Parr Rebecca D, Storey Stephen M, Mitchell Deanne M, McIntosh Avery L, Zhou Minglong, Mir Kiran D, Ball Judith M
Department of Pathobiology, Texas A&M University 4467, College Station, Texas 77843, USA.
J Virol. 2006 Mar;80(6):2842-54. doi: 10.1128/JVI.80.6.2842-2854.2006.
Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4(114-135)) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast two-hybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.
已知轮状病毒非结构蛋白4（NSP4）在内质网（ER）中作为对病毒形态发生至关重要的细胞内受体发挥作用，并且是首个被鉴定的病毒肠毒素。外源性添加的NSP4可诱导幼鼠腹泻，并刺激经尤斯灌流小室测量的跨肠段分泌性氯离子电流。圆二色性研究进一步表明，完整的NSP4以及位于延伸的C末端两亲性螺旋内的肠毒素肽（NSP4(114 - 135)）优先与小窝样模型膜相互作用。我们现在通过激光扫描共聚焦显微镜显示，在转染NSP4的和感染轮状病毒的哺乳动物细胞中，NSP4与小窝蛋白-1在细胞核周围的网状结构（可能是内质网）、细胞质以及细胞周边共定位。通过Pro-Quest酵母双杂交系统，以全长NSP4和七个重叠缺失突变体作为诱饵、小窝蛋白-1作为猎物，反之亦然，确定了NSP4第112至140位残基与小窝蛋白-1之间的直接相互作用。从感染轮状病毒的哺乳动物细胞中共免疫沉淀NSP4 - 小窝蛋白-1复合物表明，这种相互作用发生在病毒感染期间。最后，哺乳动物细胞裂解物中的小窝蛋白-1与琼脂糖珠结合的、NSP4特异性合成肽的结合证实了酵母双杂交数据，并进一步将结合域划定为第114至135位氨基酸。我们提出，NSP4与小窝蛋白-1的结合有助于NSP4从内质网到细胞表面的细胞内运输，并推测外源性添加的NSP4刺激位于小窝微区的信号分子。
