Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein.
Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein.
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DOI：
10.1371/journal.pone.0127859
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Lv Q;Guo K;Xu H;Wang T;Zhang Y
猪圆环病毒 2 型 (PCV2) 是引起猪圆环病毒相关疾病的重要传染源。迄今为止，已检测到 PCV2 的 11 种 RNA 和 5 种病毒蛋白。在这里，我们在 PCV2 基因组中发现了一个新的病毒基因，称为 ORF5，在猪肺泡巨噬细胞 3D4/2 (PAM) 中 PCV2 的有效感染过程中，该基因在转录和翻译水平上都存在。 Northern blot分析表明ORF5基因长度为180 bp，并且当以相同方向读取时与ORF1完全重叠。使用定点诱变表明 ORF5 蛋白对于 PCV2 复制不是必需的。为了研究新蛋白的生物学功能，我们构建了能够表达PCV2 ORF5的重组真核表达质粒。结果表明，带有 GFP 标记的 PCV2 ORF5 蛋白定位于内质网 (ER)，通过蛋白酶体降解，抑制 PAM 生长并延长细胞周期的 S 期。进一步的研究表明，带有 GFP 标记的 PCV2 ORF5 蛋白会诱导 ER 应激并激活 NF-κB，这一点通过 IL-6、IL-8 和 COX-2 表达的显着上调得到进一步证实。此外，通过酵母双杂交测定发现五种细胞蛋白（GPNMB、CYP1A1、YWHAB、ZNF511 和 SRSF3）与 ORF5 相互作用。这些发现为PCV2 ORF5蛋白的鉴定和功能分析提供了新的信息，并可能有助于阐明PCV2致病性的分子机制。然而，在得出任何明确的结论之前，还需要进行额外的实验来验证 PCV2 体外感染期间 ORF5 蛋白的表达和功能。
Porcine circovirus type 2 (PCV2) is the essential infectious agent responsible for causing porcine circovirus-associated diseases in pigs. To date, eleven RNAs and five viral proteins of PCV2 have been detected. Here, we identified a novel viral gene within the PCV2 genome, termed ORF5, that exists at both the transcriptional and translational level during productive infection of PCV2 in porcine alveolar macrophages 3D4/2 (PAMs). Northern blot analysis was used to demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. Site-directed mutagenesis was used to show that the ORF5 protein is not essential for PCV2 replication. To investigate the biological functions of the novel protein, we constructed a recombinant eukaryotic expression plasmid capable of expressing PCV2 ORF5. The results show that the GFP-tagged PCV2 ORF5 protein localizes to the endoplasmic reticulum (ER), is degraded via the proteasome, inhibits PAM growth and prolongs the S-phase of the cell cycle. Further studies show that the GFP-tagged PCV2 ORF5 protein induces ER stress and activates NF-κB, which was further confirmed by a significant upregulation in IL-6, IL-8 and COX-2 expression. In addition, five cellular proteins (GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3) were found to interact with ORF5 via yeast two-hybrid assay. These findings provide novel information on the identification and functional analysis of the PCV2 ORF5 protein and are likely to be of benefit in elucidating the molecular mechanisms of PCV2 pathogenicity. However, additional experiments are needed to validate the expression and function of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn.
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