A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage.
A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage.
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基于细胞的荧光共振能量转移（FRET）传感器揭示了诺如病毒蛋白酶活性和多蛋白裂解中的细胞间和细胞内变化。
DOI：
10.1074/jbc.m115.688234
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发表时间：
2015-11-13
期刊：
影响因子：
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通讯作者：
Goodfellow I
中科院分区：
文献类型：
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作者：
Emmott E;Sweeney TR;Goodfellow I
背景：诺如病毒通过病毒蛋白酶裂解多蛋白产生病毒复制酶。结果：基于细胞的FRET传感器揭示了诺如病毒基因群内部和之间底物切割的显著变化。结论：不同基因群的诺如病毒蛋白酶对底物的识别能力存在差异。意义：了解病毒蛋白酶的切割偏好对药物设计至关重要，并为产生人-鼠诺如病毒嵌合体的潜力提供了见解。病毒蛋白酶是开发抗病毒疗法的关键药物靶点。由于许多蛋白酶抑制剂模拟蛋白酶底物，蛋白酶之间底物识别的差异可能会影响它们对给定抑制剂的敏感性。在这里，我们使用基于细胞的FRET传感器来研究不同诺如病毒蛋白酶在插入分离青色荧光蛋白和黄色荧光蛋白的连接区域的各种诺如病毒切割位点切割时的活性。使用该系统，我们证明了人类诺如病毒（基因组I （GI）和II）和常用的小鼠诺如病毒（基因组V）模型的蛋白酶在底物加工方面存在差异。这些改变了基因组内部和基因组之间特定切割位点的切割效率。这些蛋白酶之间观察到的差异可能会影响对蛋白酶抑制剂的敏感性，以及MNV作为测试此类分子对抗人诺如病毒蛋白酶的模型系统的适用性。最后，我们证明，当存在MNV NS6蛋白酶而不存在GI或GII蛋白酶时，除了NS6 - 7连接外，用GI或GII等量的MNV多蛋白切割位点替代MNV多蛋白切割位点会导致传染性病毒的产生。
Background: Noroviruses generate the viral replicase through polyprotein cleavage by a viral protease. Results: A cell-based FRET sensor reveals significant variations in substrate cleavage both within and between norovirus genogroups. Conclusion: Norovirus proteases from different genogroups vary in their substrate recognition. Significance: Understanding the cleavage preferences of viral proteases is critical for drug design and provides insights into the potential for generating human-murine norovirus chimeras. The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6–7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.