Ma Yuanmei, Wei Yongwei, Zhang Xiaodong, Zhang Yu, Cai Hui, Zhu Yang, Shilo Konstantin, Oglesbee Michael, Krakowka Steven, Whelan Sean P J, Li Jianrong
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
J Virol. 2014 Mar;88(5):2913-26. doi: 10.1128/JVI.03420-13. Epub 2013 Dec 26.
One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient translation of mRNA. The role of mRNA cap ribose 2'-O methylation is enigmatic, although recent work has implicated this as a signature to avoid detection of RNA by the innate immune system (S. Daffis, K. J. Szretter, J. Schriewer, J. Q. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. P. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, P. Y. Shi, M. S. Diamond, Nature 468:452-456, 2010, doi:10.1038/nature09489). Working with vesicular stomatitis virus (VSV), we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2'-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2'-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors.
Nonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many of these viruses, there are no vaccines or antiviral drugs available. mRNA cap methylation is essential for mRNA stability and efficient translation. Our current understanding of mRNA modifications of NNS RNA viruses comes largely from studies of vesicular stomatitis virus (VSV). In this study, we showed that recombinant VSVs (rVSVs) defective in mRNA cap methylation were attenuated in vitro and in vivo. In addition, these methyltransferase (MTase)-defective rVSVs triggered high levels of antibody responses and provided complete protection against VSV infection. Thus, this study will not only contribute to our understanding of the role of mRNA cap MTase in viral pathogenesis but also facilitate the development of new live attenuated vaccines for VSV, and perhaps other NNS RNA viruses, by inhibiting viral mRNA cap methylation.
信使核糖核酸（mRNA）帽状鸟嘌呤-N-7（G-N-7）甲基化的一个作用是促进mRNA的高效翻译。mRNA帽状核糖2'-O甲基化的作用尚不明确，尽管最近的研究表明这是一种避免被天然免疫系统检测到RNA的特征（S. 达菲斯、K. J. 施雷特、J. 施里韦尔、J. Q. 李、S. 尤恩、J. 埃雷特、T. Y. 林、S. 施内勒、R. 祖斯特、H. P. 董、V. 蒂尔、G. C. 森、V. 芬斯特尔、W. B. 克利姆斯特拉、T. C. 皮尔森、R. M. 布勒、M. 盖尔、P. Y. 施、M. S. 戴蒙德，《自然》468:452 - 456，2010，doi:10.1038/nature09489）。我们之前利用水疱性口炎病毒（VSV）进行研究，发现一组在预测的甲基转移酶催化位点（重组VSV - K1651A、- D1762A和 - E1833Q）或S - 腺苷甲硫氨酸（SAM）结合位点（重组VSV - G1670A、- G1672A和 - G4A）携带突变的重组VSV在帽状甲基化方面存在缺陷，并且在细胞培养中的生长也受到减弱。在此，我们分析了这些重组体在小鼠中的毒力。我们发现，在G - N - 7和2'-O甲基化均有缺陷的重组VSV - K1651A、- D1762A和 - E1833Q在小鼠中高度减毒。这三种病毒均引发高水平的中和抗体，并能为抵御强毒VSV的攻击提供完全保护。相比之下，接种仅在G - N - 7甲基化有缺陷的重组VSV - G1670A和 - G1672A的小鼠在体内减毒，但仍保留低水平的毒力。在G - N - 7和2'-O甲基化均完全有缺陷的重组VSV - G4A在小鼠中也表现出低毒力，尽管在肺和脑中未检测到有效的病毒复制。综上所述，我们的结果表明，消除病毒mRNA帽状甲基化可作为一种使VSV以及可能的其他非节段性负链RNA病毒减毒的方法，有望用于疫苗和病毒载体。
非节段性负义（NNS）RNA病毒包括多种重要的人类、动物和植物病原体。对于其中许多病毒，尚无可用的疫苗或抗病毒药物。mRNA帽状甲基化对于mRNA稳定性和高效翻译至关重要。我们目前对NNS RNA病毒mRNA修饰的理解很大程度上来自于水疱性口炎病毒（VSV）的研究。在本研究中，我们表明在mRNA帽状甲基化方面有缺陷的重组VSV（rVSV）在体外和体内均减毒。此外，这些甲基转移酶（MTase）缺陷的rVSV引发高水平的抗体反应，并能为抵御VSV感染提供完全保护。因此，本研究不仅将有助于我们理解mRNA帽状MTase在病毒发病机制中的作用，还将通过抑制病毒mRNA帽状甲基化促进开发针对VSV以及可能的其他NNS RNA病毒的新型减毒活疫苗。
