Cryo-EM structure of a transcribing cypovirus

Abstract

Double-stranded RNA viruses in the family Reoviridae are capable of transcribing and capping nascent mRNA within an icosahedral viral capsid that remains intact throughout repeated transcription cycles. However, how the highly coordinated mRNA transcription and capping process is facilitated by viral capsid proteins is still unknown. Cypovirus provides a good model system for studying the mRNA transcription and capping mechanism of viruses in the family Reoviridae . Here, we report a full backbone model of a transcribing cypovirus built from a near-atomic-resolution density map by cryoelectron microscopy. Compared with the structure of a nontranscribing cypovirus, the major capsid proteins of transcribing cypovirus undergo a series of conformational changes, giving rise to structural changes in the capsid shell: ( i ) an enlarged capsid chamber, which provides genomic RNA with more flexibility to move within the densely packed capsid, and ( ii ) a widened peripentonal channel in the capsid shell, which we confirmed to be a pathway for nascent mRNA. A rod-like structure attributable to a partially resolved nascent mRNA was observed in this channel. In addition, conformational change in the turret protein results in a relatively open turret at each fivefold axis. A GMP moiety, which is transferred to 5’-diphosphorylated mRNA during the mRNA capping reaction, was identified in the pocket-like guanylyltransferase domain of the turret protein.

In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus

“…3f, Extended Data Figs 9, 10f-k). Consistent with previous icosahedral reconstructions, our asymmetric reconstructions show that the capsid shell of t-CPV expands outwards from q-CPV, with the maximal (~10 Å) expansion occurring at the vertex region 20,21 , to which Module A of the bracelet domain is attached (Fig. 4e,f).…”

A Non-Symmetric Reconstruction Technique for Transcriptionally-Active Viral Assemblies

“…In comparing our results to the high-resolution structures of the transcriptionally-active subviral particle of cypo virus, a member of the same viral family as RV (i.e., Reoviridae), we found an essential commonality-a series of conformational changes must occur in the viral capsid during mRNA synthesis, capping, and egress [14]. It remains unknown whether this process is being driven by the structural mobility of the internal RNA genome or by fluid protein rearrangements in response to the changing RNA landscape during transcription.…”

Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases

“…Analysis of the three-dimensional structure of BmCPV by cryoelectron microscopy (cryo-EM) revealed that mRNA capping occurred in the enzymic domains of the pentameric turret whose five unique channels guide nascent mRNA sequentially to GTase, N structural comparison via cryo-EM of transcribing and non-transcribing BmCPV showed that transfer of a GMP moiety occurred to the 59 end of the diphosphate-ended RNA after its binding to Lys234 of the GTase pocket via a phosphoamide linkage (Yang et al, 2012) and then the RNA capping reaction occurred in the active sites of different turret protein monomers (Zhu et al, 2014) In the case of viruses lacking a pentameric turret, core protein VP3 (in the case of rotavirus) or VP4 [in the case of bluetongue virus (BTV)] provides both GTase and MTase activity for capping the 59 end of viral RNA (Trask et al, 2012). In many viruses, GTase activity has been reported to be associated with RTPase activity, such as mammalian reoviral m2 (Kim et al, 2004;Noble & Nibert, 1997b) and l1 proteins Noble & Nibert, 1997a), avian reoviral mA protein (Su et al, 2007), rotaviral NSP2 protein (Vasquez-Del Carpio et al, 2006, dengue viral NS3 protein (Bartelma & Padmanabhan, 2002), aquareoviral VP5 protein (Attoui et al, 2002) and alfavirus NSP2 protein (Vesiljeva et al, 2000), which not only has RTPase activity but also GTase activity.…”