ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro
ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro
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ORF4蛋白缺陷的PCV2突变体增强病毒诱导的细胞凋亡并在体外表现出mRNA的差异表达
DOI：
10.1016/j.virusres.2014.01.024
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发表时间：
2014-04-21
期刊：
影响因子：
5
通讯作者：
Yang, Zongqi
中科院分区：
文献类型：
--
作者：
Gao, Zhangzhao;Dong, Qinfang;Yang, Zongqi
猪圆环病毒2型（PCV2）是PCV相关疾病（PCVAD）的主要传染因子。在先前的体外研究中，在pcv2感染的细胞中检测到11种rna和4种病毒蛋白。开放阅读框（ORF） 4全长180bp，已在转录和翻译水平上得到鉴定。它与ORF3完全重叠，ORF3在病毒诱导的细胞凋亡中起作用。本研究利用启动密码子突变（M1-PCV2）和帧内终止突变（M2-PCV2）构建了两种orf4蛋白缺陷病毒，旨在研究其在病毒感染中的作用。研究了M1-PCV2和M2-PCV2复制、转录、表达病毒蛋白和诱导细胞凋亡的能力。病毒DNA复制曲线支持ORF4蛋白不是病毒复制所必需的，但在感染早期抑制病毒复制。在感染的PK-15细胞中比较ORF3 mRNA在野生型和ORF4缺陷型病毒中的表达水平，发现ORF4突变体的ORF3转录均增强，提示ORF4蛋白可能通过限制ORF3转录从而阻止病毒诱导的细胞凋亡发挥重要作用。与野生型PCV2相比，M1-PCV2和M2-PCV2中caspase 3和8的活性显著提高，进一步证实了这一点。此外，ORF4在细胞凋亡中的作用以及与ORF1相关的Rep蛋白的可能相互作用可能解释了在感染早期病毒快速生长和ORF4蛋白缺陷的PCV2突变体中ORF1 mRNA的高表达水平。(c) 2014 Elsevier B.V.版权所有
Porcine circovirus type 2 (PCV2) is the essential infectious agent of PCV associated disease (PCVAD). During previous in vitro studies, 11 RNAs and four viral proteins have been detected in PCV2-infected cells. Open reading frame (ORF) 4 is 180 bp in length and has been identified at the transcription and the translation level. It overlaps completely with ORF3, which has a role in virus-induced apoptosis. In this study, start codon mutations (M1-PCV2) or in-frame termination mutations (M2-PCV2) were utilized to construct two ORF4-protein deficient viruses aiming to investigate its role in viral infection. The abilities of M1-PCV2 and M2-PCV2 to replicate, transcribe, express viral proteins, and to cause cellular apoptosis were evaluated. Viral DNA replication curves supported that the ORF4 protein is not essential for viral replication, but inhibits viral replication in the early stage of infection. Comparison of the expression level of ORF3 mRNA among wild-type and ORF4-deficient viruses in infected PK-15 cell demonstrated enhanced ORF3 transcription of both ORF4 mutants suggesting that the ORF4 protein may play an important role by restricting ORF3 transcription thereby preventing virus-induced apoptosis. This is further confirmed by the significantly higher caspase 3 and 8 activities in M1-PCV2 and M2-PCV2 compared to wild-type PCV2. Furthermore, the role of ORF4 in cell apoptosis and a possible interaction with the ORF1 associated Rep protein could perhaps explain the rapid viral growth in the early stage of infection and the higher expression level of ORF1 mRNA in ORF4 protein deficient PCV2 mutants. (c) 2014 Elsevier B.V. All rights reserved.