Use of lysolecithin-permeabilized infected-cell extracts to investigate the in vitro biochemical phenotypes of poxvirus ts mutations altered in viral transcription activity.
Use of lysolecithin-permeabilized infected-cell extracts to investigate the in vitro biochemical phenotypes of poxvirus ts mutations altered in viral transcription activity.
复制标题
使用溶血卵磷脂透化的感染细胞提取物研究病毒转录活性改变的痘病毒 ts 突变的体外生化表型。
DOI：
10.1006/viro.1996.0177
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Niles,EG
中科院分区：
文献类型：
--
作者：
Condit,RC;Lewis,JI;Quinn,M;Christen,LM;Niles,EG
采用溶菌素透化牛痘病毒感染的细胞，以制备支持在具有早期、中期和晚期病毒基因启动子的体外质粒中忠实转录起始的提取物。定义了优化每个启动子转录的条件。利用体外系统研究了多功能的病毒mRNA加帽酶，该加帽酶同时具有病毒早期基因转录终止因子（VTF）和病毒中间基因转录起始因子的功能。在体外，低水平的信号依赖性终止早期基因转录，这可以通过添加纯mRNA加帽酶来提高。VTF依赖的转录终止被发现仅限于具有早期启动子的模板。这一限制模仿了体内转录终止，并证明转录终止仅限于识别早期而不是中期或晚期基因启动子的RNA聚合酶分子。从在非允许温度下用含有编码VTF小亚基的基因D12L中的ts突变的病毒感染的细胞制备的提取物不能支持早期基因转录终止和中间基因转录起始。在添加纯化的野生型mRNA加帽酶后，两种活性均恢复。
Lysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiationin vitroon plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. Thein vitrosystem was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observedin vitrowhich could be elevated by the addition of pure mRNA capping enzyme. VTF-dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observedin vivoand demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme.