Cheung Paul, Lim Travis, Yuan Jane, Zhang Mary, Chau David, McManus Bruce, Yang Decheng
Department of Pathology and Laboratory Medicine, The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia-St. Paul's Hospital, Vancouver, British Columbia, Canada.
Cell Microbiol. 2007 Jul;9(7):1705-15. doi: 10.1111/j.1462-5822.2007.00904.x. Epub 2007 Mar 8.
Coxsackievirus B3 (CVB3) is a positive, single-stranded RNA virus. The secondary structure of the 3' untranslated region (3'UTR) of CVB3 RNA consists of three stem-loops and is followed by a poly(A) tail sequence. These stem-loop structures have been suggested to participate in the regulation of viral replication through interaction with cellular proteins that are yet to be identified. In this study, by competitive UV cross-linking using mutated 3'UTR probes we have demonstrated that the poly(A) tail is essential for promoting HeLa cell protein interactions with the 3'UTR because deletion of this sequence abolished most of the protein interactions. Unexpectedly, mutations that disrupted the tertiary loop-loop interactions without affecting the stem-loops did not apparently affect these protein interactions, indicating that secondary structure rather than the high-order structure may play a major role in recruiting these RNA binding proteins. Among the observed 3'UTR RNA binding proteins, we have confirmed a 52 kDa protein as the human La autoantigen by using purified recombinant protein and a polyclonal La antibody. This protein can interact with both the 3' and 5'UTRs independently of the poly(A) tail. Further analysis by two-stage UV cross-linking, we found that the 3' and 5'UTR sequences may share the same binding site on the La protein.
柯萨奇病毒B3(CVB3)是一种正链单链RNA病毒。CVB3 RNA的3'非翻译区(3'UTR)的二级结构由三个茎环组成,后面跟着一个聚腺苷酸(poly(A))尾序列。这些茎环结构被认为通过与尚未鉴定的细胞蛋白相互作用参与病毒复制的调控。在本研究中,通过使用突变的3'UTR探针进行竞争性紫外线交联,我们证明聚腺苷酸尾对于促进HeLa细胞蛋白与3'UTR的相互作用至关重要,因为删除该序列消除了大部分蛋白相互作用。出乎意料的是,破坏三级环-环相互作用而不影响茎环的突变显然并未影响这些蛋白相互作用,这表明二级结构而非高阶结构可能在招募这些RNA结合蛋白中起主要作用。在观察到的3'UTR RNA结合蛋白中,我们通过使用纯化的重组蛋白和多克隆La抗体确认了一种52 kDa的蛋白为人La自身抗原。该蛋白可以独立于聚腺苷酸尾与3'和5'UTR相互作用。通过两阶段紫外线交联的进一步分析,我们发现3'和5'UTR序列可能在La蛋白上共享相同的结合位点。