Polypyrimidine-tract-binding protein affects transcription but not translation of mouse hepatitis virus RNA
Polypyrimidine-tract-binding protein affects transcription but not translation of mouse hepatitis virus RNA
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DOI：
10.1006/viro.2002.1675
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发表时间：
2002-11-10
期刊：
影响因子：
3.7
通讯作者：
Lai, MMC
中科院分区：
文献类型：
--
作者：
Choi, KS;Huang, PY;Lai, MMC
多聚嘧啶区结合蛋白（PTB）已被证明能特异性结合小鼠肝炎病毒（MHV）RNA及其互补链的5'端。为了进一步明确PTB在MHV复制中的功能，我们构建了显性负性突变细胞系，这些细胞系表达全长PTB或一种截短形式的PTB，该截短形式仅包含蛋白质的N端一半，保留了其蛋白质二聚化结构域。截短形式的PTB定位于细胞质，而全长PTB主要存在于细胞核中。截短形式在体外可与全长PTB相互作用。我们观察到，无论是全长PTB还是截短PTB，在过表达时，在MHV复制中均以显性负性方式发挥作用。然而，截短形式在合胞体形成、病毒产生以及病毒RNA和病毒蛋白的合成方面表现出更严重的影响。为了阐明PTB在MHV复制中的确切功能，我们通过将含有不同报告基因的不同类型的MHV缺陷干扰（DI）RNA转染到这些稳定细胞系中，将病毒转录过程与翻译过程分离开来。在这些细胞系中，MHV感染期间DI RNA的转录受到极大抑制，这表明PTB调节MHV转录。相反，在兔网织红细胞裂解物的体外翻译中，PTB缺失对DI RNA的翻译没有影响，在MHV感染细胞的体内翻译实验中，PTB过表达也不影响DI RNA的翻译。鉴于PTB与病毒N蛋白相互作用，而病毒N蛋白是MHV复制复合物的成分之一，PTB可能通过与复制复合物中的病毒RNA以及其他病毒和细胞因子结合，在病毒复制/转录中发挥其功能。（C）2002年爱思唯尔科学（美国）
Polypyrimidine-tract-binding protein (PTB) has been shown to bind specifically to the 5' ends of mouse hepatitis virus (MHV) RNA and its complementary strand. To further characterize the function of PTB in MHV replication, we generated dominant-negative mutant cell lines that express a full-length PTB or a truncated form of PTB, which includes only the N-terminal half of the protein, retaining its protein-dimerization domain. The truncated form of PTB was localized in the cytoplasm, whereas the full-length PTB was present mainly in the nucleus. The truncated form can interact with the full-length PTB in vitro. We observed that both the full-length and the truncated PTB, when overexpressed, functioned in a dominant-negative manner in MHV replication. However, the truncated form exhibited more severe effects on syncytia formation, virus production, and synthesis of viral RNA and viral proteins. To clarify the precise function of PTB in MHV replication, we dissociated the processes of viral transcription from translation by transfecting different types of MHV defective-interfering (DI) RNA that contain various reporter genes into these stable cell lines. Transcription of the DI RNA during MHV infection was greatly inhibited in these cell lines, indicating that PTB modulates MHV transcription. In contrast, translation of the DI RNA was not affected by PTB depletion in in vitro translation in rabbit reticulocyte lysate or by PTB overexpression in in vivo translation experiments in MHV-infected cells. Given that PTB interacts with the viral N protein, which is one of the components of the MHV replication complex, PTB may exert its function on viral replication/transcription by association with viral RNA as well as other viral and cellular factors in the replication complex. (C) 2002 Elsevier Science (USA).