Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis

The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during viral RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a structured viral RNA product and a deformed vRNP associated with a looped, double-stranded RNA, composed of a template vRNA and a nascent RNA. These results suggest that while some vRNPs keep their helical structures during viral RNA synthesis, probably for the repeated cycle of transcription and/or replication, others accidentally become structurally deformed, which probably results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural basis of viral RNA synthesis and advance our knowledge of the mechanism of viral transcription and replication.