Wang Shang-Kwei, Jiang Meei Jyh, Lin Shin-Rung, Chen Mei-Yin, Wang Hung-Hsueh, Duh Chang-Yih
Department of Microbiology and Immunology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Department of Cell Biology and Anatomy, National Cheng Kung University, Tainan, Taiwan.
J Gen Virol. 2015 May;96(Pt 5):1115-1126. doi: 10.1099/vir.0.000040. Epub 2015 Jan 6.
The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50 and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26 and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50 and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes and proteasomes reduced p20, p26 and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induced the intracellular proteolysis of p34 into p20, p26 and p28 and the cleavage of p43, p50 and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50 and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26 and p28 and concurrently increased levels of nascent p43, p50 and p84 during the infectious cycle. Intriguingly, calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.
人巨细胞病毒（HCMV）UL112 - 113基因与病毒裂解复制有关。UL112 - 113蛋白p34、p43、p50和p84通过可变剪接表达。然而，共享UL112阅读框的另外三种病毒相关蛋白（p20、p26和p28）的产生机制仍然未知。生物信息学分析表明，p34、p43、p50和p84含有潜在的类PEST降解基序。在本研究中，钙蛋白酶、溶酶体和蛋白酶体的抑制剂降低了病毒感染细胞中p20、p26和p28的水平，提示蛋白水解修饰的参与。此外，可增加细胞内钙水平并激活钙蛋白酶活性的 maitotoxin诱导p34在细胞内蛋白水解为p20、p26和p28，以及p43、p50和p84裂解为p38和一种新蛋白p34c。蛋白水解分析进一步表明，p34、p43、p50和p84是钙蛋白酶 - 1和钙蛋白酶 - 2的底物，并且它们产生的蛋白水解产物与在HCMV感染期间检测到的产物相对应。此外，p34假定的钙蛋白酶切割位点的替代突变减少了蛋白水解产物的积累。通过RNA干扰对内源性钙蛋白酶 - 1和钙蛋白酶 - 2的敲低减少了p20、p26和p28的积累，并同时增加了感染周期中新生p43、p50和p84的水平。有趣的是，钙蛋白酶的缺失增强了病毒基因组的合成。此外，稳定表达p20、p26或p28的HCMV允许细胞表现出病毒基因组合成减少和成熟病毒产生减少。我们的研究结果表明，源自钙蛋白酶催化蛋白水解的同源UL112 - 113蛋白参与了HCMV复制过程。
