STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE POLIOVIRUS REPLICATION COMPLEX
STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE POLIOVIRUS REPLICATION COMPLEX
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DOI：
10.1128/jvi.66.5.2740-2747.1992
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发表时间：
1992-05-01
影响因子：
5.4
通讯作者：
TROXLER, M
中科院分区：
文献类型：
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作者：
BIENZ, K;EGGER, D;TROXLER, M
通过蔗糖梯度离心从脊髓灰质炎病毒感染的HEp-2细胞中分离出两个膜结合复制复合物群体。 这两个部分显示出相似的超微结构特征：复制复合物被封闭在病毒诱导的囊泡的玫瑰花状壳中，并且包含非常紧密包装的第二膜系统（致密膜）。 泡状部分，带在30%蔗糖，含有复制中间体（RI）和36 S RNA。 在45%蔗糖中的组分条带仅含有微量的RI，主要含有36 S RNA，其中三分之二是糖苷化的。 在体外，两个组分显示出相似的RNA合成能力，并产生36 S正链RNA。 用脱氧胆酸钠溶解合成活性复制复合物内和周围的膜，废除了36 S RNA的完成，但仍然允许RI的延伸。 我们的研究结果表明，具有新生的正链上的RI封闭在致密的膜和复制叉包裹另外在蛋白质的复制复合物的架构。 正链RNA可以通过与生物素化的核糖核酸探针在复制复合物和病毒诱导的囊泡的玫瑰花结之间进行原位杂交来定位。 发现子代RNA链在复制复合物内的致密膜与周围病毒诱导的囊泡紧密接触的位点处完成后不久从复制复合物中释放。
Two populations of membrane-bound replication complexes were isolated from poliovirus-infected HEp-2 cells by sucrose gradient centrifugation. The two fractions show similar ultrastructural features: the replication complex is enclosed in a rosettelike shell of virus-induced vesicles and contains a very tightly packed second membrane system (compact membranes). The vesicular fraction, which bands in 30% sucrose, contains replicative intermediate (RI) and 36S RNA. The fraction banding in 45% sucrose contains only minute amounts of RI and contains mainly 36S RNA, two-thirds of which is encapsidated. In vitro, the two fractions show similar RNA synthesizing capacities and produce 36S plus-strand RNA. Dissolving the membranes within and around synthetically active replication complexes with sodium deoxycholate abolishes the completion of 36S RNA but still allows elongation in the RI. Our findings suggest an architecture of the replication complex that has the nascent plus strands on the RI enclosed in the compact membranes and the replication forks wrapped additionally in protein. Plus-strand RNA can be localized by in situ hybridization with a biotinylated riboprobe between the replication complex and the rosette of the virus-induced vesicles. It was found that the progeny RNA strands are set free soon after completion from the replication complex at the sites where the compact membranes within the replication complex are in close contact with the surrounding virus-induced vesicles.