Dunn E F, Pritlove D C, Jin H, Elliott R M
Institute of Virology, University of Glasgow, Scotland, United Kingdom.
Virology. 1995 Aug 1;211(1):133-43. doi: 10.1006/viro.1995.1386.
We describe a convenient system for analyzing bunyavirus transcription using a recombinant RNA template derived from the plasmid pBUNSCAT, which comprises a negative-sense reporter gene (chloramphenicol acetyltransferase or CAT) flanked by the exact 5' and 3' untranslated regions of the Bunyamwera virus (BUN) S RNA segment. When cells which expressed bunyavirus proteins (either by recombinant vaccinia viruses or by the vaccinia virus-T7 system) were transfected with BUNSCAT RNA, CAT activity could be measured, indicating transcription of the negative-sense reporter RNA into mRNA. The system permits investigation of both the protein and RNA sequence requirements for transcription. Extensions of 2 bases at the 5' end or 11 or 35 bases at the 3' end of BUNSCAT RNA allowed transcription but a lower level than the wild-type template. Deletion of the 5 nucleotides at the 3' end of BUNSCAT RNA reduced CAT activity by > 99%. Investigation of the viral protein requirements of the system showed that only the bunyavirus L and N proteins were needed for CAT activity. The BUN L protein was also able to transcribe the reporter RNA in concert with the N proteins of closely related bunyaviruses such as Batai, Cache Valley, Maguari, Main Drain, and Northway, but only inefficiently with those of Kairi, Guaroa, or Lumbo viruses. When BUN L proteins containing specific mutations were expressed CAT activity was only observed using those mutated L proteins previously reported to be active in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, 1992, J. Gen. Virol. 73, 2235-2244). These results illustrate the utility of this system for a detailed genetic analysis of the factors involved in bunyavirus transcription.
我们描述了一种便捷的系统，用于利用源自质粒pBUNSCAT的重组RNA模板分析布尼亚病毒转录。该质粒包含一个负义报告基因（氯霉素乙酰转移酶或CAT），其两侧是布尼亚姆韦拉病毒（BUN）S RNA片段精确的5'和3'非翻译区。当用BUNSCAT RNA转染表达布尼亚病毒蛋白的细胞（通过重组痘苗病毒或痘苗病毒-T7系统）时，可检测到CAT活性，这表明负义报告RNA转录成了mRNA。该系统允许研究转录所需的蛋白质和RNA序列要求。BUNSCAT RNA的5'端延伸2个碱基或3'端延伸11个或35个碱基可进行转录，但水平低于野生型模板。删除BUNSCAT RNA 3'端的5个核苷酸会使CAT活性降低>99%。对该系统病毒蛋白需求的研究表明，CAT活性仅需要布尼亚病毒L蛋白和N蛋白。BUN L蛋白还能够与密切相关的布尼亚病毒（如巴泰、卡谢谷、马瓜里、主排水道和诺斯韦）的N蛋白协同转录报告RNA，但与凯瑞、瓜罗阿或伦波病毒的N蛋白协同转录效率较低。当表达含有特定突变的BUN L蛋白时，仅使用先前报道在核衣壳转染试验中有活性的那些突变L蛋白才能观察到CAT活性（H. Jin和R. M. Elliott，1992，《普通病毒学杂志》73，2235 - 2244）。这些结果说明了该系统在对布尼亚病毒转录相关因子进行详细遗传分析中的实用性。
