Abstract
The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein–protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit 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. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5′-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex 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 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.
Highlights
Refinement was an iterative process within the Schrödinger suite that involved sidechain and loop conformation optimization through Prime [70], minimization through Prime and Macromodel [71], and additional conformational sampling through Macromodel
3) Nsp15 EndoN is proposed to act on the template strand, likely near the 50 terminus
Its preference for uridine suggests it acts on the 50 tail of the negative strand once positive-strand synthesis is complete
Summary
Refinement was an iterative process within the Schrödinger suite that involved sidechain and loop conformation optimization through Prime [70], minimization through Prime and Macromodel [71], and additional conformational sampling through Macromodel. The forcefield employed was OPLS4 [72]. Optimization was initially limited to the residues that form the protein–protein interface of interest and was generally carried out with Prime. Once this step was completed, the “ligand” protein was allowed to fully relax. Once the initial model was constructed, all proteins were replaced with their original PDB structures and reoptimized. Primarily in the Nterm and C-term tails, were added where necessar
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