Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle

Abstract

The viral RNA segments in influenza virions were shown to be circular in conformation by using psoralen crosslinking methods. Electron microscopy of purified RNA following treatment of virus with the psoralen reagent 4'-aminomethyltrioxsalen (AMT) revealed circles with lengths corresponding to the individual segments. RNA blot analysis using polyacrylamide gels demonstrated that RNA from AMT-treated virus had a slowed migration, consistent with it being a single-stranded circle. Furthermore, nuclease S1 protection assays indicated that the termini of the RNA segments form an approximately 15-base-pair-long panhandle. This structure is consistent with the partial sequence complementarity that has been observed for the termini of all influenza virus RNAs. By RNA blot analysis, circular structures of viral sense RNA were also found in influenza virus-infected cells at early and late time points. The circular RNA was the predominant species at the time when the major transcription product is message RNA. This finding and the observation that the termination signal for mRNA synthesis directly abuts the panhandle suggest that a panhandle in the template viral RNA is a cis regulatory signal promoting the synthesis of mRNA instead of plus-sense template. Also, since the panhandle is present in high concentration in virions, we suggest that it is required for packaging and that the input RNA after infection is in the proper conformation for synthesis of primary transcripts.