非肌肉肌球蛋白重链II-A介导猪繁殖与呼吸综合征病毒感染的分子机制

Porcine reproductive and respiratory syndrome virus (PRRSV) has highly restricted cell tropism both in vitro and in vivo. It first binds cell receptors and enters the cell through receptor mediated endocytosis. Previously, we identified nonmuscle myosin heavy chain II-A (NMHC II-A) from MA-104 cells and PAM using the anti-idiotypic antibody, Mab2-5G2, specific for PRRSV GP5. The facts that NMHC II-A binds GP5 and potentially interacts with CD163 indicate it plays important roles in mediating PRRSV infection. However, the structure biology of the interaction between NMHC II-A and GP5 or NMHC II-A and CD163 and the molecular mechanism for their roles in medicating PRRSV infection are not clear. To answer these questions, we plan in this proposal to determine the colocalization of NMHC II-A and GP5 or NMHC II-A and CD163 in the cells using Co-IP and confocal microscopy, to elucidate the key interaction regions between NMHC II-A and GP5 or NMHC II-A and CD163, 3-D structure and the key amino acids using bimolecular fluorescence complementation, X-ray diffraction and site directed mutagenesis, and to evaluate the molecular mechanism of these receptor proteins in mediating PRRSV infection both in cells and pigs. The proposed studies are expected to provide additional information for better understanding PRRSV pathogenesis and generating effective strategy for the prevention and control of this important swine disease.

猪繁殖与呼吸综合征病毒（PRRSV）通过结合细胞受体及细胞内吞作用而感染易感细胞。我们用抗PRRSV GP5的抗独特型抗体从MA-104和PAM细胞中鉴定出的非肌肉肌球蛋白重链II-A（NMHC II-A）结合GP5，已知NMHC II-A结合CD163，提示NMHC II-A通过结合GP5并与CD163互作而介导PRRSV感染宿主细胞。但NMHC II-A与GP5或与CD163互作介导病毒感染的分子机制尚不清楚。因此，本项目拟通过免疫共沉淀、激光共聚焦定位等技术验证NMHC II-A与GP5或与CD163互作；通过双分子荧光互补、X射线衍射及基因定点突变等技术明确NMHC II-A与GP5或与CD163互作的关键区域、三维结构及关键位点；通过体内外实验验证NMHC II-A介导PRRSV感染细胞及靶动物的分子机制，进一步揭示PRRSV的致病机理，为有效防控PRRS提供重要的科学依据。

猪繁殖与呼吸综合征病毒（PRRSV）通过结合细胞受体，利用细胞内吞而感染细胞。我们用抗PRRSV GP5的抗独特型抗体Mab-5G2从MA-104和PAM细胞中鉴定出的非肌肉肌球蛋白重链II-A（NMHC II-A或MYH9）结合GP5，提示NMHC II-A通过结合GP5并与CD163互作，在介导PRRSV感染宿主过程中发挥重要作用。但NMHC II-A与GP5或与CD163互作的结构生物学以及介导病毒感染的分子机制尚不清楚。因此，本项目拟通过免疫共沉淀、激光共聚焦定位等技术明确了MYH9是PRRSV感染细胞不可或缺的细胞因子；证明了MYH9和CD163同时存在是PRRSV入侵细胞的必须条件。确定了MYH9与GP5，MYH9与CD163分子的互作区域：MYH9羧基端氨基酸1651-1714区域与PRRSV GP5胞外区氨基酸29-63区域结合；MYH9羧基端氨基酸1761-1860区域与CD163 SRCR1-4区域结合。阐明了PRRSV借助宿主蛋白MYH9和CD163入侵细胞的关键步骤。PRRSV利用GP2a/4结构蛋白与CD163 SRCR5结构域结合，将其固定在细胞膜上，随后，MYH9分子沿着F-actin移动到细胞膜表面与CD163 SRCR1-4结构域结合，最后，病毒粒子利用MYH9而完成内化。利用模拟PRRSV GP5的抗独特性抗体Mab2-5G2确定了与MYH9结合的关键氨基酸位点是aa1670，aa1673，aa1679，和aa1683。体内外验证了病毒受体阻断剂阻断PRRSV感染的作用，为开发基于阻断病毒受体的阻断剂奠定了基础。