Quantitative Analysis of Influenza Virus RNP Interaction with RNA Cap Structures and Comparison to Human Cap Binding Protein eIF4E

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• 作者：Lisa Hooker ; Rachel Sully ; Balraj Handa ; Naomi Ono ; Hiroshi Koyano ; and Klaus Klumpp
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：May 27, 2003
• 年：2003
• 卷：42
• 期：20
• 页码：6234 - 6240
• 全文大小：190K
• 年卷期：v.42,no.20(May 27, 2003)
• ISSN：1520-4995

文摘

Influenza virus polymerase uses capped RNA primers for transcription initiation in infectedcells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursorsin the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonucleaseat a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNAoligonucleotides function as primers for transcription initiation. The viral cap binding site has previouslybeen mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established aquantitative assay system for the analysis of cap interaction with PB2 as part of the native, viralribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affectedby the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excessuncapped RNA, indicating that under the assay conditions, the majority of the binding energy wascontributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involvesynergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode ofinteraction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contributionfrom the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction,it was possible to identify novel cap analogues, which selectively interact with influenza virus, but nothuman cap binding protein.