Translation elongation factor-1α, La, and PTB interact with the 3′ untranslated region of dengue 4 virus RNA
Translation elongation factor-1α, La, and PTB interact with the 3′ untranslated region of dengue 4 virus RNA
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DOI：
10.1006/viro.2002.1407
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发表时间：
2002-04-10
期刊：
影响因子：
3.7
通讯作者：
del Angel, RM
中科院分区：
文献类型：
--
作者：
De Nova-Ocampo, M;Villegas-Sepúveda, N;del Angel, RM
登革4型病毒（DEN4）的384-nt长的3 '非翻译区（3'UTR）不是多聚腺苷酸化的，但包含邻近的在生物学上稳定的保守的短茎环结构（L-SL）和长茎环结构（L-SL）以及保守序列CS1和CS2。后者在DEN4中是重复的（CS2A和CS2B），登革病毒的复制与其他RNA病毒的复制一样，可能涉及位于3'UTR内的顺式元件和可以与病毒复制酶相互作用以作为复制酶复合物起作用的反式作用因子。参与与登革病毒3'UTR相互作用的病毒和细胞蛋白的鉴定和表征将帮助我们理解病毒复制的细胞要求。为了确定这些要求，用未感染和DEN4感染的C6/36细胞提取物以及3'UTR的不同片段进行迁移率变化和交联测定。我们的结果表明，RNA-蛋白质复合物与RNA形成，涉及结构域CS2A，CS2B，CS1和L-SL。能够与细胞蛋白质形成特异性和稳定复合物的最小RNA序列是CS1-L-SL区域。使用UV诱导的交联，我们鉴定了8种与完整3'UTR结合的蛋白质，其分子量为34、39、51、52、56、62、72和84 kDa。翻译延伸因子-1 α（EF-1 α）与完整的3'UTR和CS1-L-SL区域结合。此外，重组GST-人La自身抗原结合到3'UTR和CS1-L-SL区域，如通过迁移率变化和交联测定所证明的。尽管不同的抗PTB抗体不能与来自C6/36的任何细胞蛋白反应，但重组His-PTB蛋白确实与完整的3'UTR和CS1-L-SL区结合。La和PTB与病毒RNA复制所必需的序列的特异性结合可能表明这些蛋白质可以作为RNA分子伴侣发挥作用，以维持有利于病毒复制的RNA结构，而EF-1a可能作为RNA解旋酶发挥作用。(C)2002 Elsevier Science（美国）。
The 384-nt long 3' untranslated region (3'UTR) of dengue 4 virus (DEN4) is not polyadenylated, but contains the adjacent thermodynamically stable conserved short and long stem-loop structures (L-SL) and the conserved sequences CS1 and CS2. The latter are duplicated (CS2A and CS2B) in DEN4, Dengue virus replication, like that of other RNA viruses, might involve the cis-elements located within the 3'UTR and the trans-acting factors that could interact with the viral replicase to function as a replicase complex. The identification and characterization of viral and cellular proteins involved in the interaction with the 3'UTR of dengue virus will help us to understand the cellular requirements for viral replication, To determine these requirements, mobility shift and cross-linking assays were performed with uninfected and DEN4-infected C6/36 call extracts as well as the different segments of the 3'UTR. Our results revealed that RNA-protein complexes were formed with the RNAs which involved the domains CS2A, CS2B, CS1, and L-SL. The minimum RNA sequence that was able to form specific and stable complexes with cellular proteins was the CS1-L-SL region. Using UV-induced cross-linking we identified eight proteins with molecular weights of 34, 39, 51, 52, 56, 62, 72, and 84 kDa that bound to the complete 3'UTR. The translation elongation factor-1alpha (EF-1alpha) bound to the complete 3'UTR and to the CS1-L-SL region. In addition, the recombinant GST-human La autoantigen bound to the 3'UTR and to the CS1-L-SL region as demonstrated by mobility shift and cross-linking assays. Although different antibodies against PTB were unable to react with any of the cellular proteins from C6/36, the recombinant His-PTB protein did bind to the complete 3'UTR and to the CS1-L-SL region. The specific binding of La and PTB to the sequences considered essential for viral RNA replication may suggest that these proteins could function as RNA chaperones to maintain RNA structure in a conformation that favors viral replication, while EF-1a may function as an RNA helicase. (C) 2002 Elsevier Science (USA).