Aminev Aleksey G, Amineva Svetlana P, Palmenberg Ann C
Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Virus Res. 2003 Sep;95(1-2):45-57. doi: 10.1016/s0168-1702(03)00162-x.
Panels of monoclonal antibodies were raised against viral non-structural proteins of encephalomyocarditis virus (EMCV) and used to probe infected cells in laser confocal microscopy experiments and Western analyses. Surprisingly, all Mengovirus and EMCV-infected cells showed strong targeting of protein 2A, 3B(VPg), 3C(pro), and 3D(pol) signals to cellular nuclei, in particular to nucleoli, from the earliest times of infection. Viral capsid proteins (1AB, 1C, and 1D) and other non-structural proteins (2B, 2C, and 3A) did not target nuclei and remained cytoplasmic throughout the infection. The cardioviral 2A protein (subject of this article) has a novel 143 amino acid sequence, terminating in a 19 amino acid COOH-terminal processing cassette (PCC) that participates in autocatalytic, co-translational primary cleavage of the viral polyprotein. The remainder of the 2A protein shares only limited similarity with other viral or cellular sequences, except for a short motif (KRvRPFRLP) near PCC resembling the nuclear localization signals (NLS) common to many yeast ribosomal proteins. Deletions within the EMCV 2A protein that impinge on this region have been reported to diminish the ability of virus to inhibit cap-dependent translation of cellular mRNAs. We have now observed that these same deletions prevented nuclear localization. Cellular expression of 2A protein from RNA transcripts or cDNAs confirmed that it does not require other viral proteins or activities for nuclear transport; even when expressed as a single protein, 2A protein effectively shuts off translation from capped reporter mRNAs. Within infected, transfected, or DNA vector-transformed cells, the 2A protein was always found in close association with the nucleolar ribosomal chaperone protein B23, which may help the traffic 2A into nucleoli like a surrogate ribosomal protein, by virtue of the putative nucleolar localization signal (NoLS). The data are consistent with a novel mechanism for virus-induced host protein shut off in cardioviruses, whereby 2A helps to upregulate the synthesis of new and modified ribosomes that have an inherent preference for internal ribosomal entry site (IRES)-dependent viral genome translation over cap-dependent host mRNA translation.
制备了针对脑心肌炎病毒（EMCV）病毒非结构蛋白的单克隆抗体面板，并用于在激光共聚焦显微镜实验和蛋白质印迹分析中检测感染的细胞。令人惊讶的是，从感染的最早阶段开始，所有感染Mengovirus和EMCV的细胞都显示出蛋白2A、3B（VPg）、3C（pro）和3D（pol）的信号强烈靶向细胞核，特别是核仁。病毒衣壳蛋白（1AB、1C和1D）和其他非结构蛋白（2B、2C和3A）不靶向细胞核，在整个感染过程中都保留在细胞质中。心肌病毒2A蛋白（本文的研究对象）具有一个新的143个氨基酸序列，其羧基末端有一个19个氨基酸的加工盒（PCC），该加工盒参与病毒多聚蛋白的自催化、共翻译初级切割。2A蛋白的其余部分与其他病毒或细胞序列仅具有有限的相似性，除了PCC附近的一个短基序（KRvRPFRLP），类似于许多酵母核糖体蛋白共有的核定位信号（NLS）。据报道，影响该区域的EMCV 2A蛋白内的缺失会降低病毒抑制细胞mRNA帽依赖性翻译的能力。我们现在观察到，这些相同的缺失阻止了核定位。从RNA转录本或cDNA中细胞表达2A蛋白证实，其核转运不需要其他病毒蛋白或活性；即使作为单一蛋白表达，2A蛋白也能有效地关闭帽状报告mRNA的翻译。在感染、转染或DNA载体转化的细胞内，总是发现2A蛋白与核仁核糖体伴侣蛋白B23紧密结合，这可能通过假定的核仁定位信号（NoLS），像替代核糖体蛋白一样帮助2A进入核仁。这些数据与心肌病毒中病毒诱导宿主蛋白关闭的新机制一致，即2A有助于上调新的和修饰的核糖体的合成，这些核糖体对内部核糖体进入位点（IRES）依赖性病毒基因组翻译比对帽依赖性宿主mRNA翻译具有内在偏好。
