Structural and Functional Characterization of K339T Substitution Identified in the PB2 Subunit Cap-binding Pocket of Influenza A Virus
Structural and Functional Characterization of K339T Substitution Identified in the PB2 Subunit Cap-binding Pocket of Influenza A Virus
复制标题
甲型流感病毒 PB2 亚基帽结合口袋中鉴定出的 K339T 取代的结构和功能特征
DOI：
10.1074/jbc.m112.392878
复制
发表时间：
2013-04-19
影响因子：
4.8
通讯作者：
Zheng, Xiaofeng
中科院分区：
文献类型：
--
作者：
Liu, Yong;Qin, Kun;Zheng, Xiaofeng
Influenza virus RNA-dependent RNA polymerase is a heterotrimer composed of PA, PB1, and PB2 subunits. RNA-dependent RNA polymerase is required for both transcription and replication of influenza viral RNA taking place in the nucleus of infected cells. A "cap-snatching" mechanism is used to generate a 5'-capped primer for transcription in which the cap-binding domain of PB2 (PB2(cap)) captures the 5' cap of the host pre-mRNA. Our statistical analysis of PB2 sequences showed that residue Lys(339) located in the cap-binding pocket of H5N1 PB2(cap) was gradually replaced by Thr(339) over the past decade. To understand the role of this amino acid polymorphism, we solved the crystal structures of PB2(cap) with or without a pre-mRNA cap analog, m(7)GTP, in the presence of Lys(339) or Thr(339). The structures showed that Lys(339) contributes to binding the gamma-phosphate group of m(7)GTP, and the replacement of Lys(339) by Thr eliminates this interaction. Isothermal titration calorimetry analysis showed that Thr(339) attenuated the PB2(cap) cap binding activity in vitro compared with Lys(339). Further functional studies confirmed that Thr(339)-PB2-containing ribonucleoprotein complex has a reduced influenza polymerase activity and RNA synthesis activity, and a reconstituted H5N1 virus containing the Thr(339) substitution exhibited a lower virulence to mice but more active replication in Madin-Darby canine kidney cells. The K339T substitution in the cap-binding pocket of PB2 modulates the polymerase activity and virulence by regulating the cap binding activity. It is informative to track variations in the cap-binding pocket of PB2 in surveillance of the evolution and spread of influenza virus.
