Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor.
Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor.
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DOI：
10.1016/j.virol.2008.11.041
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发表时间：
2009-03-01
期刊：
影响因子：
3.7
通讯作者：
Nagy, Peter D.
中科院分区：
文献类型：
--
作者：
Li, Zhenghe;Pogany, Judit;Panavas, Tadas;Xu, Kai;Esposito, Anthony M.;Kinzy, Terri Goss;Nagy, Peter D.
关键词：
宿主RNA结合蛋白可能在正链RNA病毒复制过程中发挥多重、不可或缺的作用。为了鉴定与病毒RNA结合的宿主蛋白，我们采用了基于酵母蛋白质组微阵列的全球方法，该微阵列包含4080个纯化的酵母蛋白。生物素标记的RNA探针包括两种亲缘关系较远的RNA病毒，即番茄丛矮病毒（TBSV）和雀麦花叶病毒（BMV）。总之，我们已经确定了57酵母蛋白结合TBSV RNA和/或BMV RNA。在所鉴定的宿主蛋白中，11个以高选择性结合TBSV RNA，7个以高选择性结合BMV RNA，而其余39个宿主蛋白结合两种病毒RNA。TBSV复制子RNA和五个鉴定的宿主蛋白之间的相互作用通过凝胶迁移率变化和来自酵母的共纯化实验来证实。在TBSV的模式宿主酵母中过表达宿主蛋白，发现4个宿主蛋白增强TBSV复制以及14个蛋白抑制复制。详细分析一个确定的酵母蛋白结合TBSV RNA，即翻译延伸因子eEF 1A，揭示了它是存在于高度纯化的番茄丛矮病毒复制酶复合物。我们还证明了eEF 1A与p33复制蛋白和TBSV RNA 3′端的已知顺式作用元件的结合。使用的eEF 1A的功能突变体，我们提供的证据参与TBSV复制的eEF 1A。
Host RNA-binding proteins are likely to play multiple, integral roles during replication of plus-strand RNA viruses. To identify host proteins that bind to viral RNAs, we took a global approach based on the yeast proteome microarray, which contains 4080 purified yeast proteins. The biotin-labeled RNA probes included two distantly related RNA viruses, namely Tomato bushy stunt virus (TBSV) and Brome mosaic virus (BMV). Altogether, we have identified 57 yeast proteins that bound to TBSV RNA and/or BMV RNA. Among the identified host proteins, eleven bound to TBSV RNA and seven bound to BMV RNA with high selectivity, whereas the remaining 39 host proteins bound to both viral RNAs. The interaction between the TBSV replicon RNA and five of the identified host proteins were confirmed via gel-mobility shift and co-purification experiments from yeast. Over-expression of the host proteins in yeast, a model host for TBSV, revealed that 4 host proteins that enhanced TBSV replication as well as 14 proteins that inhibited replication. Detailed analysis of one of the identified yeast proteins binding to TBSV RNA, namely translation elongation factor eEF1A, revealed that it is present in the highly purified tombusvirus replicase complex. We also demonstrate binding of eEF1A to the p33 replication protein and a known cis-acting element at the 3′ end of TBSV RNA. Using a functional mutant of eEF1A, we provide evidence on the involvement of eEF1A in TBSV replication.
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