猪繁殖与呼吸综合征病毒PRRSV引起猪肺部严重炎性损伤，危害养猪业发展。分泌到细胞外的高迁移率族蛋白1（HMGB1）是重要促炎性因子，与PRRSV致病机理有关，但病毒诱导HMGB1分泌机制不清。我们发现，蛋白激酶C（PKC）抑制剂在不影响PRRSV增殖情况下显著抑制HMGB1分泌，故推测PKC的活化与病毒诱导HMGB1分泌促进炎性应答密切相关。本课题用免疫荧光、细胞组分蛋白分离和免疫印迹等分析体外细胞和猪肺组织内PRRSV感染对PKC的活化；再用药物、表达载体和siRNA确定活化的PKC对病毒诱导HMGB1分泌促进炎性反应的作用；最后用免疫沉淀、液相色谱串联质谱和点突变等鉴定HMGB1上关键PKC催化靶点，并用免疫荧光、免疫印迹和ELISA等确定发挥作用的病毒蛋白、蛋白结构域及作用通路。本研究为深入了解PRRSV致病机理、研制有效调控HMGB1分泌以缓解病毒引起炎性损伤的药物提供理论依据。

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry across the world. Severe pulmonary inflammatory injuries, considered as its main pathology, are shown in infected pigs. Recently, in vitro studies revealed that PRRSV infection induced HMGB1 transfer from the nucleus into cytoplasm, and further secretion to the outside of cells. Extracellular HMGB1, acted as a pro-inflammatory cytokine, enhances inflammatory response. However, until now, the mechanism of PRRSV-induced HMGB1 secretion is not known. Our preliminary studies discovered that a PKC inhibitor significantly suppressed PRRSV-induced HMGB1 secretion without hampering virus replication. Therefore, we hypothesize that PKC activation plays an important role in the PRRSV-mediated induction of HMGB1 secretion. To test the hypothesis, we propose to analyze the effect of PRRSV infection on PKC activation and identify the PKC subtype which is activated in vivo and in vitro by immunofluorescence assay, cell fractionation, Western blotting, etc. Next we will determine the role of the activated PKC subtype(s) on HMGB1 secretion in PRRSV-infected cells by using PKC activator and inhibitor, over-expression and siRNA silencing of the PKC subtype. In order to further elucidate the underlying mechanisms of HMGB1 secretion mediated by PKC in PRRSV-infected cells, we will identify the PKC catalytic sites on HMGB1 nuclear localization sequence (NLS) by Co-IP, LC-MS/MS and point mutagenesis studies. Moreover, we will also determine the role of individual viral proteins and/or protein domains on HMGB1 secretion, as well as elucidate the effect of the viral protein on the PKC up-stream signaling by real-time PCR, immunofluorescence assay, cell fractionation, Western blotting and ELISA. Completion of the proposed study will provide us with informative results on HMGB1 secretion during PRRSV infection, which will contribute to our understanding on the inflammatory response in PRRSV infection and the viral pathogenesis. Results from this study will facilitate specific drug development to alleviate virus-induced inflammatory injury via modulating HMGB1 secretion.