B组柯萨奇病毒逃逸巨噬细胞病毒清除作用的分子机制

Coxsackievirus B (CVB) is the major pathogen of viral myocarditis. The pathogenesis of CVB infection is largely unknown. Macrophages, with enriched lysosomes in the cytoplasm, are the principal pathogen scavenger and immune modulator. Chloride channel protein 7 (ClC-7) is the chloride transporter localized in lysosomal membranes. ClC-7 defficiency impairs the acidification of lysosomes and causes lysosomal storage disorders. Our previous study found that there were macrophage infiltrations in the CVB-infected myocardial tissues. Macrophages could be infected by CVB. ClC-7 degraded during CVB infection. The cleavage sites of viral 3C protease were also identified in ClC-7. Lysosomal storage was observed in the CVB-infected cells. Based on these observations, we hypothesize that lysosome impairment may occur during CVB infection due to the cleavage of ClC-7 through 3C protease, and this allows CVB to circumvent the virus-clearance effect of macrophages. This study will focus on: (1) To verify that CVB can impair lysosomes through the ClC-7 cleavage by 3C protease. (2) To evaluate the effect of lysosome impairment on the function of macrophages during CVB infection. (3) To determine whether the function of lysosomes and the viral clearence ability of macrophages are comprimised by CVB infection under in vivo condition. This study aims to elucidate the molecular mechanism by which CVB circumvents the antiviral defense of macrophages, and to provide novel target for developing anti-CVB drugs. The mechanism involved in CVB infection to be studied here might be shared by other enteroviruses. Therefore, this study is also theoretically important.

B组柯萨奇病毒（CVB）是病毒性心肌炎的主要病原，尚无特异防治措施。巨噬细胞富含溶酶体，是组织中吞噬和清除病原的主要细胞。ClC-7是溶酶体膜的氯离子转运蛋白，其缺陷可导致溶酶体酸化障碍和溶酶体贮积。我们前期研究发现在CVB感染的心肌组织有巨噬细胞浸润；CVB能感染巨噬细胞；CVB感染时ClC-7被降解，ClC-7也有CVB的3C蛋白酶识别和切割位点；CVB感染的细胞可见溶酶体贮积。据此推测CVB可通过3C破坏ClC-7，导致溶酶体功能障碍，进而逃逸巨噬细胞的病毒清除作用。本项目研究包括：①证实CVB可通过其3C切割ClC-7并影响溶酶体功能；②评价CVB破坏溶酶体对巨噬细胞功能的影响；③明确体内条件下CVB是否具有干扰溶酶体和抑制巨噬细胞病毒清除的作用。综合阐明CVB逃逸巨噬细胞病毒清除作用的分子机制，为研发相关药物提供新靶点。此机制可能适用于其他肠道病毒属成员，因而有重要生物学意义。

B组柯萨奇病毒（coxsackievirus B, CVB）是病毒性心肌炎的主要病原体，致病机制不清。在某些情况下，CVB不能被有效清除而长期存在于心肌组织，导致心肌炎进展为心肌病。CVB可利用宿主细胞的生物学过程如细胞自噬促进病毒复制。线粒体自噬是细胞有选择地清除损伤的、失去功能的线粒体以维持细胞稳态的过程。溶酶体是线粒体自噬的终点，溶酶体功能的异常不仅影响线粒体自噬，也破坏线粒体的融合与分裂的动态平衡，从而影响线粒体的功能。CLC7是分布在晚期内体、溶酶体膜的氯离子-质子交换蛋白，CLC7基因突变导致骨硬化及溶酶体贮积症，但CLC7是否与CVB致病机制有关，尚不清楚。本研究结果表明，CVB感染时，CLC7被病毒编码的蛋白酶剪切，抑制了线粒体自噬，导致细胞不能有效清除损伤的线粒体；CLC7的剪切破坏了线粒体的稳态及线粒体功能，并最终抑制了巨噬细胞对病原体的清除作用。为阐明CLC7与CVB感染的关系，我们首先用CLC7稳定沉默细胞及CLC7基因敲减鼠分析了对细胞自噬的影响。结果表明，敲减CLC7导致细胞自噬能力降低。进一步研究发现，下调CLC7对心肌线粒体的影响最显著：在应激状态下，线粒体数量减少、线粒体面积缩小，并伴有肌小节破坏；在超微结构上， CLC7敲减鼠心肌细胞线粒体在应激状态出现空泡变、结构异常。在此基础上，我们发现敲减CLC7抑制了心肌细胞Parkin、PINK的表达水平，从而下调了线粒体自噬；敲减鼠心肌组织的MFN、OPA1表达水平均下降，即线粒体融合受到抑制；敲减鼠心肌组织的IDH2、ATP5A均明显下调，表明线粒体功能受损。此外，CLC7表达水平的下降影响了细胞的能量合成及对脂类的利用能力，并抑制了巨噬细胞的消化和降解功能。本研究得出以下结论：CVB通过剪切CLC7抑制心肌细胞线粒体自噬，破坏线粒体稳态及线粒体功能；病毒对 CLC7的剪切及对线粒体功能的影响抑制了巨噬细胞的消化功能，导致巨噬细胞不能有效清除病毒。本研究进一步揭示了CVB的致病机制。