Rotavirus mRNAS are released by transcript-specific channels in the double-layered viral capsid

Abstract

Rotaviruses are the single most common cause of fatal and severe childhood diarrheal illness worldwide (>125 million cases annually). Rotavirus shares structural and functional features with many viruses, such as the presence of segmented double-stranded RNA genomes selectively and tightly packed with a conserved number of transcription complexes in icosahedral capsids. Nascent transcripts exit the capsid through 12 channels, but it is unknown whether these channels specialize in specific transcripts or simply act as general exit conduits; a detailed description of this process is needed for understanding viral replication and genomic organization. To this end, we developed a single molecule assay for capturing and identifying transcripts extruded from transcriptionally active viral particles. Our findings support a model in which each channel specializes in extruding transcripts of a specific segment that in turn is linked to a single transcription complex. Our approach can be extended to study other viruses and transcription systems. ouble-stranded RNA (dsRNA) viruses comprise a wide variety of families that vary in genome complexity. These families include Reoviridae with 10-12 genomic segments; Crysoviridae with 4 segments; Cystoviridae with 3 segments; Birnaviridae, Picobirnaviridae, and Partiviridae with 2 segments; and Totiviridae with a single genomic dsRNA. They also vary in their ability to infect diverse hosts from bacteria to humans, yet they share unique features reflecting parallels in their replication; for the Reoviridae, such features include a multicomponent capsid that crosses the host cell membrane and transcription of their dsRNA segments by capsid-attached enzymes. During cell entry, the outer layers of these viruses are lost, while their inner capsids provide a compartment for genome segments (10-12 dsRNAs). Transcript export occurs via channels at the 12 vertices of an icosahedral capsid; although crucial for establishing infection (because the transcripts act as templates for both translation and genomic dsRNA synthesis), the mechanism of transcript export is unclear. In particular, it is unknown whether the nascent transcripts are selectively released through specialized channels, and if so, what the basis of selectivity is.To address these questions, we studied rotavirus, a major cause of gastroenteritis in infants and children worldwide (1, 2), and a member of the Reoviridae family, which includes many viruses of veterinary and biomedical importance (3). Rotaviruses deliver a 70-nm-diameter double-layered particle (DLP) to host cells following entry; the outer and inner protein layers package transcription complexes (TCs), proteins VP1 (RNA-dependent RNA polymerase) and VP3 (RNA capping enzyme), and 11 dsRNA genomic segments. The outer DLP layer is made of VP6 proteins (4) arranged as pentamers and hexamers forming 132 channels of three classes, including a class of 12 channels, each placed at the fivefold vertices of the icosahedral capsid. Underneath the VP6 layer is the single-lay...

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