Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, People's Republic of China.
Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, People's Republic of China.
Sci Rep. 2019 Mar 19;9(1):4851. doi: 10.1038/s41598-019-41311-0.
Thus far, there have been no reports on the molecular characterization and antiapoptotic function of the DPV Us5 gene. To perform molecular characterization of DPV Us5, RT-PCR and pharmacological inhibition tests were used to ascertain the kinetic class of the Us5 gene. Western blotting and an indirect immunofluorescence assay (IFA) were used to analyze the expression level and subcellular localization of Us5 in infected cells at different time points. Us5 in purified DPV virions was identified by mass spectrometry. The results of RT-PCR, Western blotting, and pharmacological inhibition tests revealed that Us5 is transcribed mainly in the late stage of viral replication. The IFA results revealed that Us5 was localized throughout DPV-infected cells but was localized only to the cytoplasm of transfected cells. Mass spectrometry and Western blot analysis showed that Us5 was a virion component. Next, to study the antiapoptotic function of DPV Us5, we found that DPV CHv without gJ could induce more apoptosis cells than DPV-CHv BAC and rescue virus. we constructed a model of apoptosis in duck embryo fibroblasts (DEFs) induced by hydrogen peroxide (HO). Transfected cells expressing the Us5 gene were protected from apoptosis induced by HO, as measured by a TUNEL assay, a caspase activation assay and Flow Cytometry assay. The TUNEL assay and Flow Cytometry assay results showed that the recombinant plasmid pCAGGS-Us5 could inhibit apoptosis induced by HO in DEF cells. However, caspase-3/7 and caspase-9 protein activity upregulated by HO was significantly reduced in cells expressing the recombinant plasmid pCAGGS-Us5. Overall, these results show that the DPV Us5 gene is a late gene and that the Us5 protein is a component of the virion, is localized in the cytoplasm, and can inhibit apoptosis induced by HO in DEF cells.
迄今为止，尚未有关于 DPvUs5 基因的分子特征和抗凋亡功能的报道。为了对 DPvUs5 进行分子特征分析，我们使用 RT-PCR 和药理学抑制试验来确定 Us5 基因的动力学类别。Western blot 和间接免疫荧光试验（IFA）用于分析感染细胞在不同时间点的 Us5 表达水平和亚细胞定位。通过质谱法鉴定纯化 DPv 病毒粒子中的 Us5。RT-PCR、Western blot 和药理学抑制试验的结果表明，Us5 主要在病毒复制的晚期转录。IFA 结果表明，Us5 定位于 DPv 感染的细胞中，但仅定位于转染细胞的细胞质中。质谱法和 Western blot 分析表明，Us5 是病毒粒子的组成部分。接下来，为了研究 DPvUs5 的抗凋亡功能，我们发现没有 gJ 的 DPvCHv 比 DPv-CHv BAC 和拯救病毒能诱导更多的凋亡细胞。我们构建了由过氧化氢（HO）诱导的鸭胚成纤维细胞（DEF）凋亡模型。转染表达 Us5 基因的细胞可通过 TUNEL 测定、半胱天冬酶激活测定和流式细胞术测定，防止由 HO 诱导的细胞凋亡。TUNEL 测定和流式细胞术测定结果表明，重组质粒 pCAGGS-Us5 可抑制 DEF 细胞中由 HO 诱导的凋亡。然而，表达重组质粒 pCAGGS-Us5 的细胞中 HO 上调的 caspase-3/7 和 caspase-9 蛋白活性明显降低。总体而言，这些结果表明 DPvUs5 基因是晚期基因，Us5 蛋白是病毒粒子的组成部分，定位于细胞质中，可抑制 DEF 细胞中由 HO 诱导的凋亡。
