Zafrullah M, Ozdener M H, Panda S K, Jameel S
Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
J Virol. 1997 Dec;71(12):9045-53. doi: 10.1128/JVI.71.12.9045-9053.1997.
Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.
戊型肝炎病毒（HEV）是发展中国家的一种主要人类病原体。由于缺乏体外培养系统，关于该病毒基本生物学特性的信息非常少。HEV的第三个开放阅读框编码一种功能未知的小蛋白（约13.5 kDa），即pORF3。我们在瞬时转染的COS-1和Huh-7细胞中表达了pORF3，并表明它是一种在丝氨酸残基处被修饰的磷蛋白。构建了缺失突变体和定点突变体，以确定Ser-80为磷酸化位点。该残基存在于一个保守的一级序列中，该序列显示出p34cdc2激酶（cdc2K）和丝裂原活化蛋白激酶（MAPK）的磷酸化共有位点。用在大肠杆菌或COS-1细胞中表达的六聚组氨酸标记的pORF3进行的体外实验表明，外源性添加的MAPK能有效磷酸化该蛋白。pORF3突变体在体外也表现出与MAPK相同的磷酸化模式，这与在体内观察到的情况一致。在其一级序列中，pORF3具有两个高度疏水的N端结构域。在亚细胞分级分离中，发现pORF3与细胞骨架部分共分离，而在缺失疏水结构域I（氨基酸残基1至32）后，这种与细胞骨架的关联就消失了。这些结果表明，HEV pORF3是一种与细胞骨架相关的磷蛋白，并就pORF3在HEV复制周期中可能的功能进行了讨论。
