Precious B, Young D F, Bermingham A, Fearns R, Ryan M, Randall R E
School of Biological and Medical Sciences, University of St. Andrews, Fife, Scotland.
J Virol. 1995 Dec;69(12):8001-10. doi: 10.1128/JVI.69.12.8001-8010.1995.
The P, V, and NP genes of the paramyxovirus simian virus 5 (SV5) were cloned such that their expression was regulated by the tetracycline-controlled transactivator (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992), and mammalian cell lines that inducibly expressed individually the P, V, or NP protein or coexpressed the P plus NP or V plus NP proteins were isolated. A plasmid that expresses the tetracycline-controlled transactivator linked, via the foot-and-mouth disease virus 2A cleavage peptide sequence, to the neomycin aminoglycoside phosphotransferase gene was constructed. Cells were cotransfected with this plasmid, and the appropriate responder plasmids and clonies were selected on the basis of their resistance to Geneticin (via the neomycin aminoglycoside phosphotransferase gene). The properties of these cell lines, in terms of the induction of the P, V, and NP genes, are described in detail. Both the P and V proteins were phosphorylated when expressed alone. In immunoprecipitation studies using a monoclonal antibody that recognizes both the P and V proteins, a nonphosphorylated host cell protein with an estimated molecular weight of 150,000 was coprecipitated with V but not P. Immunofluorescence data demonstrated that when expressed separately, the P protein had a diffuse cytoplasmic distribution, but the related V protein had both a nuclear and cytoplasmic distribution. The NP protein had a granular cytoplasmic distribution, giving rise to punctate and granular fluorescence. Coexpression of the NP and P proteins resulted in the accumulation of large cytoplasmic inclusion aggregates, similar to those visualized at late times in SV5-infected cells. Coexpression of V with NP led to a partial redistribution of the NP protein in that the NP protein had both a diffuse cytoplasmic and nuclear distribution in the presence of V, but no NP-V aggregates or inclusion bodies were visualized. Direct binding studies also revealed that NP bound to both P and V. For SV5, these studies suggest that V may have a role in keeping NP soluble prior to encapsidation.
对副粘病毒猴病毒5（SV5）的P、V和NP基因进行了克隆，使其表达受四环素控制的反式激活因子调控（M. 戈森和H. 布亚德，《美国国家科学院院刊》89:5547 - 5551, 1992），并分离出了可诱导分别表达P、V或NP蛋白，或共表达P加NP或V加NP蛋白的哺乳动物细胞系。构建了一个表达通过口蹄疫病毒2A切割肽序列与新霉素氨基糖苷磷酸转移酶基因相连的四环素控制反式激活因子的质粒。将该质粒与适当的反应质粒共转染细胞，并根据细胞对遗传霉素的抗性（通过新霉素氨基糖苷磷酸转移酶基因）选择克隆。详细描述了这些细胞系在P、V和NP基因诱导方面的特性。单独表达时，P和V蛋白均被磷酸化。在使用识别P和V蛋白的单克隆抗体进行的免疫沉淀研究中，一种估计分子量为150,000的未磷酸化宿主细胞蛋白与V共沉淀，但不与P共沉淀。免疫荧光数据表明，单独表达时，P蛋白呈弥漫性细胞质分布，但相关的V蛋白具有核和细胞质分布。NP蛋白呈颗粒状细胞质分布，产生点状和颗粒状荧光。NP和P蛋白的共表达导致大量细胞质包涵体聚集物的积累，类似于在SV5感染细胞后期观察到的情况。V与NP的共表达导致NP蛋白部分重新分布，即NP蛋白在有V存在时具有弥漫性细胞质和核分布，但未观察到NP - V聚集物或包涵体。直接结合研究还表明NP与P和V都结合。对于SV5，这些研究表明V可能在衣壳化之前使NP保持可溶状态中发挥作用。
