Reversible dissociation of the poliovirus replication complex: Functions and interactions of its components in viral RNA synthesis
Reversible dissociation of the poliovirus replication complex: Functions and interactions of its components in viral RNA synthesis
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DOI：
10.1128/jvi.70.12.8675-8683.1996
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发表时间：
1996-12-01
影响因子：
5.4
通讯作者：
Bienz, K
中科院分区：
文献类型：
--
作者：
Egger, D;Pasamontes, L;Bienz, K
含有转录活性复制中间体（RT）的膜结合复制复合物可以从脊髓灰质炎病毒感染的HEp-2细胞中分离，并且由围绕复制病毒RNA的病毒诱导的囊泡的玫瑰花状结构组成。在低离子强度和低温下，ROPERATURE可逆地解离成单独的管状囊泡。通过免疫电镜和免疫沉淀测定，囊泡携带一组病毒结构蛋白和非结构蛋白以及RI RNA。在30 ℃下，囊泡重新结合成在RI中合成正链RNA的RNA。通过RNA酶保护试验评估通过高度稀释保持分离的RNA和囊泡的体外转录活性。认为在正链的5'端的前178个核苷酸的合成反映了起始，并且认为在P2基因组区域中检测到530个核苷酸片段反映了延伸。可以表明，在单个囊泡上的正链的起始和延伸与在roxylate中的那些相当，其中起始在从头组装的起始复合物中进行。通过使用去污剂处理，发现引发，而不是延长，是依赖于囊泡膜。
Membrane-bound replication complexes containing transcriptionally active replicative intermediates (RT) can be isolated from poliovirus-infected HEp-2 cells and consist of rosette-like structures of virus-induced vesicles surrounding the replicating viral RNA. At low ionic strength and low temperature, the rosettes reversibly dissociate into individual tubulated vesicles. As determined by immunoelectron microscopy and immunoprecipitation, the vesicles carry a set of viral structural and nonstructural proteins as well as RI RNA, At 30 degrees C, the vesicles reassociate into rosettes synthesizing plus-strand RNA in the RI, The in vitro transcriptional activities of rosettes and vesicles kept separated by high dilution were assessed by an RNase protection assay. The synthesis of the first 178 nucleotides at the 5' end of the plus strand was considered to reflect initiation, and the detection of a 530-nucleotide fragment in the P2 genomic region was considered to reflect elongation. It could be shown that the initiation and elongation of plus strands on individual vesicles are comparable to those in rosettes, with initiation proceeding in de novo-assembled initiation complexes. By use of detergent treatment it was found that initiation, but not elongation, is dependent on vesicular membranes.