White L J, Ball J M, Hardy M E, Tanaka T N, Kitamoto N, Estes M K
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Virol. 1996 Oct;70(10):6589-97. doi: 10.1128/JVI.70.10.6589-6597.1996.
Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.
诺如病毒（NV）是一组不可培养的人类杯状病毒的原型毒株，可引发急性肠胃炎的流行爆发。虽然这些病毒无法在组织培养细胞或动物模型中生长，但衣壳蛋白在昆虫细胞中的表达会导致重组诺如病毒样颗粒（rNV VLPs）的自组装，这些颗粒在形态和抗原性上与天然NV相似。我们利用这些rNV VLPs来研究病毒与细胞的相互作用。研究了VLPs与培养的人和动物细胞系的结合及内化情况，旨在确定体外病毒增殖可能易感的细胞系，并确定复制周期中的早期事件是否导致了病毒在细胞培养中的宿主范围狭窄和生长受限。放射性标记的VLPs特异性结合到来自不同来源的13种细胞系的细胞表面上可饱和数量的结合分子上，这些细胞系包括人肠道（分化和未分化的Caco-2）和昆虫（草地贪夜蛾9）卵巢。分化的Caco-2细胞比其他细胞系结合的rNV VLPs明显更多。不同细胞系中结合的VLPs数量的差异与来源的组织或物种无关。通过与未标记的rNV VLPs进行竞争实验确定，VLP结合是特异性的；然而，只有1.4%至6.8%的特异性预结合放射性标记的VLPs内化到细胞中。使用多克隆和单克隆抗rNV血清以及特异性抗肽血清进行阻断实验，以绘制rNV VLPs上参与细胞结合的结构域。一种单克隆抗体（NV8812）可阻断rNV VLPs与人及动物细胞系的结合。通过用衣壳蛋白的截短和切割形式进行免疫沉淀，将单克隆抗体NV8812的结合位点定位到衣壳蛋白的C末端300至384个残基处。这些数据表明，衣壳蛋白的C末端区域参与rNV VLPs与细胞的特异性结合。
