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Abstract：
African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.
摘要：
非洲猪瘟病毒（ASFV）是影响野生和家猪的传染病的病原体。B169L蛋白的功能,作为潜在的完整结构膜蛋白，仍有待实验表征。使用最先进的生物信息学工具，我们在这里证实了早期的预测，表明存在完整的膜螺旋发夹，并进一步建议将这种蛋白质锚定到内质网膜上，两个末端都面向细胞器的内腔。我们的进化分析证实了在自然界中B169L进化过程中，纯化选择在保存已鉴定域中的重要性。此外，我们讨论了这种发夹跨膜结构域（HTMD）作为IIA类病毒传播蛋白的可能功能。在不存在信号肽的情况下GFP融合蛋白的表达支持B169L作为III型膜蛋白插入ER中并在其中形成寡聚体。跨越B169LHTMD的重叠肽被重建为ER样膜，并通过红外光谱分析所采用的结构。与预测一致，B169L跨膜序列在脂质双层中采用α-螺旋构象。此外，单囊泡通透性试验证明了B169L跨膜螺旋在ER样膜中组装溶解孔，通过平面双层中的离子通道活性测量证实的容量。强调这些意见的相关性，在来自EP84R的跨膜螺旋的情况下没有观察到孔形成活性，另一种ASFV蛋白预测通过α-螺旋HTMD锚定到膜上。总的来说,我们的结果支持对B169LHTMD的病毒传播蛋白样功能的预测。重要的非洲猪瘟（ASF），一种影响家猪的毁灭性疾病，在欧亚大陆广泛传播，在猪肉行业产生重大的经济问题。预防/治愈该疾病的方法主要限于关于由大型(160-170kba)病毒基因组编码的大多数基因的作用的有限信息。在这份报告中,我们提供了有关非洲猪瘟病毒（ASFV）基因B169L功能特征的实验数据。此处呈现的数据表明B169L基因编码具有病毒传播蛋白功能的必需膜相关蛋白。
