宿主线粒体通道蛋白促进干扰素应答抑制口蹄疫病毒复制的分子机制

Voltage dependent anion-selective channel 2 promotes interferon response to inhibit foot-and-mouth disease virus replication

摘要:

口蹄疫(foot-and-mouth disease, FMD)是由口蹄疫病毒(foot-and-mouth disease virus, FMDV)感染偶蹄动物引起的一种烈性动物传染病。FMDV是一种单股正链无囊膜RNA病毒，具有二十面体对称结构。FMDV入侵宿主后会引起宿主的先天性免疫和适应性免疫反应，而FMDV已经进化出多种途径来逃避免疫。FMDV的感染致病机制复杂，目前对于参与调控病毒感染复制的宿主因子的认知仍然非常有限。宿主线粒体通道蛋白(voltage dependent anion-selective channel 2, VDAC2)是一种线粒体通道蛋白，目前关于VDAC2对FMDV复制调控的具体机制尚不清楚。 目的 确证VDAC2与FMDV之间的调控作用，并揭示VDAC2抑制FMDV复制的分子机制。 方法 通过间接免疫荧光方法确定VDAC2在细胞内的定位；利用Western blotting以及荧光定量PCR方法检测FMDV对VDAC2蛋白水平和转录水平的影响；在BHK-21细胞上利用病毒滴度测定法检测过表达VDAC2对FMDV复制的影响；利用qPCR方法检测上调或下调VDAC2后FMDV感染对细胞内IL-1β、ISG15、OAS1、mtDNA和gDNA水平的影响。 结果 结果显示VDAC2亚细胞定位于细胞质中；FMDV感染可以降低VDAC2的表达；过表达VDAC2显著抑制FMDV的复制，并呈现剂量依赖关系；干扰VDAC2能够促进FMDV的复制；过表达VDAC2可增强FMDV诱导的IFN-Ⅰ反应；干扰VDAC2可抑制FMDV诱导的IFN-Ⅰ反应；过表达VDAC2可以促进mtDNA的释放。 结论 本研究表明FMDV感染可以下调VDAC2的转录与蛋白表达，而VDAC2可通过调节mtDNA的释放促进IL-1β、ISG15和OAS1等抗病毒因子的表达，从而发挥抗病毒作用。当FMDV入侵宿主细胞时，宿主细胞可以通过VDAC2增强IFN-Ⅰ反应，对病毒复制进行调控，发挥抗病毒作用。

Abstract:

Foot-and-mouth disease (FMD) is a highly contagious disease caused by foot-and-mouth disease virus (FMDV) in cloven-hoofed animals. FMDV is a single-stranded positive-sense non-enveloped RNA virus with icosahedral symmetry. FMDV can cause innate and adaptive immunity of the host, and it has evolved a variety of immune escape ways. The FMDV infection is a complicated process involving various host proteins. However, the related mechanisms remain largely unknown. Voltage dependent anion-selective channel 2 (VDAC2) is a mitochondrial channel protein, and the specific mechanism by which VDAC2 regulates FMDV replication remains unclear. Objective To confirm the regulatory effects between VDAC2 and FMDV and reveal the molecular mechanism by which VDAC2 inhibits FMDV replication. Methods The subcellular localization of VDAC2 was determined by the indirect immunofluorescence assay. The effects of FMDV on the translation and transcription levels of VDAC2 were determined by Western blotting and qPCR, respectively. The effect of VDAC2 overexpression on FMDV replication in BHK-21 cells was measured based on the virus titer. The effects of VDAC2 overexpression and knockdown on the levels of IL-1β, ISG15, OAS1, mtDNA, and gDNA during FMDV infection were evaluated by qPCR. Results VDAC2 was localized in the cytoplasm. FMDV infection down-regulated the expression of VDAC2. The overexpression of VDAC2 inhibited FMDV replication in a dose-dependent manner, while the knockdown of VDAC2 promoted FMDV replication. The overexpression of VDAC2 enhanced FMDV-induced interferon-I (IFN-I) response, while the knockdown of VDAC2 inhibited FMDV-induced IFN-I response. In addition, the overexpression of VDAC2 increased the release of mtDNA. Conclusion FMDV infection can downregulate the transcription and translation of VDAC2, as VDAC2 can exert antiviral effects by regulating mtDNA release and promoting the expression of antiviral factors such as IL-1β, ISG15, and OAS1. This study indicates that upon FMDV infection, host cells can regulate the virus replication by enhancing IFN-I response via VDAC2 to exert antiviral effects.