猪圆环病毒2型调控宿主PKR信号通路的机制研究

The protein kinase R (PKR) plays an important role in the well characterized anti-viral innate immune response. For survival, many viruses have evolved many elaborate mechanisms to antagonize the PKR-mediated immune response at virtually every step in the pathway, from activation through substrate phosphorylation. Previous studies have indicated that PCV2 may well be capable of inducing the activation of PKR at the start of virus infection but restraining the PKR response in the early stage of PCV2 infection, although the defined mechanism is still not elucidated. Thus, the first aim of this project is to explicate the mechanism of PKR activation within PAMs infected with PCV2 via determining the changes of cellular dsRNA, total PKR, total eIF2α, phosphorylated PKR and phosphorylated eIF2α after the PAMs over-expressing or downregulate-expressing the common activator proteins of PKR were established. Furthermore, the interplay between PCV2 Cap and host Hsp40 in vitro or in vivo and their biological significance on Hsp40-P58IPK complex will be confirmed by using GST pull-down, Subcellular co-localisation, Co-IP. Meanwhile, the impact and mechanism of Hsp40 protein on PCV2 replication will also be identified through shRNA, qRT-PCR and Western blot. Finally, how PCV2 manipulates the host PKR signal transduction pathway, especially how PCV2 inhibits the PKR signal pathway, will be clearly defined.

PKR蛋白激酶在机体抗病毒天然免疫应答中发挥着重要作用，而病毒为了生存也会通过多种途径拮抗PKR介导的免疫应答。研究表明 PCV2感染过程中不仅能够激活PKR而且可以通过某种机制抑制PKR效应，但具体机制尚不清楚。本项目拟在发现PCV2感染猪肺泡巨噬细胞(PAM)过程中激活PKR的基础上，在PAM细胞中过表达或抑制表达PKR常见活化物蛋白并接种PCV2，检测 PCV2对细胞dsRNA、总PKR和eIF2α蛋白及磷酸化PKR和eIF2α蛋白表达的影响，明确PCV2激活PKR的机制；应用GST pull-down、亚细胞共定位、Co-IP、shRNA、qRT-PCR和 Western blot等技术检测PCV2 Cap与宿主Hsp40蛋白之间的互作及其对Hsp40-P58IPK复合体的影响，确定Hsp40蛋白对PCV2复制的影响及机制，最终阐明PCV2病毒抑制宿主PKR信号通路的机制。

PKR在机体抗病毒天然免疫应答中发挥着重要作用，而病毒为了生存也会通过多种途径拮抗PKR介导的免疫应答。研究表明 PCV2感染过程中不仅能够激活PKR而且可以通过某种机制抑制PKR效应，但具体机制尚不清楚。本项目拟在发现PCV2感染猪肺泡巨噬细胞(PAM)过程中激活PKR的基础上，主要开展在PAM中过表达或抑制表达PKR常见活化物蛋白并接种PCV2以检测PCV2对细胞dsRNA、总PKR和eIF2α蛋白及磷酸化PKR和eIF2α蛋白表达的影响以及应用GST pull-down、亚细胞共定位、Co-IP、shRNA、qRT-PCR和Western blot等技术检测Cap与Hsp40蛋白之间的互作及其对Hsp40-p58IPK复合体影响的研究工作，旨在明确PCV2激活PKR的机制，确定Hsp40对PCV2复制的影响及机制并最终阐明PCV2抑制宿主PKR信号通路的机制。结果表明：（1）仔猪感染PCV2后，猪体内DNAJC7（Hsp40）的表达量被上调，且表现为PCV2在猪体各组织器官的分布与猪Hsp40蛋白的分布呈正相关。用Hsp40蛋白特异性抑制剂KNK437或者慢病毒介导的shRNA下调PAM中Hsp40的表达量时，PCV2的基因组复制量和病毒颗粒的产生量均显著下降，说明Hsp40整体是促进PCV2复制的。（2）亚细胞共定位、GST-pull down及双向Co-IP实验均证实猪Hsp40与Cap蛋白之间存在相互作用。（3）PCV2感染PAM过程中能够产生被鼠抗dsRNA J2所识别的免疫荧光信号，说明PCV2感染PAM过程中能够产生dsRNA，进而说明PCV2感染能够激活宿主PKR信号通路。（4）PCV2感染早期能够产生dsRNA并激活宿主PKR信号通路以抵抗PCV2的入侵，同时由于宿主Hsp40蛋白的表达而进一步加固Hsp40-p58IPK蛋白复合体，从而抑制PCV2复制。然而，随着PCV2 Cap蛋白的表达，Cap蛋白能够与p58IPK竞争结合Hsp40蛋白，导致Hsp40-p58IPK蛋白复合体解聚并使p58IPK被释放，导致PKR对PCV2复制的抑制作用被解除。该研究结果将为揭示PCV2感染初期逃避宿主天然免疫应答提供理论依据，更为PCVAD的诊断、预防和治疗提供新的策略或治疗靶点。