Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms
Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms
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DOI：
10.1128/jvi.02167-05
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发表时间：
2006-06-01
影响因子：
5.4
通讯作者：
Estes, M. K.
中科院分区：
文献类型：
--
作者：
Berkova, Z.;Crawford, S. E.;Estes, M. K.
轮状病毒是婴儿病毒性胃肠炎的主要原因。轮状病毒非结构蛋白4(NSP4)在病毒的形态发生和致病过程中具有多效性和功能。最近的报道表明，小干扰RNA抑制NSP4的表达导致其他病毒蛋白的产生和分布以及mRNA的合成发生改变，提示NSP4也通过未知的机制影响病毒的复制。本报告描述了旨在将NSP4在细胞内的定位与其功能相关联的研究。为了能够跟踪NSP4的定位，我们将全长NSP4的C端与增强型绿色荧光蛋白(EGFP)融合，并在基于HEK 293的细胞系中诱导表达该融合蛋白，以避免可能的细胞毒性。NSP4-EGFP最初定位于内质网(ER)，通过Endo H敏感的糖基化和与ER标记蛋白的共定位来证明。只有一小部分nsp4-EGFP与ER-Golgi中间室(ERGIC)标记ERGIC-53共定位。NSP4-EGFP没有进入高尔基体，这与Endo H的敏感性和先前的报道一致，即轮状病毒感染的细胞中产生的NSP4裂解产物的分泌不被布雷菲尔丁A抑制。大量表达的NSP4-EGFP分布在细胞质中的新的囊泡结构中，而不是与ER、ERGIC、高尔基体、内体或溶酶体标记物共定位，从而偏离了已知的生物合成途径。囊泡型NSP4-EGFP的出现依赖于细胞内钙离子水平，囊泡型NSP4-EGFP与自噬标记物LC3共定位。在轮状病毒感染的细胞中，NSP4与LC3共存于与病毒质相关的帽状结构中，这是新的病毒RNA复制的场所，这可能是NSP4参与病毒复制的新机制。
Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosyllation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.