REPLICATION OF NODAMURA VIRUS AFTER TRANSFECTION OF VIRAL-RNA INTO MAMMALIAN-CELLS IN CULTURE
REPLICATION OF NODAMURA VIRUS AFTER TRANSFECTION OF VIRAL-RNA INTO MAMMALIAN-CELLS IN CULTURE
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DOI：
10.1128/jvi.66.4.2326-2334.1992
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发表时间：
1992-04-01
影响因子：
5.4
通讯作者：
GARRETT, BK
中科院分区：
文献类型：
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作者：
BALL, LA;AMANN, JM;GARRETT, BK
从受感染的哺乳小鼠后肢中纯化野田村病毒（NOV），并将其作为病毒基因组RNA RNA 1和RNA 2的来源。将病毒RNA转染到培养的小仓鼠肾（BHK21）细胞中，随后发生剧烈的RNA复制，单链RNA 1和2积累，达到接近细胞RNAs的丰度。还观察到小亚基因组RNA （RNA 3）的瞬时合成，并检测到RNA 1、2和3的双链版本。三种主要的病毒蛋白在转染细胞中合成。蛋白A（约115 kDa）和蛋白B（约15 kDa）在转染后的早期短暂产生，而大量蛋白α (43 kDa)，这两种病毒外壳蛋白的前体，在感染周期后期开始连续产生。当使用极低浓度的病毒RNA进行转染时，RNA 1发生优先复制。这一结果归因于转染的病毒RNA分离到培养的独立细胞中，随后在未接受RNA 2的细胞中复制和扩增RNA 1。因此，在极限稀释条件下对病毒RNA进行多次传代，纯化出了不含RNA 2的RNA 1，并证明了RNA 1能够长时间的自主复制，同时也伴随着RNA 3的持续合成。在单独转染RNA 1的细胞中，蛋白α不合成，蛋白A和蛋白B连续生成。转染NOV RNAs 1和NOV RNAs 2 24 h后，BHK21细胞的电镜分析显示，大量病毒颗粒在细胞质中积聚，并在某些区域形成准晶阵列。从转染的BHK21细胞中纯化的整个NOV对哺乳小鼠具有传染性，其电泳迁移率与从感染小鼠肌肉中纯化的NOV相似，但不完全相同。NOV的高产量、简单的遗传组成和不同寻常的基因组策略使其成为研究动物细胞中病毒RNA复制的一个有吸引力的系统。
Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.