Leibowitz J L, DeVries J R
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston 77225.
Virology. 1988 Sep;166(1):66-75. doi: 10.1016/0042-6822(88)90147-x.
We have developed a permeabilized cell system for assaying mouse hepatitis virus-specific RNA polymerase activity. This activity was characterized as to its requirements for mono- and divalent cations, requirements for an exogenous energy source, and pH optimum. This system faithfully reflects MHV-specific RNA synthesis in the intact cell, with regard to both its time of appearance during the course of infection and the products synthesized. The system is efficient and the RNA products were identical to those observed in intact MHV-infected cells as judged by agarose gel electrophoresis and hybridization. Permeabilized cells appear to be an ideal system for studying coronavirus RNA synthesis since they closely mimic in vivo conditions while allowing much of the experimental flexibility of truly cell-free systems.
我们开发了一种通透细胞系统，用于检测小鼠肝炎病毒特异性RNA聚合酶活性。该活性在对单价和二价阳离子的需求、对外源能量来源的需求以及最适pH值方面进行了表征。就感染过程中其出现的时间和合成的产物而言，该系统忠实地反映了完整细胞中MHV特异性RNA的合成。通过琼脂糖凝胶电泳和杂交判断，该系统效率高，RNA产物与在完整的MHV感染细胞中观察到的产物相同。通透细胞似乎是研究冠状病毒RNA合成的理想系统，因为它们紧密模拟体内条件，同时具有真正无细胞系统的许多实验灵活性。
