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- PDB-9bi9: GII.4 Sydney 2012 Polymerase domain of ProPol precursor -

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Basic information

Entry
Database: PDB / ID: 9bi9
TitleGII.4 Sydney 2012 Polymerase domain of ProPol precursor
ComponentsRNA-dependent RNA polymerase domain of Protease-Polymerase precursor
KeywordsVIRAL PROTEIN / RNA-dependent RNA polymerase / RdRp / Norovirus / GII.4 HuNV / ProPol / Protease-Polymerase
Function / homology
Function and homology information


host cell / RNA helicase activity / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding
Similarity search - Function
Viral polyprotein, Caliciviridae N-terminal / Viral polyprotein N-terminal / Norovirus 3C-like protease (NV 3CLpro) domain profile. / Norovirus peptidase C37 / Southampton virus-type processing peptidase / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / RNA-directed RNA polymerase, C-terminal domain ...Viral polyprotein, Caliciviridae N-terminal / Viral polyprotein N-terminal / Norovirus 3C-like protease (NV 3CLpro) domain profile. / Norovirus peptidase C37 / Southampton virus-type processing peptidase / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesNorovirus Hu/GII.4/Sydney/NSW0514/2012/AU
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsEruera, A. / Krause, K.
Funding support New Zealand, 1items
OrganizationGrant numberCountry
Royal Society of New Zealand21-UOO-003-CSG New Zealand
CitationJournal: J Virol / Year: 2024
Title: Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.
Authors: Alice M McSweeney / Alice-Roza Eruera / Geena M McKenzie-Goldsmith / James C Bouwer / Simon H J Brown / Louise A Stubbing / Jonathan G Hubert / Rinu Shrestha / Kevin J Sparrow / Margaret A ...Authors: Alice M McSweeney / Alice-Roza Eruera / Geena M McKenzie-Goldsmith / James C Bouwer / Simon H J Brown / Louise A Stubbing / Jonathan G Hubert / Rinu Shrestha / Kevin J Sparrow / Margaret A Brimble / Lawrence D Harris / Gary B Evans / Mihnea Bostina / Kurt L Krause / Vernon K Ward /
Abstract: Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor ...Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol enzymatic efficiencies (/) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC) values of 247.5 µM and 3.8 µM. In both instances, the IC obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.
History
DepositionApr 23, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA-dependent RNA polymerase domain of Protease-Polymerase precursor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,1612
Polymers78,1371
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein RNA-dependent RNA polymerase domain of Protease-Polymerase precursor


Mass: 78137.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Norovirus Hu/GII.4/Sydney/NSW0514/2012/AU
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: K4L8Z7
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Mg
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Protease-Polymerase precursor protein with N-terminal His6-tag.
Type: COMPLEX
Details: ProPol expressed as a fusion protein with N-terminal His6 tag and non-cleavage mutation at the Pro/Pol linker.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Norovirus / Strain: Norovirus Hu/GII.4/Sydney/NSW0514/2012/AU
Source (recombinant)Organism: unidentified baculovirus / Cell: Trichoplusia ni / Plasmid: pacpol-
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRION
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2PHENIX1.19.1_4122:model refinement
3EPUimage acquisition
CTF correctionType: NONE
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 997801 / Symmetry type: POINT

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