Influenza virus polymerase uses capped
RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped
mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated
endonuclease at a position 10-15
nucleotides downstream of the cap structure. The resulting capped
RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric
influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral
ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the
RNase pretreatment of the capped
RNA substrate and cap binding was not inhibited by excess uncapped
RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to
7-methyl-GTP was found to involve synergistic interaction with 7-methyl
guanosine and
triphosphate binding subsites. A similar mode of interaction with
7-methyl-GTP was found for
human cap binding protein eIF4E. However, the potency of
7-methyl-GTP for cap binding inhibition was 200-fold stronger with
eIF4E and had a higher contribution from 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 not
human cap binding protein.