scholarly journals The nucleotide addition cycle of the SARS-CoV-2 polymerase

2021 ◽  
Author(s):  
Subhas Chandra Bera ◽  
Mona Seifert ◽  
Robert N. Kirchdoerfer ◽  
Pauline van Nies ◽  
Yibulayin Wubulikasimu ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Polymerase ◽  
Human Health ◽  
High Throughput ◽  
Magnetic Tweezers ◽  
Power Stroke ◽  
Rna Dependent Rna Polymerase ◽  
The Core ◽  
Nucleotide Addition ◽  
Important Structure

AbstractCoronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

scholarly journals Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

Cell Reports ◽  
2017 ◽  
Vol 21 (4) ◽  
pp. 1063-1076 ◽  
Author(s):  
David Dulin ◽  
Jamie J. Arnold ◽  
Theo van Laar ◽  
Hyung-Suk Oh ◽  
Cheri Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput ◽  
Magnetic Tweezers ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analog

scholarly journals High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

2010 ◽  
Vol 107 (45) ◽  
pp. 19151-19156 ◽  
Author(s):  
C.-Y. Su ◽  
T.-J. R. Cheng ◽  
M.-I. Lin ◽  
S.-Y. Wang ◽  
W.-I. Huang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput ◽  
Polymerase Activity ◽  
Rna Dependent Rna Polymerase ◽  
Rna Polymerase Activity

scholarly journals Indels in SARS-CoV-2 occur at template-switching hotspots

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Brianna Sierra Chrisman ◽  
Kelley Paskov ◽  
Nate. Stockham ◽  
Kevin Tabatabaei ◽  
Jae-Yoon Jung ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Polymerase ◽  
Evolutionary Dynamics ◽  
De Novo ◽  
Template Switching ◽  
Nucleotide Polymorphisms ◽  
Rna Dependent Rna Polymerase ◽  
Consensus Sequences ◽  
Insertions And Deletions

AbstractThe evolutionary dynamics of SARS-CoV-2 have been carefully monitored since the COVID-19 pandemic began in December 2019. However, analysis has focused primarily on single nucleotide polymorphisms and largely ignored the role of insertions and deletions (indels) as well as recombination in SARS-CoV-2 evolution. Using sequences from the GISAID database, we catalogue over 100 insertions and deletions in the SARS-CoV-2 consensus sequences. We hypothesize that these indels are artifacts of recombination events between SARS-CoV-2 replicates whereby RNA-dependent RNA polymerase (RdRp) re-associates with a homologous template at a different loci (“imperfect homologous recombination”). We provide several independent pieces of evidence that suggest this. (1) The indels from the GISAID consensus sequences are clustered at specific regions of the genome. (2) These regions are also enriched for 5’ and 3’ breakpoints in the transcription regulatory site (TRS) independent transcriptome, presumably sites of RNA-dependent RNA polymerase (RdRp) template-switching. (3) Within raw reads, these indel hotspots have cases of both high intra-host heterogeneity and intra-host homogeneity, suggesting that these indels are both consequences of de novo recombination events within a host and artifacts of previous recombination. We briefly analyze the indels in the context of RNA secondary structure, noting that indels preferentially occur in “arms” and loop structures of the predicted folded RNA, suggesting that secondary structure may be a mechanism for TRS-independent template-switching in SARS-CoV-2 or other coronaviruses. These insights into the relationship between structural variation and recombination in SARS-CoV-2 can improve our reconstructions of the SARS-CoV-2 evolutionary history as well as our understanding of the process of RdRp template-switching in RNA viruses.

scholarly journals Working Mechanism of Remdesivir and Discovering New Potential Inhibitors to SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp) using High-Throughput Virtual Screening and Molecular Dynamics Simulations .

2021 ◽  
Author(s):  
Dylan Brunt ◽  
Phillip Lakernick ◽  
CHUN WU
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Virtual Screening ◽  
Rna Polymerase ◽  
High Throughput ◽  
Binding Free Energy ◽  
Free Energy Calculations ◽  
Rna Dependent Rna Polymerase ◽  
Zinc Database ◽  
High Throughput Virtual Screening

Abstract RNA-dependent RNA polymerase (RdRp), is an enzyme essential component in the RNA replication within the life cycle of the severely acute respiratory coronavirus-2 (SARS-CoV-2), causing the deadly respiratory induced sickness COVID-19. Remdesivir is a prodrug that has seen some success in inhibiting this enzyme, however there is still the pressing need for effective alternatives. In this study, we present the discovery of four non-nucleoside small molecules that bind favorably to RdRp over adenosine-triphosphate (ATP) and active-form remdesivir-triphosphate (RTP) using high-throughput virtual screening (HTVS) coupled with extensive (total 4800 ns) molecular dynamics (MD) simulations with using the ZINC compounds database against SARS-CoV-2 RdRp (PDB: 7BV2). We found that the simulations with both ATP and RTP remained stable for the duration of their trajectories, and it was revealed that the phosphate tail of RTP was stabilized by a positive amino acid pocket near the entry channel of RTP and magnesium ions containing residues K551, R553, R555 and K621. It was also found that residues D623, D760, and N691 further stabilized the ribose portion of RTP with U10 on the template RNA strand forming hydrogen pairs with the adenosine motif. Using these models of RdRp, we employed them to screen the ZINC database of ~17 million molecules. Using docking and drug properties scoring, we narrowed down our selection to fourteen candidates. These were subjected to 200 ns simulations each underwent free energy calculations. We identified four hit compounds from the ZINC database that have similar binding poses to RTP while possessing lower overall binding free energies, with ZINC097971592 having a binding free energy two times lower than RTP.

