Garigliany M-M, Cloquette K, Leroy M, Decreux A, Goris N, De Clercq K, Desmecht D
Department of Pathology, Faculty of Veterinary Medicine, University of Liège, 4000 Liège, Belgium.
Transgenic Res. 2009 Oct;18(5):719-32. doi: 10.1007/s11248-009-9268-x. Epub 2009 Apr 23.
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.
Mx蛋白是由干扰素诱导产生的大分子鸟苷三磷酸酶动力蛋白超家族成员。这些蛋白备受关注,因为其中一些对致病性RNA病毒具有抗病毒活性,如正粘病毒科、布尼亚病毒科和弹状病毒科的成员。在目前已检测的多种哺乳动物Mx蛋白中,我们最近在体外证明,牛的异构体1(boMx1)具有卓越的抗狂犬病病毒活性。这一发现促使我们寻找合适的体内模型来确认和评估基因治疗策略。利用细菌人工染色体(BAC)转基因技术,我们构建了转基因小鼠品系,这些品系在其天然启动子以及短程和长程调控元件的控制下表达抗病毒的boMx1蛋白和boMx2蛋白。从mRNA测序和蛋白质印迹法推断,表达的boMx1和boMx2能够正确组装。通过免疫组织化学在各种器官中检测到了poly-I/C介导的boMx1表达,并且根据ELISA测定的组织boMx1浓度,转基因品系很容易被分为高表达或低表达品系。发现poly-I/C诱导的Madin-Darby牛肾细胞、牛鼻甲细胞以及来自转基因小鼠高表达品系的培养细胞中boMx1浓度大致相同,这表明该蛋白是在接近生理水平下产生的。此外,牛Mx系统的插入使转基因小鼠对水疱性口炎病毒相关疾病和死亡具有抗性,并且来自高表达转基因小鼠的胚胎成纤维细胞对该病毒的敏感性要低得多。这些结果表明,牛Mx系统在体内是一种强大的抗VSV因子,并且表明这里构建的转基因小鼠品系构成了一个良好的模型,可用于在体内研究牛Mx蛋白发挥的各种已知和未知的抗病毒功能,重点是boMx1的抗狂犬病功能。