Division of Global Epidemiology, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan.
Division of Risk Analysis and Management, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan.
Viruses. 2021 Nov 20;13(11):2316. doi: 10.3390/v13112316.
Viral protein 35 (VP35) of Ebola virus (EBOV) is a multifunctional protein that mainly acts as a viral polymerase cofactor and an interferon antagonist. VP35 interacts with the viral nucleoprotein (NP) and double-stranded RNA for viral RNA transcription/replication and inhibition of type I interferon (IFN) production, respectively. The C-terminal portion of VP35, which is termed the IFN-inhibitory domain (IID), is important for both functions. To further identify critical regions in this domain, we analyzed the physical properties of the surface of VP35 IID, focusing on hydrophobic patches, which are expected to be functional sites that are involved in interactions with other molecules. Based on the known structural information of VP35 IID, three hydrophobic patches were identified on its surface and their biological importance was investigated using minigenome and IFN-β promoter-reporter assays. Site-directed mutagenesis revealed that some of the amino acid substitutions that were predicted to disrupt the hydrophobicity of the patches significantly decreased the efficiency of viral genome replication/transcription due to reduced interaction with NP, suggesting that the hydrophobic patches might be critical for the formation of a replication complex through the interaction with NP. It was also found that the hydrophobic patches were involved in the IFN-inhibitory function of VP35. These results highlight the importance of hydrophobic patches on the surface of EBOV VP35 IID and also indicate that patch analysis is useful for the identification of amino acid residues that directly contribute to protein functions.
埃博拉病毒（EBOV）的病毒蛋白 35（VP35）是一种多功能蛋白，主要作为病毒聚合酶辅因子和干扰素拮抗剂发挥作用。VP35 分别与病毒核蛋白（NP）和双链 RNA 相互作用，以进行病毒 RNA 转录/复制和抑制 I 型干扰素（IFN）的产生。VP35 的 C 端部分，即干扰素抑制结构域（IID），对于这两种功能都很重要。为了进一步确定该结构域中的关键区域，我们分析了 VP35 IID 表面的物理特性，重点关注预计与其相互作用的其他分子有关的疏水性斑块。基于 VP35 IID 的已知结构信息，在其表面上鉴定出三个疏水性斑块，并通过 minigenome 和 IFN-β启动子报告基因检测来研究其生物学重要性。定点突变揭示，一些预测会破坏斑块疏水性的氨基酸取代由于与 NP 的相互作用减少而显著降低了病毒基因组复制/转录的效率，这表明疏水性斑块可能通过与 NP 的相互作用对于复制复合物的形成至关重要。还发现疏水性斑块参与了 VP35 的 IFN 抑制功能。这些结果突出了 EBOV VP35 IID 表面上的疏水性斑块的重要性，并且表明斑块分析对于鉴定直接影响蛋白质功能的氨基酸残基很有用。
