NMR studies of the panhandle structure of the influenza virus RNA

Abstract

The influenza A viruses which is the most severe and common among the influenza viruses have highly conserved 3`` and 5`` terminal sequences of genomic RNA called panhandle. The conserved panhandle RNA linked by UUCG tetraloop was studied by using NMR spectroscopy. The panhandle is partially double-stranded and involved in initiation and termination of transcription, switching from transcription to replication as well as packaging. The panhandle structure was studied by NMR and molecular modeling by Cheong et al. but the conserved sequences were modified and the results were argued against. This study was initiated to certify this point by using the unmodified RNA which was available by the trans cleaving hammerhead ribozyme. Overall conformation looks like that of Cheong et al. U3AG/U27UG(I) and A7ACAA/U22GCU(II) have been known to important binding sites of the influenza RNA polymerase. In region (I), U3:G29, A4:U28, G5:U27 are base paired, and the base pair steps seem to deviate slightly from the canonical A form helix. In region (II), there seem to be no base pairs or very weak base pairs, which cannot be identified. All base pairs except UUCG tetraloop stem melt between 30 and 35 degree in temperature varying experiment implying that the both region (I) and (II) are single-stranded in physiological conditions which were proposed by the fork model. However, since the influenza virus RNA polymerase would stabilize both regions, the in vivo panhandle structure might be different from the in vitro case so it needs further investigation at the stage of polymerase binding.