College of Veterinary Medicine, Northwest A&F University, Yangling, China.
Center for Retrovirus Research, The Ohio State University, Columbus, Ohio, USA.
J Virol. 2018 May 14;92(11). doi: 10.1128/JVI.00100-18. Print 2018 Jun 1.
Porcine circovirus type 2 (PCV2) capsid protein (Cap) is a unique structure protein that plays pivotal roles in the process of viral replication and pathogenesis. Herein, we characterized a putative porcine Makorin RING finger protein 1 (pMKRN1) variant, an N-terminal-truncated variant of putative full-size porcine MKRN1 which has a unique expression pattern resulting from the porcine gene and which interacts with PCV2 Cap. A domain mapping assay showed that the C terminus of pMKRN1 and fragments (amino acids 108 to 198) of Cap are required for this interaction. PCV2 transiently upregulated pMKRN1 in PK-15 cells, but persistent viral infection downregulated pMKRN1 in major pathological tissues of PCV2-infected piglets. Overexpression of pMKRN1 significantly inhibited the generation of progeny PCV2 via ubiquitination and degradation of Cap, whereas knockout of pMKRN1 blocked Cap degradation and promoted progeny virus replication. pMKRN1 specifically targeted PCV2 Cap lysine residues 164, 179, and 191 to induce polyubiquitination and subsequent degradation. Mutation of either of the three lysine residues in the Cap protein or mutation of the histidine at residue 243 within the RING finger domain of pMKRN1 abrogated the E3 ligase activity of pMKRN1, rendering cells incapable of inducing Cap ubiquitination and degradation. Consistent with this finding, a Cap ubiquitination-deficient PCV2 strain showed enhanced virus replication and produced severe histological lesions in the lung and lymph node tissues compared with wild-type PCV2. Taken together, the results presented here suggest that PCV2 downregulates the pMKRN1 variant to avoid pMKRN1-mediated Cap ubiquitination and degradation, thus promoting viral replication and pathogenesis in its targeted tissues. Porcine circovirus type 2 is the pathogen to which pigs are the most susceptible, causing immense economic losses in the global swine industry, but whether host cells have developed some strategies to prevent viral replication is still unclear. Here, we found that porcine MKRN1 (pMKRN1) was upregulated in the early stage of PCV2 infection and mediated the polyubiquitination and degradation of Cap protein to block PCV2 replication, yet persistent PCV2 infection downregulated pMKRN1 levels to avoid degradation, promoting viral replication and pathogenesis in its targeted tissues. These data present new insight into the molecular mechanisms underlying the antiviral effects of pMKRN1 E3 ligase during PCV2 infection and also suggest potential new control measures for PCV2 outbreaks.
猪圆环病毒 2 型（PCV2）衣壳蛋白（Cap）是一种独特的结构蛋白，在病毒复制和发病机制过程中发挥关键作用。在此，我们对一种假定的猪 Makorin RING 指蛋白 1（pMKRN1）变体进行了表征，该变体是假定全长猪 MKRN1 的 N 端截断变体，由于猪基因的存在，具有独特的表达模式，并且与 PCV2 Cap 相互作用。结构域映射分析表明，pMKRN1 的 C 末端和 Cap 的片段（氨基酸 108 至 198）是这种相互作用所必需的。PCV2 瞬时上调 PK-15 细胞中的 pMKRN1，但持续性病毒感染下调 PCV2 感染仔猪主要病理组织中的 pMKRN1。过表达 pMKRN1 通过泛素化和降解 Cap 显著抑制后代 PCV2 的产生，而 pMKRN1 的敲除则阻止 Cap 降解并促进后代病毒复制。pMKRN1 特异性靶向 PCV2 Cap 的赖氨酸残基 164、179 和 191 以诱导多泛素化和随后的降解。Cap 蛋白中这三个赖氨酸残基中的任何一个或 pMKRN1 的 RING 指结构域内的组氨酸 243 的突变都消除了 pMKRN1 的 E3 连接酶活性，使细胞无法诱导 Cap 泛素化和降解。与这一发现一致的是，与野生型 PCV2 相比，Cap 泛素化缺陷型 PCV2 株显示出增强的病毒复制并在肺和淋巴结组织中产生严重的组织病理学损伤。总之，这里提出的结果表明，PCV2 下调 pMKRN1 变体以避免 pMKRN1 介导的 Cap 泛素化和降解，从而促进其靶组织中的病毒复制和发病机制。猪圆环病毒 2 是对猪最易感的病原体，给全球养猪业造成了巨大的经济损失，但宿主细胞是否已经开发出一些策略来阻止病毒复制仍不清楚。在这里，我们发现猪 MKRN1（pMKRN1）在 PCV2 感染的早期阶段被上调，并介导 Cap 蛋白的多泛素化和降解以阻断 PCV2 的复制，但持续性 PCV2 感染下调了 pMKRN1 水平以避免降解，从而促进其靶组织中的病毒复制和发病机制。这些数据为 PCV2 感染期间 pMKRN1 E3 连接酶的抗病毒作用的分子机制提供了新的见解，并为 PCV2 爆发提供了新的潜在控制措施。
