1Department of Biotechnology and Bioinformatics, Korea University, Sejong 339-700, Republic of Korea.
†Present address: Konkuk University, Seoul, Republic of Korea.
J Gen Virol. 2018 Nov;99(11):1482-1493. doi: 10.1099/jgv.0.001151. Epub 2018 Sep 28.
Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg-dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg or Mn in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.
RNA 结构的调节在 RNA 病毒的生命周期中至关重要。感染后立即进行复制需要 RNA 解链，以确保 RNA 模板不会处于分子内或分子间双链形式。杯状病毒 NS3 是高度保守的非结构 (NS) 蛋白之一，在六个基因群中具有与螺旋酶超家族 3 共同的保守基序。然而，其生物学功能尚未完全阐明。在这项研究中，我们报告了来自鼠诺如病毒 (MNV) 的重组全长 NS3 的寡聚状态以及核苷酸三磷酸酶 (NTPase) 和 RNA 分子伴侣活性。MNV NS3 具有依赖 Mg 的 NTPase 活性，并且保守 NTPase 基序的定点突变阻断了酶活性和细胞中的病毒复制。此外，通过荧光共振能量转移 (FRET) 基测定法发现 NS3 以 NTP 独立的方式在存在 Mg 或 Mn 的情况下使双链 RNA 不稳定。然而，RNA 不稳定活性不受 NTPase 保守基序突变的影响。这些结果表明，MNV NS3 具有 NTPase 非依赖性的 RNA 分子伴侣样活性，并且基于 FRET 的 RNA 失稳测定法有可能鉴定出针对 NS3 的新型抗病毒药物。
