自噬通过外泌体途径影响山羊副流感病毒3型复制的研究

Caprine parainfluenza virus type 3 (CPIV3) is one of the important viral respiratory pathogens. Our previous study had identified that induction of autophagy enhanced intracellular and extracellular viral RNA in MDBK cells infected with CPIV3, contrarily, the viral proteins expression was significantly inhibited, further research found that viral proteins and RNA mediated by exosomes were increased. Some studies have confirmed that autophagy promotes formation and secretion of exosomes and the information materials entry into the exosomes. Therefore, we speculated that induction of autophagy could facilitate the loading of viral proteins and RNA into exosomes, and promote the formation and secretion of exosomes, resulting to the increasing release of viral components outside the cells, and finally inhibit the intracellular virus replication. On the above foundation, we will study the exosomes regulation by autophagy from two aspects: the exosomal formation process including regulation of multivesicular bodies (MVBs) formation and CPIV3 proteins and RNA loading into the MVBs, and the secretion process involving the relaxation of accumulated MVBs and increasing secretion of MVBs to outside. Furthermore, the mechanism of exosomes regulation function, lysosomal dysfunction, is analyzed. In this research, we keep a watchful eye on the vector, exosomes, a new pathway playing an important role in the autophagy related viral replication. The results will provide a theoretical reference for the research and development of antiviral drugs and the prevention and control measures of CPIV3.

山羊副流感病毒3型（CPIV3）是导致羊呼吸道疾病的重要病原。前期研究中，MDBK细胞经诱导自噬后感染CPIV3，检测病毒复制水平发现胞内、胞外病毒RNA水平上调，病毒蛋白水平却明显降低，而外泌体中病毒RNA和蛋白水平都显著增加。有研究发现，自噬促进外泌体形成和分泌，并促进信息物质进入外泌体。由此推测，诱导自噬通过促进CPIV3病毒蛋白和RNA装载入外泌体，增加外泌体的分泌进而将病毒成分释放到细胞外，影响病毒在细胞内的复制。鉴于此，本项目从自噬调控外泌体的形成和分泌出发，分别验证自噬对MVBs形成和CPIV3蛋白/RNA进入MVBs的调控作用，诱导自噬对缓解MVBs的积累和促进MVBs释放到细胞外的作用；并进一步分析自噬调控外泌体形成和分泌的机制—溶酶体功能失调的作用。综合阐述外泌体“载体”是自噬影响CPIV3复制的一种途径，以期为抗病毒药物研发和制定CPIV3的防控措施提供理论参考。

山羊副流感病毒3型（CPIV3）是导致羊呼吸道疾病的重要病原。有研究发现，自噬可影响外泌体形成和分泌，调控信息物质进入外泌体。由此推测，自噬可调控CPIV3病毒蛋白和RNA装载入外泌体，影响外泌体的分泌进而将病毒成分释放到细胞外，从而影响病毒在细胞内的复制。鉴于此，本项目从自噬调控外泌体的形成和分泌出发，分别验证自噬对MVBs形成和CPIV3蛋白/RNA进入MVBs的调控作用，诱导自噬对缓解MVBs的积累和促进MVBs释放到细胞外的作用；并进一步分析自噬调控外泌体形成和分泌的机制，以期为抗病毒药物研发和制定CPIV3的防控措施提供理论参考。.研究发现，诱导细胞自噬后感染CPIV3，细胞分泌的外泌体减少，而外泌体中携带的病毒蛋白和RNA增加。诱导自噬的细胞内CD63和HSP70表达水平均显著下降，且与CPIV3滴度呈正相关。说明诱导细胞自噬后，不但使CPIV3复制水平降低，且抑制外泌体的形成和释放，促进病毒蛋白和RNA进入MVBs。.既然诱导自噬可抑制外泌体分泌，是否自噬抑制了细胞内MVBs形成。CPIV3感染细胞后，分别用PBS对照、雷帕霉素、3-MA处理发现，诱导自噬后，细胞内病毒蛋白水平显著下降，细胞内MVBs和细胞外外泌体分子标志CD63和HSP70水平均有所下降。说明诱导自噬可显著抑制外泌体形成。而CPIV3感染可抑制细胞自噬，细胞内MVBs水平和胞外外泌体水平均显著上升。.在CPIV3感染过程中，自噬与外泌体是两个分别平衡的途径。用外泌体抑制剂GW4869处理CPIV3感染细胞，抑制外泌体的分泌，发现外泌体携带的病毒蛋白和RNA水平也随之降低。但CD63和病毒F蛋白蓄积在细胞内，且二者可共定位。说明抑制细胞外泌体的释放，使病毒蛋白随MVBs滞留于细胞内。在CPIV3感染情况下抑制外泌体分泌，也可促进细胞自噬。.溶酶体在CPIV3感染中的自噬与外泌体平衡中发挥重要作用。通过荧光染色及电镜切片都发现，CPIV3感染组细胞中自噬溶酶体数量显著降低，而GW4869处理组的自噬溶酶体无显著变化，与CPIV3感染组相比，GW4869处理组自噬溶酶体数量上升。都说明自噬溶酶体在调控外泌体与自噬水平中发挥作用。