Comparative Structural Analysis of Transcriptionally Competent and Incompetent Rotavirus-antibody Complexes

During genome transcription in rotavirus, as with many segmented double-stranded RNA viruses, mRNA is transcribed within the intact subviral particle and translocated through specific channels in the capsid. To understand how the conformation of the capsid affects the efficiency of transcriptional events in the viral core, we carried out a series of comparative structural and biochemical studies to characterize four different structural forms of the virus exhibiting differing transcriptional behavior. Two of these were virus-antibody complexes having contrasting transcriptional capabilities, and two were variant structural forms of the virus that exist during the life cycle and also exhibit contrasting transcriptional behavior. Three-dimensional structural studies using electron cryomicroscopy showed that the binding of one Fab (8H2/G5) does not affect the conformation of the capsid, and the efficiency of mRNA production is similar to that of the native subviral particle. The other Fab (2A11/E9) introduces conformational changes in the capsid similar to those seen in the transcriptionally incompetent mature particle. In both of the transcriptionally incompetent particle types, mRNA synthesis was arrested after limited elongation with the resulting oligonucleotide transcripts remaining trapped inside the particles. Our results indicate that the continuous translocation of nascent mRNA through the capsid is critical for efficient transcript elongation and that the blockage of translocation causes premature termination of transcription.