Kochs G, Trost M, Janzen C, Haller O
Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Hermann-Herder-Strasse 11, Freiburg, D-79008, Germany.
Methods. 1998 Jul;15(3):255-63. doi: 10.1006/meth.1998.0629.
MxA protein is an interferon-induced GTPase of human cells that inhibits the multiplication of several RNA viruses, including influenza viruses and bunyaviruses. Studies on MxA transgenic mice have shown that MxA is a powerful antiviral agent in vivo. It has been suggested that this cellular protein also protects humans from viral disease, but the mechanism(s) by which MxA exerts its antiviral action is still poorly understood. Using an in vitro cosedimentation assay, we now demonstrate that MxA tightly interacts with components of the ribonucleoprotein complex of Thogoto virus, an influenza-like virus transmitted by ticks. This assay demonstrates for the first time a physical interaction between MxA GTPase and a viral target structure. It is based on three elements, namely, highly active MxA GTPases as effector molecules, viral ribonucleoprotein particles as viral targets, and GTPgammaS as a stabilizing factor. Furthermore, using a simple nuclear translocation assay, we show that human MxA protein forms oligomers in vivo. This assay provides a stringent test for tight association of partner molecules in intact mammalian cells. It not only will be useful for studying physical interactions of MxA with partner molecules, but may also be applicable to other studies on protein-protein interactions in living cells.
Mx A蛋白是一种由干扰素诱导产生的人类细胞GTP酶，可抑制包括流感病毒和布尼亚病毒在内的多种RNA病毒的增殖。对Mx A转基因小鼠的研究表明，Mx A在体内是一种强大的抗病毒剂。有人提出，这种细胞蛋白也能保护人类免受病毒疾病侵害，但Mx A发挥其抗病毒作用的机制仍知之甚少。通过体外共沉降试验，我们现在证明Mx A与Thogoto病毒（一种由蜱传播的类流感病毒）核糖核蛋白复合物的成分紧密相互作用。该试验首次证明了Mx A GTP酶与病毒靶标结构之间的物理相互作用。它基于三个要素，即作为效应分子的高活性Mx A GTP酶、作为病毒靶标的病毒核糖核蛋白颗粒以及作为稳定因子的GTPγS。此外，通过一个简单的核转位试验，我们表明人类Mx A蛋白在体内形成寡聚体。该试验为完整哺乳动物细胞中伙伴分子的紧密结合提供了一个严格的测试。它不仅将有助于研究Mx A与伙伴分子的物理相互作用，也可能适用于对活细胞中蛋白质-蛋白质相互作用的其他研究。
