内部核糖体进入位点决定口蹄疫病毒毒力的分子机制

Virulence is one of the most intriguing phenotype for viruses. Previously, we have found that the domain 4 of internal ribosomal entry site (IRES), which located in the 5′ untranslated region of foot-and-mouth disease virus (FMDV), is a determinant of virulence. Importantly, the sub-domain K of IRES domains 4 replaced with the corresponding domain of bovine rhinitis B virus (BRBV) in FMDV genome yielded a highly attenuated IRES-chimeric virus FMDV(K) in sucking mice, which was correlated with the decreased translational activity of the IRES in this chimeric virus. As cellular proteins such as eukaryotic initiation factors and IRES trans-acting factors are required for viral IRES function, thus a hypothesis is proposed that the sub-domain K of FMDV IRES contains a critical site for binding to an key cellular protein, which would be responsible for the IRES-mediated translation initiation to sustain viral replication. In this study, the crucial site in the IRES sub-domain K that determined the virulence phenotype of FMDV will be defined by utilizing a reverse-genetics approach. In addition, cellular protein with different binding activity to the wt IRES and site-mutated IRES will be screened by RNA Pulldown assay using biotinylated RNA and further identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Besides, the role of the target protein played in the IRES-mediated translation initiation will be determined through knockdown or over-expression of the protein. Furthermore, the effect of the crucial site mutation on the binding capacity of the target protein to IRES and IRES-mediated translation initiation efficiency will be confirmed. These studies will elucidate the molecular basis and its mechanism of FMDV virulence determined by IRES , and enrich the theory of picornavirus pathogenesis.

毒力是病毒的一个重要生物学表型。在前期研究中我们发现，内部核糖体进入位点（IRES）的结构域4决定口蹄疫病毒（FMDV）的毒力，而且IRES亚结构域K的置换能够使FMDV对乳鼠的毒力几乎完全丧失。由于IRES介导的翻译启始需要宿主细胞中翻译起始因子和反式作用因子的参与，因此我们提出假设：FMDV IRES的亚结构域K中存在关键的蛋白结合位点，通过与细胞蛋白相互作用行使IRES的功能、维持病毒的复制活性和毒力。有鉴于此，本立项设定以下两方面研究内容：1）利用FMDV反向遗传操作系统，通过嵌合及定点突变方法确定IRES的亚结构域K中决定FMDV毒力的关键位点；2）以生物素标记的IRES为“诱饵”，通过RNA pulldown技术结合质谱分析筛选与野生型IRES和点突变IRES的结合存在差异的宿主蛋白，并确定毒力决定位点突变对宿主蛋白与病毒IRES结合能力以及IRES翻译启始效率的影响。上述研究结果将阐明IRES决定FMDV毒力的分子基础及其发生机制，丰富微RNA病毒致病作用的理论。

口蹄疫（Foot-and-mouth disease, FMD）是由口蹄疫病毒（Foot-and-mouth disease virus, FMDV）引起的猪、牛、羊等偶蹄动物感染的一种急性、热性、高度接触性传染病。该病的发生与流行不但严重危害畜牧业生产，也严重影响畜产品的进出口贸易。世界动物卫生组织（World Organization For Animal Health）呼吁在全球范围内控制和根除口蹄疫。减毒活疫苗因其能够诱导强大而持久的保护性免疫而被认为是预防、控制甚至消灭传染病的最有效策略。然而，目前口蹄疫尚无安全有效的减毒活疫苗。本项目研究发现，以牛鼻病毒IRES（内部核糖体进入位点）结构域4置换O型FMDV IRES结构域4的嵌合病毒FMDV(R4)，具有温度敏感减毒（ts&att）表型。通过IRES的结构与功能研究确定，IRES结构域4 的亚结构域K区环结构上的C351G突变是导致FMDV获得ts&att表型的分子决定因素。序列比对分析发现，IRES的351位核苷酸C在GenBank公布的七个血清型的所有FMDV毒株中绝对保守。重要的是，将IRES C351G突变引入到A型或Asia1型FMDV感染性克隆中，均可赋予拯救病毒ts&att表型，这表明IRES C351G突变决定FMDV的温度敏感减毒表型具有普遍性。分子机制研究确定，IRES的C351G突变减弱了其与细胞蛋白PTB的结合致使IRES温度依赖性翻译缺陷，从而赋予FMDV ts&att表型。rC351G突变株接种口蹄疫病毒自然宿主猪或牛均不表现任何口蹄疫临床症状，也不排毒和传播，但可产生强的中和抗体反应且对异源强毒攻击提供完全保护。另外，rC351G突变株在体内、体外传代过程中遗传均稳定且毒力不返强。以上研究结果表明，IRES是FMDV关键的毒力决定因子，修饰IRES，尤其是C351G突变是开发安全有效的FMDV减毒活疫苗的可行策略。