Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-β3 and fibronectin
Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-β3 and fibronectin
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DOI：
10.1128/jvi.78.18.10045-10053.2004
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发表时间：
2004-09-01
影响因子：
5.4
通讯作者：
Einerhand, AWC
中科院分区：
文献类型：
--
作者：
Boshuizen, JA;Rossen, JWA;Einerhand, AWC
轮状病毒是幼儿病毒性胃肠炎和脱水腹泻的最重要原因。轮状病毒非结构蛋白 4 (NSP4) 是一种肠毒素，被确定为症状性轮状病毒感染的重要病原体。为了鉴定与 NSP4 相互作用的细胞蛋白，使用了酿酒酵母的双杂交技术。 NSP4 cDNA 源自人轮状病毒株 Wa，被克隆到酵母穿梭载体 pGBKT7 中。从克隆到酵母穿梭载体 pGAD10 中的 Caco-2 细胞衍生的肠道 cDNA 文库中筛选了与 NSP4 相互作用的蛋白质。通过免疫共沉淀和免疫组织化学共定位在体内证实了蛋白质相互作用。经过双杂交文库筛选后，我们反复分离出编码细胞外基质（ECM）蛋白层粘连蛋白-β3（氨基酸[aa] 274至878）的cDNA和编码ECM蛋白纤连蛋白（氨基酸1755至1884）的cDNA。使用NSP4的缺失突变体，我们绘制了与ECM蛋白相互作用的区域，位于氨基酸87和145之间。层粘连蛋白-β3的缺失分析表明，包含层粘连蛋白-β3的氨基酸726至875的区域与NSP4相互作用。通过免疫共沉淀证实了 NSP4 与层粘连蛋白-β3 或纤连蛋白的相互作用。 NSP4 存在于受感染的肠细胞和受感染的新生小鼠的基底膜 (BM) 中，并与层粘连蛋白-β3 共定位，表明存在生理相互作用。总之，用 NSP4 进行双杂交筛选产生了两种潜在的靶蛋白：层粘连蛋白-β3 和纤连蛋白，它们与肠毒素 NSP4 相互作用。 NSP4 从受感染上皮细胞的基底侧释放以及随后与位于 BM 的 ECM 蛋白结合可能标志着轮状病毒疾病形成的新机制。
Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as An important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.