isoforms was evaluated in vitro using transfection of Vero cells followed by flow cytometric determination of the fraction of influenza virus-infected cells among MX-producing and MX-nonproducing cell populations. A significant difference in the anti-influenza activity brought by the two MX1 isoforms was demonstrated, which suggests that a significant improvement of innate resistance of pigs by genetic selection might be feasible provided the differences found here in vitro are epidemiologically relevant in vivo. [less ▲]

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See detailThe Mus musculus Mx1 gene confers strong protection against highly pathogenic influenzavirus A H5N1
Garigliany, Mutien-Marie ULg; Lambrecht, Bénédicte; Leroy, Michaël et al

Poster (2007)

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See detailLes interférons de type 1 et leur fonction antivirale
Leroy, Michael; Desmecht, Daniel ULg

in Annales de Médecine Vétérinaire (2006), 150(2), 73-107

Type I interferons are part of the innate immune system involved in the first line defense against invasion, replication and spreading of pathogens such as viruses. Through specific membrane or ... [more ▼]

Type I interferons are part of the innate immune system involved in the first line defense against invasion, replication and spreading of pathogens such as viruses. Through specific membrane or intracellular receptors, virus entry induces the expression of type I interferons which stimulates the cells through an autocrine and paracrine manner. Interferons are polypeptides that stimulate cells through membrane receptors. They induce a cascade of events leading to the synthesis of many proteins some of which having specific antiviral properties. The most characterized are the MX proteins, the 2’5’ oligoadenylate synthetase/ ribonuclease L pathway and the double-stranded RNA-dependent protein kinase R system. Recently, the protein ISG20 has been involved in the specific antiviral function of type I interferons. The interferons act on multiple steps of the viral cycle including penetration, decapsidation, mRNA transcription, protein synthesis, genome replication, assembling and excretion of viral particles. Nowadays, due to their major efficiency, type I interferons are used in therapeutics against some viral infections and cancers. [less ▲]

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See detailExpression of the interferon-alpha/beta-inducible bovine Mx1 dynamin interferes with replication of rabies virus
Leroy, Michael; Pire, Grégory; Baise, Etienne ULg et al

in Neurobiology of Disease (2006), 21(3), 515-521

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 ... [more ▼]

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. [less ▲]

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See detailResistance of paramyxoviridae to type I interferon-induced Bos taurus Mx1 dynamin
Leroy, Michael; Baise, Etienne ULg; Pire, Grégory et al

in Journal of Interferon & Cytokine Research (2005), 25(4), 192-201

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 ... [more ▼]

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. [less ▲]

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See detailConditional expression of type I interferon-induced bovine Mx1 GTPase in a stable transgenic vero cell line interferes with replication of vesicular stomatitis virus
Baise, Etienne ULg; Pire, Grégory; Leroy, Michael et al

in Journal of Interferon & Cytokine Research (2004), 24(9), 513-521

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 ... [more ▼]

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. [less ▲]

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See detailGenomic structure, organisation, and promoter analysis of the bovine (Bos taurus) Mx1 gene
Gérardin; Baise, Etienne ULg; Pire, Grégory et al

in Gene (2004), 326

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 ... [more ▼]

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. [less ▲]

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