State Key Lab of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.
J Biol Chem. 2013 Apr 19;288(16):11013-23. doi: 10.1074/jbc.M112.392878. Epub 2013 Feb 22.
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 (PB2cap) 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 PB2cap 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 PB2cap 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 γ-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 PB2cap 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.
流感病毒 RNA 依赖性 RNA 聚合酶是由 PA、PB1 和 PB2 亚基组成的异三聚体。RNA 依赖性 RNA 聚合酶是在感染细胞的细胞核中进行流感病毒 RNA 的转录和复制所必需的。一种“帽子抢夺”机制被用于产生用于转录的 5'-加帽引物，其中 PB2（PB2cap）的帽子结合结构域捕获宿主前 mRNA 的 5'帽。我们对 PB2 序列的统计分析表明，位于 H5N1 PB2cap 帽子结合口袋中的残基 Lys（339）在过去十年中逐渐被 Thr（339）取代。为了了解该氨基酸多态性的作用，我们在存在 Lys（339）或 Thr（339）的情况下，分别解析了 PB2cap 与或不与 pre-mRNA 帽类似物 m(7)GTP 结合的晶体结构。结构表明 Lys（339）有助于结合 m(7)GTP 的 γ-磷酸基团，而 Lys（339）被 Thr（339）取代消除了这种相互作用。等温滴定量热法分析表明，与 Lys（339）相比， Thr（339）在体外削弱了 PB2cap 的帽子结合活性。进一步的功能研究证实，含有 Thr（339）的 PB2 核糖核蛋白复合物的流感聚合酶活性和 RNA 合成活性降低，并且含有 Thr（339）取代的重配 H5N1 病毒对小鼠的毒力降低，但在 Madin-Darby 犬肾细胞中的复制更活跃。PB2 帽子结合口袋中的 K339T 取代通过调节帽子结合活性来调节聚合酶活性和毒力。跟踪 PB2 帽子结合口袋中的变异情况对于监测流感病毒的进化和传播具有重要意义。
