Data_Sheet_4_Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization.PDF

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they ‘chaperone’ p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

目前尚无针对兔出血症病毒（Rabbit haemorrhagic disease virus, RHDV）及其他兔病毒属（Lagovirus）病毒的稳定细胞培养体系，因此兔病毒属非结构蛋白p23的确切功能尚未明确。为此，学界已借助一系列体外（in vitro）及计算机模拟（in silico）模型对p23展开研究，结果显示p23可发生寡聚化、定位于细胞质，并拥有一段包含两个两亲性螺旋的保守C端区域。此外，其他杯状病毒中p23的位置同源蛋白已被证实具有病毒孔蛋白（viroporin）活性。本研究阐明了p23寡聚化的分子机制：定点诱变实验证实，N端半胱氨酸残基对p23二聚化至关重要。本研究还通过基于细胞培养氨基酸稳定同位素标记（stable isotope labeling with amino acids in cell culture, SILAC）的蛋白质组学技术，鉴定出p23的细胞互作蛋白；在转染细胞中，热休克蛋白Hsp70与Hsp110可与p23结合，提示二者可在p23整合至细胞膜前对其发挥分子伴侣功能。我们对感染病毒的兔肝组织进行了全转录组与蛋白质组分析，观察到错误折叠蛋白应答、钙信号通路以及内质网（endoplasmic reticulum, ER）网络调控均发生了显著变化。最后，流式细胞术实验结果显示，转染p23的细胞其细胞质内钙浓度略有升高。综上，现有证据表明p23是一种病毒孔蛋白，可在内质网膜上形成钙离子传导通道。