Journal: 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.
Entire : Protease-Polymerase precursor protein with N-terminal His6-tag.
Entire
Name: Protease-Polymerase precursor protein with N-terminal His6-tag.
Components
Complex: Protease-Polymerase precursor protein with N-terminal His6-tag.
Protein or peptide: RNA-dependent RNA polymerase domain of Protease-Polymerase precursor
Ligand: MAGNESIUM ION
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Supramolecule #1: Protease-Polymerase precursor protein with N-terminal His6-tag.
Supramolecule
Name: Protease-Polymerase precursor protein with N-terminal His6-tag. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: ProPol expressed as a fusion protein with N-terminal His6 tag and non-cleavage mutation at the Pro/Pol linker.
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Basic information
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Sample
Keywords
Function and homology information
Norovirus / 
Authors
New Zealand, 1 items 
Citation
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emd_44577.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-44577
Z (Sec.)
Y (Row.)
X (Col.)
Sample components
Trichoplusia ni (cabbage looper)
Processing
Electron microscopy
FIELD EMISSION GUN
