非洲猪瘟病毒毒力因子MGF360-4L靶向降解RIPK3促进病毒复制的研究

Virulence factor MGF360-4L of African swine fever virus facilitates viral replication by degrading RIPK3

本研究首先利用CellTiter-Glo试剂盒测定MGF360-4L对细胞活力的影响;通过蛋白免疫印迹(Western-blot)检测MGF360-4L及其突变体对细胞坏死通路关键接头分子表达的影响;通过同源重组技术构建MGF360-4L缺失毒株(ASFV-ΔMGF360-4L);利用实时荧光定量PCR(RT-qPCR)分析ASFV野生毒株(ASFV-WT)与其缺失毒株(ASFV-ΔMGF360-4L)复制水平的差异;通过Western-blot检测MGF360-4L缺失对ASFV诱导的细胞坏死的影响。结果表明,MGF360-4L包含1个RHIM,并通过溶酶体途径降解RIPK3,从而抑制细胞坏死通路的活化;MGF360-4L发挥上述功能依赖其RHIM结构域;ASFV-ΔMGF360-4L毒株复制水平低于野生毒株,且该缺失毒株能够诱导更高水平的细胞坏死。本研究证实ASFV毒力因子MGF360-4L依赖其RHIM结构域靶向降解RIPK3,并促进病毒自身复制,为ASFV致病机理的阐明提供了科学数据。

In the present study,the effects of MGF360-4L on cell viability were first determined by Cell Titer-Glo kit.The effects of MGF360-4L and its mutants on the expression of key adaptors of necrosis pathway were detected by Western-blot.MGF360-4L-deficient ASFV(ASFV-ΔMGF360-4L)was constructed by homologous recombination technique.Real-time quantitative fluorescent PCR(RT-q PCR)was used to analyze the replication difference between wild-type ASFV(ASFV-WT)and ASFV-ΔMGF360-4L.The effects of MGF360-4L deletion on ASFV-induced necrosis were detected by Western blot.These results indicated that MGF360-4L contained a RHIM and degraded RIPK3 via the lysosomal pathway,thereby inhibiting the activation of necrosis signal.MGF360-4L promoted the degradation of RIPK3 in a RHIM domain-dependent manner.ASFV-ΔMGF360-4L had a lower replication level than that of ASFV-WT,and the deficient strain induced higher levels of necrosis.Altogether,our study suggests that ASFV virulence factor MGF360-4L degrades RIPK3 relied on its RHIM motif and promotes viral replication,which provides scientific data for the elucidation of ASFV pathogenesis.