抗菌肽cathelicidins抵御非包膜病毒CVB3复制和病毒性心肌炎的功能及分子机制

Cathelicidin antimicrobial peptides are a family of gene-encoded peptide effectors of innate immunity for antimicrobial defense and, more recently, immunomodulation. Cathelicidins show broad-spectrum antiviral activity against enveloped virus. However, the role and mechanism of cathelicidins against nonenveloped virus infection remain elusive. Preliminary results showed that the expression of cathelicidin (CRAMP) was the most significantly up-regulated in the heart of CVB3-induced VMC mice. And CVB3 replication in heart and viral myocarditis were significantly inhibited by intraperitoneal injection of cathelicidins. Furthermore, cathelicidins inhibited the replication of CVB3 in a direct manner. In addition, we found that CVB3 infection significantly induced the up-regulation of exosome secretions in cardiomyocytes and mice, and the purified exosomes could infect cardiomyocytes. But cathelicidins could inhibit the up-regulated secretion of exosome in cardiomyocytes and mice and their infection in cardiomyocytes. On the basis of these results, we hypothesize that cathelicidins can bind to the envelope membrane of exosomes through amphipathic structure and inhibit exosome-mediated CVB3 infection. Next, we will use CRAMP-deficient mice to further confirm the antiviral and immunoregulatory effects of cathelicidins against nonenveloped CVB3 replication and CVB3 infection induced VMC. Besides, we will explore the relative mechanisms in cardiomyocytes and VMC mice. We will investigate the effects of cathelicidins on exosome-mediated virus transmission, immune escape and immune injury. And we will explore other possible mechanisms, including the activation of CVB3 replication-dependent autophagy, apotosis and ERK, as well as the antiviral responses of type I IFNs/CD8+ T cells. Hopefully, we will propose an innovative understanding for the role of cathelicidins against nonenveloped virus infection, and provide cathelicidin antimicrobial peptides as novel therapeutic targets for VMC therapy.

抗菌肽cathelicidins具广谱抗包膜病毒功能，但其对非包膜病毒的功能尚不明确。在非包膜病毒CVB3感染早期，我们发现cathelicidin在小鼠心脏上调最为显著，并以间接的方式显著抑制了CVB3复制；CVB3感染可诱导心肌细胞和小鼠上调分泌外泌体，且该外泌体可感染心肌细胞，人或鼠源cathelicidin可抑制其上调和感染。我们推测cathelicidins可通过抑制外泌体介导的病毒感染而发挥抗病毒作用。为证实该推测，我们拟以cathelicidin敲除小鼠确证其抑制CVB3感染的功能；在心肌细胞和小鼠中，阐明其是否通过两亲性螺旋结构与外泌体脂质外膜结合并致其穿孔，从而抑制外泌体介导的病毒传播、免疫逃避和机体免疫损伤；探索其是否调控病毒感染中的细胞自噬、凋亡、ERK激酶和宿主抗病毒应答。本课题将提供抗菌肽cathelicidins抵抗非包膜病毒的新机制和病毒性心肌炎防治的新策略。

抗菌肽cathelicidins（小鼠：CRAMP，人：LL-37）具有广谱抗包膜病毒的功能，但其对非包膜病毒的作用与机制尚不明确。B3型柯萨奇病毒（Coxsackievirus B3，CVB3）是一种非包膜病毒，属于肠道病毒属、小RNA病毒科。CVB3的感染是引起病毒性心肌炎和扩张型心肌病的一个重要诱因。我们发现在非包膜病毒CVB3感染后，小鼠心脏中的CRAMP表达水平显著上调。给小鼠腹腔注射CRAMP或LL-37可以抑制CVB3的感染。CRAMP的敲除可以增加小鼠对CVB3的易感性。CRAMP和LL-37也可以在原代心肌细胞中抑制CVB3的复制。CRAMP和LL-37对CVB3感染的抑制作用不是通过直接灭活CVB3，也不是通过调控心肌细胞的抗病毒应答，而是通过抑制外泌体介导的CVB3传播，但不影响受体介导的CVB3传播。CRAMP和LL-37可以与外泌体中的热休克蛋白60（HSP60）结合，破坏外泌体的完整性，进而抑制外泌体中的病毒组分向受体细胞传播，最终抑制外泌体中的病毒组分在受体细胞中建立感染。此外，CRAMP和LL-37与HSP60的相互作用还可以抑制HSP60诱导的心肌细胞凋亡，进而抑制HSP60促进的CVB3复制。本课题揭示了抗菌肽cathelicidins抵抗病毒的新机制，同时为CVB3诱导的病毒性心肌炎提供新的治疗策略。