宿主细胞DEAD-box RNA解旋酶DDX3和DDX5在猪繁殖与呼吸综合征病毒感染中的作用机制研究

Porcine reproductive and respiratory syndrome virus continues to cause economically significant impacts on the swine industry worldwide. Unfortunately, the conventional vaccines fail to provide sustainable disease control. Therefore, the successful control of the disease relies tremendously on the basic knowledge of host-virus interactions. DEAD-box RNA helicases are expressed ubiquitously in host cell. Members of this family of protein DDX3 and DDX5 have been shown to participate in several viruses replication, including HIV, HBV, HIV and influenza virus. Our preliminary data showed that overexpression of these two proteins inhibit PRRSV propagation, while decrease of DDX3 and DDX5 expression by RNA interference facilitates virus propagation. Therefore, in this project, we plan to continue the studies on the interactions between DDX3,DDX5 and virus and on how the interactions between viral protein(s) and DDX3 influence interferon production in MARC-145 and PAM-pCD163 cells by using virology and cell biology methods. Furthermore, we will determine the crystal structures of DDX3 or DDX5 with viral proteins to provide more information on the interactions between these proteins. The studies on DDX3 and DDX5 will help us to clarify their roles in PRRSV infection,which will also provide more insights in the molecular mechanisms of PRRSV replication and the novel strategies to control PRRSV infection.

猪繁殖与呼吸综合征病毒感染对世界养猪业造成重大经济损失。目前疫苗不能有效控制该病的发生。对该病的有效控制很大程度上依赖对病毒和宿主相互作用机制的了解。DEAD-box RNA解旋酶是宿主细胞中广泛存在的蛋白。该家族的DDX3和DDX5与HIV、HBV、HCV、流感病毒感染有关，但具体机制仍不清楚。我们的初步研究结果表明这两个蛋白与PRRSV病毒的感染有关，过量表达会抑制病毒增殖，而抑制表达会促进病毒增殖。本项目拟借助病毒学和细胞生物学的方法在MARC-145和PAM-pCD163细胞上研究DDX3和DDX5与病毒相互作用影响病毒增殖的机制，以及病毒蛋白与DDX3相互作用对干扰素产生的影响。并借助蛋白质结晶学方法研究DDX3或DDX5与病毒蛋白复合物的空间结构，从结构上明确宿主蛋白与病毒蛋白的相互作用方式。本研究将促进对PRRSV增殖机制的了解，对寻找新的抗PRRSV感染的方法具有重要意义。

(DEAD)-box RNA helicase 3 (DDX3)是ATP依赖的RNA解旋酶并参与许多RNA的代谢过程。病毒感染过程中，DDX3参与先天免疫的调节。目前，关于DDX3参与先天免疫调节的研究主要集中在其对IFN-β信号通路的调节。本研究中我们发现DDX3参与了NF-κB信号通路的调节。.细胞因子的产生是先天免疫反应激活的重要表现特征。通过干扰DDX3和qRT-PCR实验，发现poly(I:C)或TNF-α刺激HELA造成的促炎症细胞因子上升（IL-1，IL-6，IL-8和TNF-α）会受到DDX3表达降低的影响。这说明DDX3参与先天免疫的调节并影响炎症细胞因子的产生。我们通过 Western Blot、核质分离和间接免疫荧光等技术对DDX3沉默状态下NF-κB信号通路上的信号分子进行检测发现，DDX3沉默的情况下，poly(I:C)或TNF-α刺激造成的IKK-β，p65和IκBα的磷酸化水平均下降；IκBα的降解减少；p65的入核减少。进一步证实DDX3参与NF-κB信号通路调节。DDX3对NF-κB信号通路的调节与其ATPase和解旋酶活性无关。.通过质谱和免疫共沉淀实验发现DDX3与PP2A-C相互作用，表明DDX3可能通过PP2A-C来调节NF-κB信号通路。当我们沉默DDX3后，PP2A-C与IKK-β的相互作用明显增强，说明DDX3调节PP2A-C与IKK-β的相互作用。IKK-β的组成性激活突变体S177/181E可以回补DDX3沉默造成的p65的磷酸化下降，进一步证明DDX3通过PP2A-C来调控NF-κB信号通路。.Tyr307被磷酸化会使得PP2A失去活性。DDX3沉默后PP2A-C的磷酸化明显下调。Y307D是PP2A磷酸化模拟突变体，无磷酸酶活性。过表达Y307D突变体可以增强IKK-β 和 p65的磷酸化。过表达Y307D突变体回补由于沉默DDX3造成的p65的磷酸化下调。这些结果说明DDX3通过调解PP2A-C的磷酸化来调节NF-κB信号通路。