College of Veterinary Medicine, Northwest A & F University, Yangling, 712100, Shaanxi, China.
Department of Preventive Medicine and Public Health, Faculty of Veterinary Science, University of Nyala, P.O Box: 155, Nyala, Sudan.
Vet Res. 2018 Oct 5;49(1):102. doi: 10.1186/s13567-018-0599-6.
Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis.
许多病毒蛋白与抑制靶细胞中的细胞凋亡有关，因此有利于病毒复制。新城疫病毒（NDV）的 V 蛋白就是这样一种蛋白，它以种属特异性的方式在抑制细胞凋亡中发挥重要作用。然而，迄今为止，还没有报道阐明 V 蛋白的抗细胞凋亡机制。本研究旨在确定 V 蛋白在鸡胚成纤维细胞系（DF-1 细胞）中的凋亡潜能，并阐明其作用的分子机制。在这里，使用酵母双杂交系统筛选与 V 蛋白相互作用的宿主蛋白，并鉴定硫氧还蛋白样蛋白 1（TXNL1）为潜在的结合伴侣。V 蛋白和 TXNL1 蛋白在 DF-1 细胞中的免疫共定位进一步验证了这两种蛋白的相互作用。通过在 DF-1 细胞中转染 TXNL1 蛋白和敲低 TXNL1 蛋白，检测 NDV 复制和细胞凋亡的影响。通过流式细胞术检测细胞凋亡。通过定量实时 PCR（Q-PCR）和 Western blot 检测 Bax、Bcl-2 和 Caspase-3 的 mRNA 和蛋白表达水平。通过 Q-PCR 和噬斑试验检测 NDV 表达。结果表明，TXNL1 蛋白诱导 DF-1 细胞凋亡并抑制 NDV 复制。此外，Western blot 和 Q-PCR 结果表明，TXNL1 通过涉及 Bcl-2\Bax 和 Caspase-3 的途径诱导细胞凋亡。最后，这项工作深入了解了 V 蛋白抑制细胞凋亡的机制。
