In Situ Structures of the Segmented Genome and RNA Polymerase Complex Inside a dsRNA Virus

Abstract

Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented dsRNA inside the viral capsid and carry out endogenous mRNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoEM and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the 10 segmented dsRNAs are organized with 10 TECs in a specific, non-symmetric manner, with a dsRNA segment attached directly to each TEC. TEC consists of two extensively-interacting subunits: an RNA-dependent RNA polymerase (RdRP) subunit and an NTPase subunit, VP4. By comparing the TEC structures in q-CPV and t-CPV, we find that the bracelet domain of RdRP undergoes significant conformational change, leading to formation of the RNA template entry channel and access to the polymerase active site. The N-terminal helix from each of the two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the missing link between sensing of external cues by the CSP and activation of endogenous RNA transcription by the TEC inside the virus.