猪δ冠状病毒激活钙离子通道SOCE的分子机制

钙离子是细胞的第二信使，在细胞的各种生理功能和信号转导中发挥重要作用。病毒常常通过调控细胞的钙离子水平促进自身增殖和致病。因此，病毒靶向的钙离子通道或钙离子信号是抗病毒药物设计的良好靶标。猪δ冠状病毒（PDCoV）是一种新发现的肠道冠状病毒，我们的前期研究证实PDCoV感染显著上调胞浆钙离子浓度，促进病毒增殖；而且还发现PDCoV感染诱导细胞内质网钙离子向胞浆转运，激活钙池控制的钙离子内流（SOCE）通道，但其作用的分子机制尚不清楚。本项目拟采用基因编码荧光钙离子探针等技术系统分析PDCoV诱导钙离子从内质网向胞浆转运、激活SOCE通道、促进细胞外钙离子内流的分子细节；确定调控SOCE通道的病毒编码蛋白并揭示其作用机制和关键位点；构建关键位点突变的重组病毒，通过动物实验揭示PDCoV激活SOCE通道对其致病性的影响，为阐明PDCoV的致病机理奠定基础，并为抗PDCoV的药物开发提供靶标。

Ionic calcium (Ca2+) is a versatile intracellular second messenger, which modulates various important physiological functions and signal transductions of cells. Virus usually regulates cellular Ca2+ channels or intracellular Ca2+ levels to promote viral replication and pathogenicity. Thus, the Ca2+ channel(s) or Ca2+ signaling(s) targeted by virus are drug targets to develop antiviral drugs. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes serious vomiting and diarrhea in suckling piglets. Our previous studies have showed that PDCoV infection significantly upregulates intracellular Ca2+ concentrations to favor its replication. We also found that PDCoV infection depletes endoplasmic reticulum (ER) Ca2+ to activate the store-operated calcium entry (SOCE) channel. However, the molecular mechanisms remain largely unclear. In this project, we will use the genetically-encoded calcium indicators (GECIs) to image the transfer of ER Ca2+ to cytoplasm and the activation of SOCE channel in live cells after PDCoV infection. The viral protein associated with SOCE activation will be identified and the key function domains or amino acids will be mapped. The recombinant viruses with deletion or mutation of key function domains or amino acids will be generated by using the established reverse genetics system of PDCoV, and animal experiments will be performed to reveal the role of SOCE channel for the pathogenicity of PDCoV. Taken together, the studies of this project will lay the foundation to elucidate the pathogenesis of PDCoV and provide the potential drug target for development of anti-PDCoV drugs.