Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit
Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit
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DOI：
10.1099/0022-1317-77-5-1025
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发表时间：
1996-05-01
影响因子：
3.8
通讯作者：
Digard, P
中科院分区：
文献类型：
--
作者：
Blok, V;Cianci, C;Digard, P
流感病毒RNA聚合酶由PB1、PB2和PA蛋白组成的异源三聚体复合体，其中PB2亚基负责识别宿主细胞RNA上的5‘帽结构，作为病毒mRNA合成的引物。为了进一步研究PB2在mRNA合成中的作用，我们测试了一组针对PB2蛋白特定区域的多克隆抗血清对病毒粒子转录酶的抑制能力。所有5份血清都具有足够的滴度来免疫沉淀PB2，4份血清能够识别含有PB1和PA的聚合酶复合体。然而，只有针对PB2(F5)羧基末端的血清才能显著抑制聚合酶活性。这种血清大大减少了由珠蛋白mRNA启动的合成，但仅部分抑制了由二核苷酸APG或APG和CAP类似物启动的转录。优先抑制珠蛋白诱导的合成并不是由于干扰帽识别，因为在光亲和帽结合试验中，血清F5不减少PB2的标记。然而，F5的Ig G和Fab片段对病毒粒子内切酶活性有抑制作用。这表明PB2的C末端在转录启动过程中起着关键作用，并与核酸内切酶活性有关。
The influenza virus RNA polymerase consists of a heterotrimeric complex of the PB1, PB2 and PA proteins, with the PB2 subunit responsible for recognizing 5' cap structures on the host cell RNAs used as primers for virus mRNA synthesis. To investigate further the role PB2 plays in mRNA synthesis, a set of polyclonal antisera raised against defined regions of the protein were tested for their ability to inhibit the virion transcriptase. All five sera were of sufficient titre to immunoprecipitate PB2 and four were capable of recognizing polymerase complexes containing PB1 and PA. However, only the serum raised against the carboxy terminus of PB2 (F5) substantially inhibited polymerase activity. This serum drastically reduced synthesis primed by globin mRNA, but only partially inhibited transcription primed by the dinucleotide ApG, or ApG and cap analogue. The preferential inhibition of globin-primed synthesis did not result from interference with cap recognition, as serum F5 did not reduce labelling of PB2 in a photoaffinity cap-binding assay. However, IgG and Fab fragments from F5 were found to inhibit virion endonuclease activity. This suggests that the C terminus of PB2 plays a crucial role in transcription initiation and implicates PB2 in endonuclease activity.