Gao J, Cheng A C, Wang M S, Jia R Y, Zhu D K, Chen S, Liu M F, Liu F, Yang Q, Sun K F, Chen X Y
Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu, Sichuan, China.
Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu, Sichuan, China
Genet Mol Res. 2015 Oct 29;14(4):13779-90. doi: 10.4238/2015.October.28.40.
The US2 protein has been reported to contribute to duck enteritis virus (DEV) infection; however, its kinetics and localization during infection, and whether it is a component of virion, have not been previously reported. To elucidate the function of DEV US2, US2 was amplified by polymerase chain reaction (PCR) and inserted into pET-32a(+); this was expressed, the recombinant US2 protein was purified, and a polyclonal antibody generated. In addition, the kinetics and localization of the US2 gene and protein were determined by quantitative real-time fluorescent PCR, ganciclovir (GCV), and cycloheximide (CHX) treatment, western-blot, and indirect immunofluorescence assay. The packaging of US2 into DEV virions was revealed by a protease protection assay. US2 was found to be transcribed 24 h post-infection (pi) and peaked at 72 h pi; the US2 protein was detected 48 h pi, except in the presence of GCV or CHX. US2 was packed into virions and also localized to the plasma membrane and cytoplasm in infected cells. The results showed that the DEV US2 is a late gene, and that its encoding protein could be a tegument component localized mainly in the cytoplasm. This study provides useful data for the further analysis of DEV US2, including an explanation for the genetic conservation among alphaherpesviruses.
据报道，US2蛋白有助于鸭肠炎病毒（DEV）感染；然而，其在感染过程中的动力学和定位，以及它是否是病毒粒子的组成部分，此前尚未见报道。为阐明DEV US2的功能，通过聚合酶链反应（PCR）扩增US2并将其插入pET-32a(+)；进行表达，纯化重组US2蛋白，并制备多克隆抗体。此外，通过定量实时荧光PCR、更昔洛韦（GCV）和放线菌酮（CHX）处理、蛋白质免疫印迹和间接免疫荧光试验确定US2基因和蛋白的动力学及定位。通过蛋白酶保护试验揭示了US2包装进DEV病毒粒子的情况。发现US2在感染后24小时（pi）转录，并在72小时pi达到峰值；在48小时pi检测到US2蛋白，但在存在GCV或CHX的情况下除外。US2被包装进病毒粒子，并且也定位于感染细胞的质膜和细胞质中。结果表明，DEV US2是一个晚期基因，其编码蛋白可能是主要定位于细胞质中的被膜成分。本研究为进一步分析DEV US2提供了有用的数据，包括对α疱疹病毒间遗传保守性的解释。
