山羊副流感病毒3型靶向STAT1拮抗I型干扰素抗病毒应答的分子机制

Caprine parainfluenza virus type 3 (CPIV3), an emerging pathogen from the Respirovirus of Paramyxoviridae with high pathogenicity and infectiousness, has led to much economic loss in goat industry. To promote viral colonization and infection, many viruses have evolved different strategies to counter host innate immune signaling. Our preliminary study found that CPIV3 infection targeted JAK/STAT pathway to inhibit type I interferon signaling transduction by inhibiting STAT1 expression, phosphorylation and nuclear translocation, while the underlying mechanism remains unclear. In this proposed study, we will focus on the STAT1 intracytoplasmic activation and nuclear transport, from the perspectives of protein interaction, degradation and kinase activity, to explore how the CPIV3 antagonizes the type I interferon antiviral response within the infection model of goat tracheal epithelial cell. Furthermore, by monitoring the regulation process of each CPIV3 encoding protein to STAT1, we aim to identify the viral protein inhibiting STAT1 activation and its functional sites, understand the molecular details of viral proteins regulating STAT1, and finally elucidate the molecular mechanism how CPIV3 hijacks STAT1 to antagonize the antiviral response of type I interferon. This study expands the cognition of CPIV3 modulating on host innate immune response, and lays a foundation for the development of effective CPIV3 control measures.

山羊副流感病毒3型（CPIV3）为羊呼吸道疫病重要病原，致病力及传播性强，给养羊业造成较大危害。许多病毒为了自身的定殖与感染，进化出多种逃避宿主先天免疫应答的策略。前期试验发现CPIV3感染拮抗I型干扰素抗病毒效应，显著下调STAT1表达、磷酸化激活及核转运效率，提示其具有劫持JAK/STAT通路的潜能，但具体作用机制尚不明确。本研究计划以STAT1胞质内激活、核转运等调控过程为切入点，利用山羊气管上皮细胞模型，通过蛋白互作、降解及激酶活性等分析，探究CPIV3感染拮抗STAT1激活的分子过程。进一步通过逐个监测CPIV3编码蛋白对STAT1的调控作用，鉴定抑制STAT1激活的病毒蛋白及其功能位点，解析病毒蛋白调控STAT1的作用过程，阐明其辅助CPIV3靶向STAT1拮抗I型干扰素抗病毒应答的分子机制。上述研究将扩展CPIV3调制宿主先天免疫应答的认知，为开发有效防治措施提供参考。

CPIV3是引起山羊呼吸道疫病的重要病原体，是副黏病毒科呼吸道病毒属的新成员，其可有效感染山羊和绵羊。前期研究发现CPIV3感染可拮抗I型干扰素的抗病毒效应，本项目进一步证实山羊IFN-α提前孵育或与CPIV3同时孵育均可有效抑制CPIV3体外感染，但对于已建立的感染不存在抗病毒活性。利用原代山羊气管上皮细胞及MDBK细胞模型，发现CPIV3感染抑制STAT1的表达，下调IFN-α诱导的STAT1的表达和磷酸化水平，抑制STAT1核转运过程，进而辅助病毒逃避IFN抗病毒信号传导。通过单克隆抗体制备及western blot分析确定CPIV3感染过程中可编码非结构蛋白V、D及C，其中非结构蛋白V和C显著抑制IFN-α对下游ISGs的转录激活效应，发挥免疫拮抗作用。进一步研究发现非结构蛋白V不影响IFN-α诱导pSTAT1的表达，但其可与STAT1直接互作；非结构蛋白C可拮抗IFN-α诱导pSTAT1的表达，其VR2区域可参与该调控过程，同时其可与STAT1直接互作，共同辅助CPIV3逃避I型干扰素抗病毒应答。通过Co-IP结合质谱分析结果发现，CPIV3非结构蛋白V可与宿主蛋白RTN4发生互作，而RTN4可调控干扰素通路激活过程；同时非结构蛋白C可与核转运蛋白IPO7互作，而IPO7蛋白参与CPIV3感染对I型干扰素抗病毒效应的拮抗过程，进一步说明病毒通过其编码产物调控多种宿主蛋白的生物功能而促进自身的复制与感染。本项目解析了I型干扰素与CPIV3体外复制间的动态关系，初步探讨了CPIV3非结构蛋白V和C辅助病毒阻断I型干扰素抗病毒反应的免疫逃逸机制，为阐明CPIV3的致病性及开发新的抗病毒策略奠定理论基础。