Spaan W J, Rottier P J, Horzinek M C, van der Zeijst B A
Virology. 1981 Jan 30;108(2):424-34. doi: 10.1016/0042-6822(81)90449-9.
We have determined the kinetics of virus production and virus-specific RNA synthesis in Sac(−) cells infected with mouse hepatitis virus strain A59 (MHV-A59). Immunofluorescence showed that all cells became infected at a multiplicity of 10 PFU/cell. The virus was concentrated and purified to obtain the high titered stocks needed for these one-step growth experiments. Release of virus into the culture medium started 4 hr after infection (pi) and was complete at 10 hr pi. Synthesis of virus-specific RNA, measured by the incorporation of [H]uridine in the presence of 1 μg/ml actinomycin D, also started at 4 hr pi and its maximum rate occurred between 6 and 8 hr pi. RNA labeled during this period was isolated from infected cells. About 50% of this RNA bound to oligo(dT)-cellulose; this material was denatured with glyoxal-dimethyl sulfoxide and analyzed by electrophoresis in 1% agarose gels. Seven RNA species with the following molecular weights were present: 5.6 × 10 (RNA1), 4.0 × 10 (RNA2), 3.0 × 10 (RNA3), 1.4 × 10 (RNA4), 1.2 × 10 (RNA5), 0.9 × 10 (RNA6), and 0.6 × 10 (RNA7). RNA1 comigrated with the viral genome. Artifacts caused by defective interfering particles or breakdown of RNA were excluded. To determine whether these RNA species were functional as messengers in infected cells, virus-specific RNAs present in polyribosomes were analyzed. EDTA treatment was used to discriminate between RNA present in polyribosomes and in EDTA-resistant, presumably ribonucleoprotein, particles. Most (91%) of RNA1 was present in EDTA-resistant particles; the remainder and all other RNAs synthesized between 6 and 8 hr pi were present in polyribosomes. We conclude that MHV-A59 has six subgenomic mRNAs. Since the total molecular mass (11.1 × 10 daltons) of these messengers is about twice that of the viral genome, sequence homologies must exist between the mRNAs. The position of these homologous regions and the translation products of each of the mRNAs remain to be determined.
我们已经确定了感染小鼠肝炎病毒A59株（MHV - A59）的Sac(−)细胞中病毒产生和病毒特异性RNA合成的动力学。免疫荧光显示，在感染复数为10 PFU/细胞时，所有细胞均被感染。病毒经过浓缩和纯化，以获得这些一步生长实验所需的高滴度病毒储备液。病毒在感染后4小时开始释放到培养基中，并在感染后10小时释放完毕。通过在1 μg/ml放线菌素D存在下掺入[H]尿苷来测量的病毒特异性RNA合成也在感染后4小时开始，其最大合成速率出现在感染后6至8小时之间。在这一时期标记的RNA从感染细胞中分离出来。大约50%的这种RNA与寡聚（dT）-纤维素结合；该物质用乙二醛 - 二甲基亚砜变性，并在1%琼脂糖凝胶中进行电泳分析。存在七种具有以下分子量的RNA种类：5.6×10（RNA1）、4.0×10（RNA2）、3.0×10（RNA3）、1.4×10（RNA4）、1.2×10（RNA5）、0.9×10（RNA6）和0.6×10（RNA7）。RNA1与病毒基因组迁移位置相同。排除了由缺陷干扰颗粒或RNA降解引起的假象。为了确定这些RNA种类在感染细胞中是否作为信使发挥功能，对多核糖体中存在的病毒特异性RNA进行了分析。使用EDTA处理来区分多核糖体中存在的RNA和存在于EDTA抗性颗粒（推测为核糖核蛋白颗粒）中的RNA。大部分（91%）的RNA1存在于EDTA抗性颗粒中；其余部分以及在感染后6至8小时之间合成的所有其他RNA存在于多核糖体中。我们得出结论，MHV - A59有六种亚基因组mRNA。由于这些信使的总分子量（11.1×10道尔顿）约为病毒基因组的两倍，因此mRNA之间必定存在序列同源性。这些同源区域的位置以及每个mRNA的翻译产物仍有待确定。
