An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15

Autor: Elizabeth A. Campbell, Uli Schmitz, John P. Bilello, Joy Y. Feng, Jason K. Perry, Todd C. Appleby
Rok vydání: 2021
Předmět:
Models
Molecular
Guanylyltransferase
Transcription
Genetic
MST
microscale thermophoresis
coronavirus
Viral Nonstructural Proteins
Random hexamer
Virus Replication
Biochemistry
Transcription (biology)
proofreading
TRS-B
body TRS
Polymerase
MTase
methyltransferase
Subgenomic mRNA
biology
Chemistry
EndoN
endonuclease
TRS-L
leader TRS
MD
molecular dynamics
CTD
C-terminal domain
Cell biology
Molecular Docking Simulation
nsp
nonstructural viral protein
Proofreading
NTPase
nucleoside triphosphatase
Dimerization
Research Article
Exonuclease
GTase
guanylyltransferase
RTC
replication-transcription complex
Endoribonucleases
Humans
TRS
transcription regulatory sequence
viral transcription
NTD
N-terminal domain
protein structure
Protein Structure
Quaternary
Molecular Biology
RNA
Double-Stranded
structure model
Binding Sites
SARS-CoV-2
molecular modeling
C-terminus
COVID-19
Helicase
Cell Biology
nsp15
Coding strand
Transcription preinitiation complex
Biophysics
biology.protein
viral replication
MHV
murine hepatitis virus
N
nucleocapsid
Zdroj: The Journal of Biological Chemistry
ISSN: 0021-9258
DOI: 10.1016/j.jbc.2021.101218
Popis: Using available cryo-EM and x-ray crystal structures of the nonstructural proteins that are responsible for SARS-CoV-2 viral RNA replication and transcription, we have constructed an atomistic model of how the proteins assemble into a functioning superstructure. Our principal finding is that the complex is hexameric, centered around nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, where nsp14 is seen to undergo a large conformational change between its two domains. This conformational change facilitates binding of six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Two of the six polymerase subunits are each proposed to carry dimers of nsp13, while two others are proposed to carry monomers. The polymerase subunits that coordinate nsp13 dimers also bind the nucleocapsid, which positions the 5’-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analyzing the path of the viral RNA indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping (the nsp12 NiRAN and the nsp14 and nsp16 methyltransferases). The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping and the role of the endonuclease. It provides a platform to guide biochemical and structural research to address the stoichiometric and spatial configuration of the replication-transcription complex.Author SummaryThe replication of the coronavirus genome and the synthesis of subgenomic mRNA is a complex process involving multiple viral proteins. Despite a fairly complete structural picture of the individual proteins that are believed to coalesce into a larger replication-transcription complex, there is no clear model of how these proteins interact. Here we present the first detailed atomistic model of a complete replication-transcription complex for SARS-CoV-2, made up of the non-structural proteins nsp7-nsp16, as well as the nucleocapsid. Forming a large, hexameric superstructure centered around nsp15, the model provides new perspective on the function of its individual components, including the exonuclease, the endonuclease, the NiRAN site, the helicase, the multiple zinc fingers, and the nucleocapsid. It offers a cohesive view of replication, proofreading, template switching and mRNA capping, which should serve as a guide for future experimental exploration.
Databáze: OpenAIRE
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