Studies of the replication of a bovine enterovirus RNA.
Studies of the replication of a bovine enterovirus RNA.
复制标题
牛肠道病毒 RNA 复制的研究。
DOI：
10.1099/0022-1317-43-1-75
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
S. Martin
中科院分区：
文献类型：
--
作者：
D. Todd;S. Martin
用盐沉淀和凝胶过滤技术从感染牛肠道病毒的BHK细胞中分离出复制中间体（RI）、复制型（RF）和单链（SS）RNA。动力学实验表明，在感染后（p.i.）RF的量是否超过RI或SS RNA的量。RF在1.5%聚丙烯酰胺-琼脂糖凝胶上的电泳显示，至少存在三种双链RNA，其中一种与可接近的含poly（A）的道相关。所有的RF通过99%二甲基亚砜（DMSO）变性，尽管当样品返回到水性条件时，快速发生再退火。使用硫酸铯密度梯度含有溴化乙锭没有发现RF分子群体中的环状结构的证据。用核糖核酸酶处理RI产生双链RNA分子，其中一些分子的大小小于完整的RF。用DMSO变性并在99%DMSO蔗糖梯度上分析表明，RI不含有比病毒体RNA更长的单链。RI的一部分与poly（U）-Sepharose 4 B柱结合。所涉及的多聚腺苷酸束仅存在于具有最大沉降系数（30至35 S）的新生RNA链中。牛肠道病毒诱导的SS RNA与纯化的病毒体RNA相比，在蔗糖梯度沉降和丙烯酰胺凝胶上的流动性方面是异质的。造成这种差异的原因从来没有得到令人满意的解决。通过99%DMSO-蔗糖梯度沉降表明，异质性是由于聚集，而不是任何变化的链长或构象差异。我们的研究结果支持单链模板模型，而不是一个小核糖核酸病毒RNA复制的环形模型。
Replicative intermediate (RI), replicative form (RF) and single-stranded (SS) RNA have been isolated from BHK cells infected with a bovine enterovirus by salt precipitation and gel filtration techniques. Kinetic experiments showed that at no time up to 16 h post-infection (p.i.) did the amount of RF exceed that of RI or SS RNA. Electrophoresis of RF on 1.5% polyacrylamide-agarose gels showed that at least three species of double-stranded RNA were present, one of which was associated with an accessible poly(A)-containing tract. All of the RF was denatured by 99% dimethylsulphoxide (DMSO), although reannealling occurred rapidly when samples were returned to aqueous conditions. No evidence for circular structures in the RF molecular population was found by use of caesium sulphate density gradients containing ethidium bromide. Treatment of RI with ribonuclease produced double-stranded RNA molecules, some of which were smaller in size than intact RF. Denaturation with DMSO and analysis on 99% DMSO sucrose gradients showed that the RI did not contain single strands of greater length than virion RNA. A portion of the RI bound to poly(U)-Sepharose 4B columns. The poly(A) tracts involved were present only in the nascent RNA strands with greatest sedimentation coefficients (30 to 35S). Bovine enterovirus induced SS RNA was heterogeneous with regard to both sedimentation through sucrose gradients and mobility on acrylamide gels compared to purified virion RNA. The reason for this difference has never been satisfactorily resolved. Sedimentation through 99% DMSO-sucrose gradients showed that the heterogeneity was due to aggregation rather than any variation in chain length or conformational differences. Our results support the single-stranded template model rather than a circular model for picornavirus RNA replication.