|Entry||Database: PDB / ID: 6m99|
|Title||In situ structure of transcriptional enzyme complex and asymmetric inner capsid protein of aquareovirus at primed state|
|Keywords||VIRAL PROTEIN / Reovirus / transcriptional enzyme complex / polymerase / RdRp / NTPase|
|Function / homology||C2H2-type zinc finger / Reovirus RNA-dependent RNA polymerase lambda 3 / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / Reovirus RNA-dependent RNA polymerase lambda 3 / Reovirus minor core protein Mu-2 / Zinc finger C2H2-type / Reovirus minor core protein, Mu-2 / RNA-directed RNA polymerase, reovirus ...C2H2-type zinc finger / Reovirus RNA-dependent RNA polymerase lambda 3 / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / Reovirus RNA-dependent RNA polymerase lambda 3 / Reovirus minor core protein Mu-2 / Zinc finger C2H2-type / Reovirus minor core protein, Mu-2 / RNA-directed RNA polymerase, reovirus / viral genome replication / viral capsid / nucleic acid binding / viral nucleocapsid / RNA-directed 5'-3' RNA polymerase activity / structural molecule activity / RNA binding / Putative core protein NTPase/VP5 / VP3 / VP2|
Function and homology information
|Specimen source||Grass carp reovirus|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.4 Å resolution|
|Authors||Ding, K. / Zhou, Z.H.|
|Citation||Journal: J. Virol. / Year: 2018|
Title: Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating.
Authors: Ke Ding / Lisa Nguyen / Z Hong Zhou
Abstract: Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The structures of the transcriptional ...Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how the RNA genome and TEC respond to priming in reoviruses is unknown. Here, we determined the near-atomic-resolution asymmetric structure of ARV in the primed state by cryo-electron microscopy (cryo-EM), revealing the structures of 11 TECs inside each capsid and their interactions with the 11 surrounding double-stranded RNA (dsRNA) genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. The RdRp VP2 and the NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states showed translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel was open in both states, suggesting that channel blocking is not a regulating mechanism between these states in ARV. Instead, the NTPase C-terminal domain appears to regulate RNA translocation between the quiescent and primed states. Taking the data together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining similar mechanisms to coassemble their genome segments, TEC, and capsid proteins into infectious virions. Viruses in the family are characterized by the ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genomes with transcriptional enzymes into a multilayered virion and for priming such a virion for transcription are poorly understood. By cryo-EM and novel asymmetric reconstruction, we determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase. The NTPase has a newly discovered domain that translocates the 5' end of plus-sense RNA in segmented dsRNA genomes from the NTPase to polymerase VP2 when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.
SummaryFull reportAbout validation report
|Date||Deposition: Aug 23, 2018 / Release: Sep 5, 2018|
|Structure viewer||Molecule: |
Downloads & links
B: Putative core protein NTPase/VP5
|#1: Protein/peptide|| |
Mass: 141685.438 Da / Num. of mol.: 1 / Source: (natural) Grass carp reovirus / References: UniProt: Q9E3V9
|#2: Protein/peptide|| |
Mass: 80381.516 Da / Num. of mol.: 1 / Source: (natural) Grass carp reovirus / References: UniProt: Q8JU68
Mass: 132203.312 Da / Num. of mol.: 10 / Source: (natural) Grass carp reovirus / References: UniProt: Q9E3V8
|#4: Chemical|| ChemComp-PO4 / ||#5: Chemical|| ChemComp-ZN / |
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: transcriptional enzyme complex and asymmetric inner capsid protein|
Type: VIRUS / Entity ID: 1,
|Molecular weight||Experimental value: NO||Source (natural)||Organism: Grass carp reovirus||Details of virus||Empty: NO / Enveloped: NO / Virus isolate: STRAIN / Virus type: VIRION||Natural host||Organism: Ctenopharyngodon idella||Buffer solution||pH: 7.5||Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES||Specimen support||Grid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3||Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 25 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 73472 / Algorithm: FOURIER SPACE / Symmetry type: POINT|
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