猪繁殖与呼吸综合征病毒通过激活PKC诱导HMGB1分泌促进炎性应答的作用机制研究

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.

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。

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。本项目研究发现PRRSV能够通过激活蛋白激酶Cδ（PKCδ）诱导细胞内HMGB1分泌并促进炎性应答。在PRRSV感染细胞内HMGB1的分泌与PKCδ的活化水平呈正相关，且都表现出随感染剂量和感染时间的增加而升高。用PKCδ抑制剂或siRNA能有效抑制PRRSV诱导的HMGB1向胞浆的迁移和分泌；且抑制PKCδ能显著降低细胞内炎性因子的表达。筛选发现，PRRSV结构蛋白E和ORF5a与病毒诱导PKCδ活化引起HMGB1分泌密切相关。进一步探究机制，我们发现PRRSV主要是通过磷酸化修饰细胞内HMGB1 NLS序列上的苏氨酸位点（Thr）诱导HMGB1的分泌；且NLS上第51位Thr与HMGB1分泌密切相关。PRRSV感染细胞内PKCδ与HMGB1并没有直接的相互结合；通过蛋白质谱和免疫共沉淀分析发现PRRSV感染细胞内与HMGB1发生相互作用的蛋白是核糖体蛋白S3（RPS3）。另外，我们通过对猪感染不同PRRSV毒株发现，PRRSV感染猪体内PKCδ活化、HMGB1分泌和肺组织炎性反应水平相一致。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。