Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.
Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China; Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, China.
Vet Microbiol. 2021 Jul;258:109099. doi: 10.1016/j.vetmic.2021.109099. Epub 2021 May 4.
Poxviruses have evolved multiple strategies to modulate host-derived factors to create an optimal environment for viral efficient replication. Our previous study indicated that cyclophilin B (CypB) is a critical factor for ORFV replication in MDBK cells. However, the precise molecular mechanism by which CypB facilitates ORFV replication remains less understood. In the present study, the function of CypB in ORFV replication is further evaluated. The overexpression of CypB was observed to facilitate ORFV replication in OFTu cells and HeLa cells, however, RNA interference (RNAi)-mediated reduction of endogenous CypB decreased the levels of ORFV replication. Coimmunoprecipitation experiments revealed that the CypB interacted with ORFV ORF058 protein, a late protein involved in virus entry. The interaction of host factor CypB and ORF058 protein was further confirmed by confocal microscopy analysis and GST-pull down. In addition, the 52-55 aa was identified as the critical binding sites for CypB on ORF058 protein by GST-pull down with OFTu cells overexpressing CypB and purified GST-tagged truncated ORF058. In conclusion, we demonstrate that CypB is a critical host factor for ORFV replication in vitro by interacting with ORF058 protein, providing new insights into ORFV pathogenesis.
痘病毒进化出多种策略来调节宿主来源的因子，以创造有利于病毒高效复制的最佳环境。我们之前的研究表明，亲环素 B（CypB）是 ORFV 在 MDBK 细胞中复制的关键因素。然而，CypB 促进 ORFV 复制的确切分子机制仍知之甚少。在本研究中，进一步评估了 CypB 在 ORFV 复制中的功能。我们观察到 CypB 的过表达促进了 OFTu 细胞和 HeLa 细胞中的 ORFV 复制，而 RNA 干扰（RNAi）介导的内源性 CypB 减少则降低了 ORFV 的复制水平。共免疫沉淀实验表明，CypB 与 ORFV ORF058 蛋白相互作用，ORF058 蛋白是一种参与病毒进入的晚期蛋白。通过共聚焦显微镜分析和 GST 下拉实验进一步证实了宿主因子 CypB 与 ORF058 蛋白的相互作用。此外，通过 GST 下拉实验，我们确定了 CypB 与 ORF058 蛋白相互作用的 52-55aa 是关键结合位点，该实验是用过表达 CypB 的 OFTu 细胞和纯化的 GST 标记的截断 ORF058 进行的。总之，我们通过 CypB 与 ORF058 蛋白相互作用，证明 CypB 是体外 ORFV 复制的关键宿主因子，为 ORFV 的发病机制提供了新的见解。
