Momose F, Handa H, Nagata K
Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Biochimie. 1996;78(11-12):1103-8. doi: 10.1016/s0300-9084(97)86736-3.
Transcription and replication of the influenza virus RNA genome take place in the nuclei of infected cells. Ribonucleoprotein (RNP) complexes consisting of viral RNA, RNA polymerase, and nucleocapsid protein (NP) are proven to be the catalytic unit for RNA synthesis, while it has been indicated that the viral RNA polymerase activity is modulated by host-derived nuclear factors. Here we have identified such host factors present in nuclear extracts prepared from uninfected HeLa cells with biochemical complementation assays using the in vitro RNA synthesis system. The stimulatory activity was not absorbed to phosphocellulose but was tightly bound to Q-Sepharose. The eluate recovered from Q-Sepharose was able to stimulate the RNA synthesis catalyzed by both RNP complexes and purified RNA polymerase and NP. The stimulatory activity was further separated into two distinct fractions, designated RAF-1 (RNA polymerase activating factor-1) and RAF-2 fractions, through phenyl-Sepharose column chromatography. When these fractions were fractionated through a gel filtration column, RAF-1 and RAF-2 activities were recovered in fractions corresponding to the molecular mass of 350 kDa and 60 kDa, respectively. Furthermore, the RAF-2 fraction was shown to contain an inhibitory activity, tentatively designated RIF-1 (RNA polymerase inhibitory factor-1). RIF-1 sedimented as fast as bovine serum albumin in glycerol density gradient centrifugation. Roles of these host factors are discussed in the context of viral RNA transcription and replication.
流感病毒RNA基因组的转录和复制在受感染细胞的细胞核中进行。由病毒RNA、RNA聚合酶和核衣壳蛋白（NP）组成的核糖核蛋白（RNP）复合物被证明是RNA合成的催化单位，同时有迹象表明病毒RNA聚合酶活性受宿主来源的核因子调节。在这里，我们通过使用体外RNA合成系统的生化互补试验，在从未感染的HeLa细胞制备的核提取物中鉴定出了此类宿主因子。刺激活性不被磷酸纤维素吸附，但与Q-琼脂糖紧密结合。从Q-琼脂糖中回收的洗脱液能够刺激由RNP复合物以及纯化的RNA聚合酶和NP催化的RNA合成。通过苯基琼脂糖柱色谱法，刺激活性进一步被分离成两个不同的组分，分别命名为RAF-1（RNA聚合酶激活因子-1）和RAF-2组分。当这些组分通过凝胶过滤柱进行分级分离时，RAF-1和RAF-2活性分别在对应于350 kDa和60 kDa分子量的组分中回收。此外，RAF-2组分显示含有一种抑制活性，暂定为RIF-1（RNA聚合酶抑制因子-1）。在甘油密度梯度离心中，RIF-1沉降速度与牛血清白蛋白一样快。本文在病毒RNA转录和复制的背景下讨论了这些宿主因子的作用。
