Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.活化T细胞核因子5（Nuclear factor of activated T cells 5, NFAT5）/张力增强子结合蛋白（Tonicity enhancer binding protein, TonEBP）是一种在肾脏中由高渗应激诱导的转录因子。尽管NFAT5在全身各组织广泛表达，但其在其他器官中的功能却鲜有研究。尽管已有研究证实NFAT5对心脏发育与功能至关重要，但其在感染性心脏疾病中的作用仍未明确。本研究旨在阐明NFAT5干扰柯萨奇病毒B3（Coxsackievirus B3, CVB3）感染的分子机制——柯萨奇病毒B3是病毒性心肌炎的主要致病病原体。本研究初始结果显示，在CVB3感染过程中，NFAT5的mRNA水平保持稳定，但该蛋白会发生切割，导致其蛋白表达量下降。通过生物信息学预测结合定点诱变实验对预测切割位点进行验证，证实NFAT5蛋白可被CVB3 2A蛋白酶在甘氨酸503位点切割。该切割事件会导致NFAT5功能失活，其产生的70kDa N端切割产物（p70-NFAT5）可对全长NFAT5蛋白发挥显性负调控作用。我们进一步研究发现，过表达NFAT5以对抗病毒蛋白酶的切割作用，尤其是过表达无法被切割的NFAT5突变体，可显著抑制CVB3的复制。异位表达NFAT5可上调诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）的表达，而该因子已被证实可抑制CVB3复制。iNOS对于NFAT5发挥抗CVB3效应的必要性，得到了以下实验结果的支持：抑制iNOS的活性可阻断NFAT5的抗CVB3作用。在病毒性心肌炎小鼠模型中，我们观察到高渗盐水或甘露醇溶液处理可上调NFAT5与iNOS的表达，抑制CVB3复制并减轻心脏组织损伤。综上，本研究数据表明，在CVB3感染期间，NFAT5的抗CVB3活性会因2A蛋白酶介导的NFAT5切割而受损。因此，通过高渗试剂诱导NFAT5的表达，有望成为开发抗CVB3治疗药物的潜在策略。