Calpain-1与CD163互作介导PRRSV脱壳及其分子机制研究

CD163是PRRSV脱壳的必需受体，但病毒脱壳的具体分子机制未知。Calpain-1通过重构细胞骨架参与囊泡运输。前期数据发现：wild-type Marc-145能感染PRRSV，其CD163与Calpain-1互作；而SRCR5缺失的细胞不能感染病毒，该编辑的CD163不与Calpain-1互作，提示Calpain-1可能在病毒脱壳中发挥重要的作用。在此基础上，本项目拟通过co-IP验证质谱数据，分析PRRSV感染对Calpain-1的影响，验证Calpain-1在病毒脱壳阶段发挥作用；阐明其与CD163 SRCR5互作的关键结构，制备Calpain-1敲除细胞系研究其对病毒感染的作用，揭示其调节病毒脱壳的分子机制；最后通过活体攻毒进一步验证Calpain-1对病毒感染的作用。预期结果不仅可确证Calpain-1可调节PRRSV感染，还可为挖掘PRRSV抗病/易感基因奠定基础。

CD163 is required for PRRSV uncoating, but the molecular mechanism of its uncoating remains unknown. Calpain-1 reconstructs cytoskeleton to regulate vesicular trafficking. Our previous data of LC-MS/MS showed that, wild-type Marc-145 cells can be infected with PRRSV, and its CD163 interacts with Calpain-1, while SRCR5-deleted cells resist PRRSV infection and the gene-edited CD163 cannot interacts with Calpain-1. These data suggest that Calpain-1 may play a crucial role in virus infection. Therefore, we plan to confirm the LC-MS/MS data using co-IP, investigate the effect of PRRSV infection on Calpain-1, and demonstrate Calpain-1 functions in virus uncoating rather than in virus absorption and entry. Then we address the mechanism of the interaction of Calpain-1 with SRCR5, test its role in virus infection using a Calpain-1 knock out cell line, thus illustrating the molecular mechanism of its regulation on virus uncoating. Finally we further analyze the influence of Calpain-1 on infection in vivo. The data not only confirm the role of Calpain-1 in PRRSV replication, but also provide new theoretical basis for screening susceptibility or resistance genes against the virus.