Lv Qizhuang, Guo Kangkang, Xu Han, Wang Tao, Zhang Yanming
Laboratory of Veterinary Public Health and Food Safety, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, PR China.
PLoS One. 2015 Jun 2;10(6):e0127859. doi: 10.1371/journal.pone.0127859. eCollection 2015.
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.
猪圆环病毒2型（PCV2）是导致猪发生猪圆环病毒相关疾病的主要感染因子。迄今为止，已检测到PCV2的11种RNA和5种病毒蛋白。在此，我们在PCV2基因组中鉴定出一个新的病毒基因，命名为ORF5，在PCV2感染猪肺泡巨噬细胞3D4/2（PAMs）的生产性感染过程中，该基因在转录和翻译水平均存在。Northern印迹分析表明，ORF5基因长度为180 bp，与ORF1同向阅读时完全重叠。定点诱变表明，ORF5蛋白对PCV2复制并非必需。为了研究该新蛋白的生物学功能，我们构建了一个能够表达PCV2 ORF5的重组真核表达质粒。结果表明，绿色荧光蛋白（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。进一步研究表明，GFP标记的PCV2 ORF5蛋白诱导内质网应激并激活核因子κB（NF-κB），白细胞介素6（IL-6）、白细胞介素8（IL-8）和环氧化酶2（COX-2）表达显著上调进一步证实了这一点。此外，通过酵母双杂交试验发现5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信息，可能有助于阐明PCV2致病的分子机制。然而，在得出任何明确结论之前，还需要额外的实验来验证体外PCV2感染过程中ORF5蛋白的表达和功能。
