NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, PR China.
Swiss Light Source at the Paul Scherrer Institute, CH-5232 Villigen, Switzerland.
Nucleic Acids Res. 2022 Sep 9;50(16):9470-9489. doi: 10.1093/nar/gkac671.
The HAV nonstructural protein 2C is essential for virus replication; however, its precise function remains elusive. Although HAV 2C shares 24-27% sequence identity with other 2Cs, key motifs are conserved. Here, we demonstrate that HAV 2C is an ATPase but lacking helicase activity. We identified an ATPase-independent nuclease activity of HAV 2C with a preference for polyuridylic single-stranded RNAs. We determined the crystal structure of an HAV 2C fragment to 2.2 Å resolution, containing an ATPase domain, a region equivalent to enterovirus 2C zinc-finger (ZFER) and a C-terminal amphipathic helix (PBD). The PBD of HAV 2C occupies a hydrophobic pocket (Pocket) in the adjacent 2C, and we show the PBD-Pocket interaction is vital for 2C functions. We identified acidic residues that are essential for the ribonuclease activity and demonstrated mutations at these sites abrogate virus replication. We built a hexameric-ring model of HAV 2C, revealing the ribonuclease-essential residues clustering around the central pore of the ring, whereas the ATPase active sites line up at the gaps between adjacent 2Cs. Finally, we show the ribonuclease activity is shared by other picornavirus 2Cs. Our findings identified a previously unfound activity of picornavirus 2C, providing novel insights into the mechanisms of virus replication.
甲型肝炎病毒非结构蛋白 2C 对于病毒复制是必需的；然而，其确切功能仍不清楚。尽管 HAV 2C 与其他 2C 共享 24-27%的序列同一性，但关键基序是保守的。在这里，我们证明 HAV 2C 是一种 ATP 酶，但缺乏解旋酶活性。我们发现 HAV 2C 具有依赖于 ATP 的核酸酶活性，对多聚尿嘧啶单链 RNA 具有偏好性。我们确定了 HAV 2C 片段的晶体结构，分辨率为 2.2 Å，包含一个 ATP 酶结构域、一个与肠道病毒 2C 锌指 (ZFER) 等效的区域和一个 C 末端两性螺旋 (PBD)。HAV 2C 的 PBD 占据相邻 2C 中的一个疏水口袋 (Pocket)，我们表明 PBD-Pocket 相互作用对于 2C 功能至关重要。我们确定了对核糖核酸酶活性至关重要的酸性残基，并证明这些位点的突变会导致病毒复制的丧失。我们构建了 HAV 2C 的六聚体环模型，揭示了核糖核酸酶必需的残基聚集在环的中心孔周围，而 ATP 酶活性位点则排列在相邻 2C 之间的间隙中。最后，我们表明其他小核糖核酸病毒 2C 具有核糖核酸酶活性。我们的发现鉴定了小核糖核酸病毒 2C 的一种以前未发现的活性，为病毒复制的机制提供了新的见解。
