The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo.
The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo.
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轮状病毒非结构蛋白 NS35 在体外和体内均具有 RNA 结合活性。
DOI：
10.1016/0042-6822(92)90245-k
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发表时间：
1992
期刊：
影响因子：
3.7
通讯作者：
Patton,JT
中科院分区：
文献类型：
--
作者：
Kattoura,MD;Clapp,LL;Patton,JT
为了确定和表征轮状病毒RNA结合蛋白的目标，我们使用了凝胶阻滞试验和蛋白质-RNA交联紫外线（UV）光检查从SA 11感染的细胞制备的细胞质裂解物中RNA结合蛋白的存在。在凝胶阻滞试验中产生的带移分析表明，感染的细胞含有大量的病毒蛋白，其对单链和双链RNA都具有亲和力，但缺乏序列特异性。在体外将该蛋白与放射性标记的RNA交联，然后用RNA酶处理和用抗NS 35单克隆抗体免疫沉淀，结果表明RNA结合活性与NS 35相关。此外，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析从天然凝胶分离的蛋白质-RNA复合物显示，NS 35是该复合物的唯一病毒蛋白组分。由于在兔网织红细胞裂解物中通过翻译表达的NS 35表现出对聚（U）-琼脂糖的亲和力，因此NS 35必须具有内在的RNA结合活性，其能够在不存在其他病毒蛋白的情况下发挥作用。免疫沉淀的RNase处理的交联形成在完整的细胞暴露于紫外线后证实，NS 35是密切相关的ssRNA在感染的细胞。基于其结合RNA的能力，并且鉴于先前的研究已经表明NS 35定位于感染细胞中的病毒质，对于RNA复制是必需的，并且是复制酶颗粒的组分，我们提出NS 35的功能是将病毒mRNA集中在病毒质中，并且NS 35-mRNA复合物作为基因组分类和复制的底物。
Toward the goal of identifying and characterizing rotavirus RNA-binding proteins, we have used a gel retardation assay and protein-RNA cross-linking by ultraviolet (uv) light to examine cytoplasmic lysates prepared from SA11-infected cells for the presence of RNA-binding proteins. Analysis of band shifts produced in the gel retardation assay indicated that infected cells contained significant amounts of a viral protein which had affinity for both single-stranded and double-stranded RNA but lacked sequence specificity. Cross-linking of this protein to radiolabeled RNAin vitrofollowed by RNase treatment and immunoprecipitation with an anti-NS35 monoclonal antibody revealed that the RNA-binding activity was associated with NS35. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein-RNA complex isolated from native gels revealed that NS35 was the only viral protein component of the complex. Since NS35 expressed by translation in rabbit reticulocyte lysates exhibited affinity for poly(U)-Sepharose, NS35 must possess intrinsic RNA-binding activity that is able to function in the absence of other viral proteins. Immunoprecipitation of RNase-treated cross-links formed in intact cells following exposure to uv light confirmed that NS35 was intimately associated with ssRNA in the infected cell. On the basis of its ability to bind RNA and given that previous studies have shown that NS35 localizes to the viroplasm in infected cells, is essential for RNA replication, and is a component of replicase particles, we propose that NS35 functions to concentrate viral mRNAs in the viroplasm and that NS35-mRNA complexes serve as substrates for genome assortment and replication.