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

Abstract

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.

The Splicing Factor Proline-Glutamine Rich (SFPQ/PSF) Is Involved in Influenza Virus Transcription

“…The apparent Kd for the interaction of the isolated PB2 cap-binding domain with 7mGTP is around 170 µM [10], in good agreement with the inhibition data reported for cap-RNA crosslinking to influenza virus RNPs [59], whereas the affinity of binding of eIF4E or CBC to cap-analogues is much higher [60], [61]. This is in contradistinction to the elution profiles of eIF4F and influenza polymerase-template complexes on a 7mGTP-Sepharose resin [14] which show a stronger binding of the latter.…”

Preclinical Activity of VX-787, a First-in-Class, Orally Bioavailable Inhibitor of the Influenza Virus Polymerase PB2 Subunit

“…Other investigators have examined PB2 as a target (47,48), but no cellular or animal antiviral activity was reported to our knowledge. We initiated a search for new anti-influenza agents using a phenotypic-based approach (28), aware that this represented a high bar for screening because influenza produces a rapid and clear CPE followed by cell death in vitro (49)(50)(51)(52)(53).…”

“…These differences could explain the lack of activity of VX-787 against influenza B virus. There is significant amino acid divergence between influenza A virus PB2 and the other characterized eukaryotic host cell cap-binding proteins eIF4E, nuclear cap-binding complex protein CBC, or vaccinia virus VP39 (47), reducing the likelihood of undesired effects in uninfected host cells.…”

Analysis of Flavivirus NS5 Methyltransferase Cap Binding

“…The flavivirus MTase is a new and potentially valuable antiviral target to develop drugs against flavivirus replication. Drugs that interfere with influenza virus RNA cap binding have been previously described 29, indicating that viral cap binding is a viable drug target. Our increased understanding of how the flavivirus MTase binds RNA cap structures will aid in the rational development of highly potent and specific drugs for the treatment of flavivirus infections.…”