scholarly journals Potential Novel Thioether-Amide or Guanidine-Linker Class of SARS-CoV-2 Virus RNA-Dependent RNA Polymerase Inhibitors Identified by High-Throughput Virtual Screening Coupled to Free-Energy Calculations

2021 ◽  
Vol 22 (20) ◽  
pp. 11143
Author(s):  
Marko Jukič ◽  
Dušanka Janežič ◽  
Urban Bren
Keyword(s):  
Virtual Screening ◽  
Rna Polymerase ◽  
High Throughput ◽  
Biological Evaluation ◽  
Free Energy Calculations ◽  
Rna Dependent Rna Polymerase ◽  
Linear Interaction ◽  
Energy Calculations ◽  
High Throughput Virtual Screening

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new pathogen from the family of Coronaviridae that caused a global pandemic of COVID-19 disease. In the absence of effective antiviral drugs, research of novel therapeutic targets such as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) becomes essential. This viral protein is without a human counterpart and thus represents a unique prospective drug target. However, in vitro biological evaluation testing on RdRp remains difficult and is not widely available. Therefore, we prepared a database of commercial small-molecule compounds and performed an in silico high-throughput virtual screening on the active site of the SARS-CoV-2 RdRp using ensemble docking. We identified a novel thioether-amide or guanidine-linker class of potential RdRp inhibitors and calculated favorable binding free energies of representative hits by molecular dynamics simulations coupled with Linear Interaction Energy calculations. This innovative procedure maximized the respective phase-space sampling and yielded non-covalent inhibitors representing small optimizable molecules that are synthetically readily accessible, commercially available as well as suitable for further biological evaluation and mode of action studies.

scholarly journals Crystal Structure and Thermostability Characterization of Enterovirus D68 3Dpol

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Chunnian Wang ◽  
Caiyan Wang ◽  
Qing Li ◽  
Zhong Wang ◽  
Wei Xie
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Critical Role ◽  
Limited Proteolysis ◽  
Core Structure ◽  
Rna Dependent Rna Polymerase ◽  
Viral Pathogen ◽  
The Core ◽  
Number Of Patients ◽  
Enterovirus D68

ABSTRACT Enterovirus D68 (EV-D68) is one of the many nonpolio enteroviruses that cause mild to severe respiratory illness. The nonstructural protein 3Dpol is an RNA-dependent RNA polymerase (RdRP) of EV-D68 which plays a critical role in the replication of the viral genome and represents a promising drug target. Here, we report the first three-dimensional crystal structure of the RdRP from EV-D68 in complex with the substrate GTP to 2.3-Å resolution. The RdRP structure is similar to structures of other viral RdRPs, where the three domains, termed the palm, fingers, and thumb, form a structure resembling a cupped right hand. Particularly, an N-terminal fragment (Gly1 to Phe30) bridges the fingers and the thumb domains, which accounts for the enhanced stability of the full-length enzyme over the truncation mutant, as assessed by our thermal shift assays and the dynamic light scattering studies. Additionally, the GTP molecule bound proximal to the active site interacts with both the palm and fingers domains to stabilize the core structure of 3Dpol. Interestingly, using limited proteolysis assays, we found that different nucleoside triphosphates (NTPs) stabilize the polymerase structure by various degrees, with GTP and CTP being the most and least stabilizing nucleosides, respectively. Lastly, we derived a model of the core structure of 3Dpol stabilized by GTP, according to our proteolytic studies. The biochemical and biophysical characterizations conducted in this study help us to understand the stability of EV-D68-3Dpol, which may extend to other RdRPs as well. IMPORTANCE Enterovirus D68 (EV-D68) is an emerging viral pathogen, which caused sporadic infections around the world. In recent years, epidemiology studies have reported an increasing number of patients with respiratory diseases globally due to the EV-D68 infection. Moreover, the infection has been associated with acute flaccid paralysis and cranial nerve dysfunction in children. However, there are no vaccines and antiviral treatments specifically targeting the virus to date. In this study, we solved the crystal structure of the RNA-dependent RNA polymerase of EV-D68 and carried out systematic biophysical and biochemical characterizations on the overall and local structural stability of the wild-type (WT) enzyme and several variants, which yields a clear view on the structure-activity relationship of the EV-D68 RNA polymerase.

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