Etkind P R, Krug R M
J Virol. 1975 Dec;16(6):1464-75. doi: 10.1128/JVI.16.6.1464-1475.1975.
Influenza viral complementary RNA (cRNA) was purified free from any detectable virion-type RNA (vRNA), and its genetic content and activity in wheat germ cell-free extracts were examined. After phenol-chloroform extraction of cytoplasmic fractions from infected cells, poly(A)-containing viral cRNA is found in two forms: in single-stranded RNA and associated with vRNA in partially and fully double-stranded RNA. To purify single-stranded cRNA free of these double-stranded forms, it was necessary to employ, as starting material, RNA fractions in which cRNA was predominantly single stranded. Two RNA fractions were successfully employed as starting material: polyribosomal RNA and the total cytoplasmic RNA from infected cells treated with 100 mug of cycloheximide (CM) per ml at 3 h after infection. In WSN virus-infected canine kidney (MDCK) cells, the addition of CM at 3 h after infection stimulates the production of cRNA threefold and causes a very large increase in the proportion of the cytoplasmic cRNA which is single stranded; double-stranded RNA forms are greatly reduced in amount. Total cRNA was obtained by oligo(dT)-cellulose chromatography, and single-stranded cRNA was separated from double-stranded forms by Sepharose 4B chromatography. The cRNA preparation purified from polyribosomes consists of 95% single-stranded cRNA, with the remaining 5% apparently being double-stranded RNA forms. The cRNA preparation purified from CM-treated cells (CM cRNA) is even more pure: 100% of the radiolabeled RNA is single-stranded cRNA. Annealing experiments, in which a limited amount of 32P-labeled genome RNA was annealed to the cRNA, indicate that the purified cRNA contains at least 84 to 90% of the genetic information in the vRNA genome. Purified viral cRNA (CM cRNA) is very active in directing the synthesis of virus-specific proteins in wheat germ cell-free extracts.
流感病毒互补RNA（cRNA）被纯化，不含任何可检测到的病毒粒子型RNA（vRNA），并检测了其在小麦胚无细胞提取物中的遗传内容和活性。在用苯酚-氯仿提取感染细胞的细胞质组分后，发现含poly（A）的病毒cRNA有两种形式：单链RNA形式以及与vRNA形成部分和完全双链RNA的形式。为了纯化不含这些双链形式的单链cRNA，有必要使用cRNA主要为单链的RNA组分作为起始材料。有两种RNA组分成功用作起始材料：多核糖体RNA和感染后3小时用每毫升100微克环己酰亚胺（CM）处理的感染细胞的总细胞质RNA。在WSN病毒感染的犬肾（MDCK）细胞中，感染后3小时添加CM可使cRNA的产量增加三倍，并导致细胞质cRNA中单链形式的比例大幅增加；双链RNA形式的数量大大减少。通过寡聚（dT）-纤维素柱层析获得总cRNA，通过琼脂糖4B柱层析将单链cRNA与双链形式分离。从多核糖体纯化的cRNA制剂由95%的单链cRNA组成，其余5%显然是双链RNA形式。从CM处理细胞纯化的cRNA制剂（CM cRNA）纯度更高：100%的放射性标记RNA是单链cRNA。用有限量的32P标记的基因组RNA与cRNA进行退火实验，结果表明纯化的cRNA包含vRNA基因组中至少84%至90%的遗传信息。纯化的病毒cRNA（CM cRNA）在指导小麦胚无细胞提取物中病毒特异性蛋白质的合成方面非常活跃。
