Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, PR China; Key Laboratory of Animal Diseases Diagnostic and Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Ministry of Agriculture, Nanjing 210095, PR China.
Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, PR China.
Virus Res. 2015 Feb 2;197:17-25. doi: 10.1016/j.virusres.2014.12.004. Epub 2014 Dec 10.
ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for virus viability, but all serine mutant clones produced infectious progeny viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.
ORF5a 蛋白最近被鉴定为猪繁殖与呼吸综合征病毒 (PRRSV) 的一种新型结构蛋白。ORF5a 蛋白在 2 型 PRRSV 中具有两个位于位置 29 和 30 的高度保守的半胱氨酸。在这项研究中，基于 2 型 PRRSV cDNA 克隆 (pAJXM)，确定了 ORF5a 蛋白半胱氨酸残基对病毒复制的意义。每个半胱氨酸均被丝氨酸或甘氨酸取代，并将突变引入 pAJXM。我们发现，位置 30 的半胱氨酸突变为甘氨酸对病毒活力是致命的，但所有的丝氨酸突变体克隆都产生了感染性的子代病毒。这表明 ORF5a 蛋白中的半胱氨酸残基不是 2 型 PRRSV 复制所必需的。双分子荧光互补 (BiFC) 和免疫共沉淀 (Co-IP) 实验用于研究 ORF5a 蛋白与其他包膜蛋白的相互作用。这些结果表明，ORF5a 蛋白与自身非共价相互作用，并与 GP4 和 2b 蛋白相互作用。位置 30 的半胱氨酸突变为甘氨酸会影响 ORF5a 蛋白与自身的非共价相互作用，这可能是携带位置 30 的半胱氨酸突变为甘氨酸的突变体表现出致死表型的原因。
