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

2021 ◽  
Author(s):  
Jason K Perry ◽  
Todd C Appleby ◽  
John P Bilello ◽  
Joy Y Feng ◽  
Uli C Schmitz ◽  
...  
Keyword(s):  
Conformational Change ◽  
Spatial Configuration ◽  
Viral Rna ◽  
Template Switching ◽  
Atomistic Model ◽  
Nonstructural Proteins ◽  
Template Strand ◽  
Mrna Capping ◽  
Large Conformational Change ◽  
Principal Finding

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.

scholarly journals X-Ray Crystallography Reveals a Large Conformational Change during Guanyl Transfer by mRNA Capping Enzymes

Cell ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 545-553 ◽  
Author(s):  
Kjell Håkansson ◽  
Aidan J. Doherty ◽  
Stewart Shuman ◽  
Dale B. Wigley
Keyword(s):  
Conformational Change ◽  
X Ray ◽  
X Ray Crystallography ◽  
Mrna Capping ◽  
Large Conformational Change ◽  
Capping Enzymes

scholarly journals ORF1a-Encoded Replicase Subunits Are Involved in the Membrane Association of the Arterivirus Replication Complex

1998 ◽  
Vol 72 (8) ◽  
pp. 6689-6698 ◽  
Author(s):  
Yvonne van der Meer ◽  
Hans van Tol ◽  
Jacomine Krijnse Locker ◽  
Eric J. Snijder
Keyword(s):  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Membrane Association ◽  
Nonstructural Proteins ◽  
Reading Frame ◽  
Replication Complex ◽  
Viral Proteases ◽  
Double Label ◽  
Membrane Bound ◽  
Triton X

ABSTRACT Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.

scholarly journals Light and heat control over secondary structure and amyloid-like fiber formation in an overcrowded-alkene-modified Trp zipper

2015 ◽  
Vol 6 (12) ◽  
pp. 7311-7318 ◽  
Author(s):  
Claudia Poloni ◽  
Marc C. A. Stuart ◽  
Pieter van der Meulen ◽  
Wiktor Szymanski ◽  
Ben L. Feringa
Keyword(s):  
Secondary Structure ◽  
Conformational Change ◽  
Fiber Formation ◽  
Major Influence ◽  
Heat Control ◽  
Large Conformational Change

The use of an overcrowded alkene photoswitch to control a model β-hairpin peptide is described. The light-induced, large conformational change has major influence on the secondary structure and the aggregation of the peptide, permitting the triggered formation of amyloid-like fibrils.

scholarly journals A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)

2018 ◽  
Vol 29 (9) ◽  
pp. 1060-1074 ◽  
Author(s):  
Tomohiro Kubo ◽  
Yuqing Hou ◽  
Deborah A. Cochran ◽  
George B. Witman ◽  
Toshiyuki Oda
Keyword(s):  
Conformational Change ◽  
Molecular Mechanism ◽  
Wild Type ◽  
Sliding Direction ◽  
Large Conformational Change ◽  
Dynein Arms ◽  
Biochemical Analyses

Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.

scholarly journals Requirement of RAD52 Group Genes for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (21) ◽  
pp. 7758-7764 ◽  
Author(s):  
Venkateswarlu Gangavarapu ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Template Switching ◽  
Template Strand ◽  
Fork Regression ◽  
Strand Annealing ◽  
Almost All ◽  
Annealing Mode ◽  
Damaged Dna

ABSTRACT In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases η and ζ and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.

scholarly journals Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus Type 39 Growth in Mouse Cells

2005 ◽  
Vol 79 (9) ◽  
pp. 5363-5373 ◽  
Author(s):  
Julie R. Harris ◽  
Vincent R. Racaniello
Keyword(s):  
Amino Acid ◽  
Viral Replication ◽  
Host Cell ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Viral Receptor ◽  
Viral Growth ◽  
L Cells ◽  
Cell Components ◽  
Mouse Cells

ABSTRACT Many steps of viral replication are dependent on the interaction of viral proteins with host cell components. To identify rhinovirus proteins involved in such interactions, human rhinovirus 39 (HRV39), a virus unable to replicate in mouse cells, was adapted to efficient growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L cells). Amino acid changes were identified in the 2B and 3A proteins of the adapted virus, RV39/L. Changes in 2B were sufficient to permit viral growth in mouse cells; however, changes in both 2B and 3A were required for maximal viral RNA synthesis in mouse cells. Examination of infected HeLa cells by electron microscopy demonstrated that human rhinoviruses induced the formation of cytoplasmic membranous vesicles, similar to those observed in cells infected with other picornaviruses. Vesicles were also observed in the cytoplasm of HRV39-infected mouse cells despite the absence of viral RNA replication. Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A is known to be required for formation of membranous vesicles. We suggest that productive HRV39 infection is blocked in ICAM-L cells at a step posttranslation and prior to the formation of a functional replication complex. The observation that changes in HRV39 2B and 3A proteins lead to viral growth in mouse cells suggests that one or both of these proteins interact with host cell proteins to promote viral replication.

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