Structural Basis of the Influenza A Virus RNA Polymerase PB2 RNA-binding Domain Containing the Pathogenicity-determinant Lysine 627 Residue
Structural Basis of the Influenza A Virus RNA Polymerase PB2 RNA-binding Domain Containing the Pathogenicity-determinant Lysine 627 Residue
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DOI：
10.1074/jbc.c800224200
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发表时间：
2009-03-13
影响因子：
4.8
通讯作者：
Tsuge, Hideaki
中科院分区：
文献类型：
--
作者：
Kuzuhara, Takashi;Kise, Daisuke;Tsuge, Hideaki
由于甲型流感病毒具有RNA基因组，其RNA依赖性RNA聚合酶（包括PA、PB 1和PB 2亚基）对于病毒转录和复制至关重要。RNA引物/启动子与聚合酶的结合是病毒转录的起始步骤。在我们目前的研究中，我们通过X射线晶体学揭示了PB 2的C-末端RNA结合结构域的2.7 A三级结构。该结构域包含PB 2的赖氨酸627，并且该残基与甲型流感病毒的高致病性和宿主范围限制相关。我们发现，从我们目前的分析，这种赖氨酸是位于一个独特的“phi”形结构，包括一个螺旋和一个环绕环内的PB 2结构域。通过静电分析，我们确定了一个高度碱性的沟沿着这个phi环，并发现赖氨酸627位于phi环。其中在位置627处谷氨酸被取代的PB 2结构域突变体显示出显著较低的RNA结合活性。这是第一个报告，以显示RNA结合活性和致病性决定因子赖氨酸627之间的关系。使用Matras程序进行蛋白质三维结构比较，我们进一步发现PB 2结构域中的螺旋束与激活剂1的螺旋束相似，激活剂1是DNA复制钳装载器（复制因子C）的40 kDa亚基，也是RNA结合蛋白。这表明，在甲型流感病毒转录和细胞DNA复制的基础上的RNA结合机制之间存在功能和结构关系。因此，我们目前的研究结果为开发靶向病毒RNA聚合酶的引物/启动子RNA结合的新药提供了重要的新信息。
Because the influenza A virus has an RNA genome, its RNA-dependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNAprimers/promoters to the polymerases is an initiation step in viral transcription. In our current study, we reveal the 2.7 A tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2, and this residue is associated with the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique "phi"-shaped structure consisting of a helix and an encircled loop within the PB2 domain. By electrostatic analysis, we identified a highly basic groove along with this phi loop and found that lysine 627 is located in the phi loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. This is the first report to show a relationship between RNA binding activity and the pathogenicity-determinant lysine 627. Using the Matras program for protein three-dimensional structural comparisons, we further found that the helix bundles in the PB2 domain are similar to that of activator 1, the 40-kDa subunit of DNA replication clamp loader (replication factor C), which is also an RNA-binding protein. This suggests a functional and structural relationship between the RNA-binding mechanisms underlying both influenza A viral transcription and cellular DNA replication. Our present results thus provide important new information for developing novel drugs that target the primer/promoter RNA binding of viral RNA polymerases.