ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.
ORF3 蛋白是猪流行性腹泻病毒 (Porcine epidemic diarrhea virus，PEDV) 基因组编码的唯一的辅助蛋白，与病毒毒力相关。为确定PEDV ORF3 细胞质定位信号，文中构建了系列PEDV DR13wt ORF3 蛋白全长或截短肽重组表达载体，转染Vero 细胞并利用激光共聚焦显微镜分析与EGFP 融合表达的全长ORF3 蛋白和其系列截短肽在细胞内的分布。结果表明，全长ORF3 蛋白或所有包含2 个跨膜域的40–91 aa 基序的ORF3 截短肽均只定位于细胞质中，而不包含40–91aa 基序的ORF3 截短肽分布于整个细胞中 (细胞质和细胞核均有分布)。这表明40–91 aa是猪流行性腹泻病毒ORF3 蛋白细胞质定位的关键结构域。PEDV ORF3 蛋白细胞质定位结构域的明确为进一步研究其细胞内转运和生物学功能提供了参考。.
Keywords: ORF3 protein; cytoplasmic location; location signal; porcine epidemic diarrhea virus; transmembrane domain.