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- PDB-7eei: Structure of Rift Valley fever virus RNA-dependent RNA polymerase -

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Basic information

Entry
Database: PDB / ID: 7eei
TitleStructure of Rift Valley fever virus RNA-dependent RNA polymerase
ComponentsReplicaseRNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN / Polymerase / Complex / Replicate
Function / homology
Function and homology information


nucleoside binding / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription
Similarity search - Function
RNA-directed RNA polymerase, phlebovirus / RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
Biological speciesRift valley fever virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWang, X. / Hu, C.X.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: J Virol / Year: 2022
Title: Structure of Rift Valley Fever Virus RNA-Dependent RNA Polymerase.
Authors: Xue Wang / Cuixia Hu / Wei Ye / Jia Wang / Xiaofei Dong / Jie Xu / Xiaorong Li / Manfeng Zhang / Hongyun Lu / Fanglin Zhang / Wei Wu / Shaodong Dai / Hong-Wei Wang / Zhongzhou Chen /
Abstract: Rift Valley fever virus (RVFV) belongs to the order and is the type species of genus , which accounts for over 50% of family species. RVFV is mosquito-borne and causes severe diseases in both ...Rift Valley fever virus (RVFV) belongs to the order and is the type species of genus , which accounts for over 50% of family species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus , the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus . Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.
History
DepositionMar 18, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Mar 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Assembly

Deposited unit
A: Replicase


Theoretical massNumber of molelcules
Total (without water)202,2611
Polymers202,2611
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area54320 Å2

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Components

#1: Protein Replicase / RNA-dependent RNA polymerase / Transcriptase


Mass: 202260.734 Da / Num. of mol.: 1 / Mutation: K322S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rift valley fever virus / Production host: Pichia aff. alni PL5W1 (fungus) / References: UniProt: A2SZS3, RNA-directed RNA polymerase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structural insights into Rift Valley fever virus replication machinery
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.238 MDa / Experimental value: YES
Source (natural)Organism: Rift Valley fever virus
Source (recombinant)Organism: Pichia aff. alni PL5W1 (fungus)
Buffer solutionpH: 8.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 554102 / Symmetry type: POINT
RefinementHighest resolution: 3.6 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610424
ELECTRON MICROSCOPYf_angle_d1.07514156
ELECTRON MICROSCOPYf_dihedral_angle_d20.0493572
ELECTRON MICROSCOPYf_chiral_restr0.0551615
ELECTRON MICROSCOPYf_plane_restr0.0071817

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