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    Dimeric form of SARS-CoV-2 polymerase


    doi:10.1101/2021.03.23.436644 Date: 2021-03-24 Source: bioRxiv

    The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) to replicate and transcribe its genome. Structures of the RdRp PROTEIN revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the coronavirus polymerase and resolve its structure at 4.8 [A] resolution. In this structure, the two RdRps PROTEIN contain only one copy of nsp8 and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp PROTEIN dimer facilitates template switching during production of sub-genomic RNAs for transcription.

    Protein-primed RNA synthesis in SARS-CoVs MESHD and structural basis for inhibition by AT-527

    Authors: Ashleigh Shannon; Veronique Fattorini; Bhawna Sama; Barbara Selisko; Mikael Feracci; Camille Falcou; Pierre Gauffre; Priscila El Kazzi; Etienne Decroly; Nadia Rabah; Karine Toulon; Cecilia Eydoux; Jean-Claude Guillemot; Mathieu Noel; Francoise Debart; Jean-Jacques Vasseur; Adel Moussa; Steven Good; Kai Lin; Jean-Pierre Sommadossi; Yingxiao Zhu; Xiaodong Yan; Hui Shi; Francois Ferron; Bruno Canard

    doi:10.1101/2021.03.23.436564 Date: 2021-03-23 Source: bioRxiv

    How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and -2 Nidovirus RdRp PROTEIN-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP- Nsp HGNC8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp PROTEIN active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5'-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 A resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp PROTEIN active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and -2, representing a promising drug for COVID-19 MESHD treatment.

    The SARS-CoV-2 replication-transcription complex is a priority target for broad-spectrum pan-coronavirus drugs

    Authors: Setayesh Yazdani; Nicola De Maio; Yining Ding; Vijay Shahani; Nick Goldman; Matthieu Schapira

    doi:10.1101/2021.03.23.436637 Date: 2021-03-23 Source: bioRxiv

    In the absence of effective treatment, COVID-19 MESHD is likely to remain a global disease burden. Compounding this threat is the near certainty that novel coronaviruses with pandemic potential will emerge in years to come. Pan-coronavirus drugs - agents active against both SARS-CoV-2 and other coronaviruses - would address both threats. A strategy to develop such broad-spectrum inhibitors is to pharmacologically target binding sites on SARS-CoV-2 proteins that are highly conserved in other known coronaviruses, the assumption being that any selective pressure to keep a site conserved across past viruses will apply to future ones. Here, we systematically mapped druggable binding pockets on the experimental structure of fifteen SARS-CoV-2 proteins and analyzed their variation across twenty-seven - and {beta}-coronaviruses and across thousands of SARS-CoV-2 samples from COVID-19 MESHD patients. We find that the two most conserved druggable sites are a pocket overlapping the RNA binding site of the helicase nsp13, and the catalytic site of the RNA-dependent RNA polymerase PROTEIN nsp12, both components of the viral replication-transcription complex. We present the data on a public web portal (https://www.thesgc.org/SARSCoV2_pocketome/) where users can interactively navigate individual protein structures and view the genetic variability of drug binding pockets in 3D.

    Identification of guanylyltransferase activity in the SARS-CoV-2 RNA polymerase

    Authors: Alexander P Walker; Haitian Fan; Jeremy R Keown; Jonathan Grimes; Ervin Fodor

    doi:10.1101/2021.03.17.435913 Date: 2021-03-18 Source: bioRxiv

    SARS-CoV-2 is a positive-sense RNA virus that is responsible for the ongoing Coronavirus Disease MESHD Coronavirus Disease 2019 MESHD ( COVID-19 MESHD) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease MESHD and death MESHD in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 PROTEIN (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors that are involved in RNA proofreading and 5' capping of viral mRNAs. The formation of the 5' cap-1 HGNC structure is known to require a guanylyltransferase (GTase) as well as 5' triphosphatase and methyltransferase activities. However, the mechanism of SARS-CoV-2 mRNA capping remains poorly understood. Here we show that the SARS-CoV-2 RNA polymerase nsp12 functions as a GTase. We characterise this GTase activity and find that the nsp12 NiRAN (nidovirus RdRP PROTEIN-associated nucleotidyltransferase) domain is responsible for carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We also show that remdesivir triphosphate, the active form of the antiviral drug remdesivir, inhibits the SARS-CoV-2 GTase reaction as efficiently as RNA polymerase activity. These data improve understanding of coronavirus mRNA cap synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

    Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex

    Authors: Brandon Malone; James Chen; Qi Wang; Eiiza Llewellyn; Young Joo Choi; Paul Dominic B. Olinares; Xinyun Cao; Carolina Hernandez; Edward T. Eng; Brian T. Chait; David E. Shaw; Robert Landick; Seth A. Darst; Elizabeth A. Campbell

    doi:10.1101/2021.03.13.435256 Date: 2021-03-14 Source: bioRxiv

    Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp MESHD RdRp PROTEIN bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp MESHD RdRp PROTEIN backtracking. The results establish that the single-stranded 3'-segment of the product-RNA generated by backtracking extrudes through the RdRp PROTEIN NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3'-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp PROTEIN backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

    Dual targeting of cytokine storm and viral replication in COVID-19 MESHD by plant-derived steroidal pregnanes in silico

    Authors: Gideon A. Gyebi; Oludare M. Ogunyemi; Ibrahim M. Ibrahim; Saheed O. Afolabi; Joseph O. Adebayo

    doi:10.21203/rs.3.rs-329239/v1 Date: 2021-03-14 Source: ResearchSquare

    The high morbidity and mortality rate of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection MESHD arises majorly from the Acute Respiratory Distress Syndrome MESHD and “cytokine storm” syndrome, which is sustained by an aberrant systemic inflammatory response and elevated pro-inflammatory cytokines. Thus, phytocompounds with broad-spectrum anti-inflammatory activity that target multiple SARS-CoV-2 proteins will enhance the development of effective drugs against the disease. In this study, an in-house library of 106 steriodal plant-derived pregnanes (PDPs) was docked in the active regions of human glucocorticoid receptors (hGRs) in a comparative molecular docking analysis. Based on the minimal binding energy and a comparative dexamethason binding mode analysis, a list of top twenty ranked PDPs docked in the agonist conformation of hGR, with binding energies ranging between -9.8 and -11.2 Kcal/mol, was obtained and analyzed for interactions with the human Janus kinases 1 and Interleukins-6 and SARS-CoV-2 3-chymotrypsin- like protease, Papain PROTEIN-like protease and RNA-dependent RNA polymerase PROTEIN. For each target protein, the top three ranked PDPs were selected. Eight PDPs (bregenin, hirundigenin, anhydroholantogenin, atratogenin A, atratogenin B, glaucogenin A, glaucogenin C and glaucogenin MESHD D) with high binding tendencies to the catalytic residues of multiple targets were identified. A high degree of structural stability was observed from the 100 ns molecular dynamics simulation analyses of glaucogenin C MESHD and hirundigenin complexes of hGR. The selected top-eight ranked PDPs demonstrated favourable druggable and in silico ADMET properties. Thus, the therapeutic potentials of glaucogenin C MESHD and hirundigenin can be explored for further in vitro and in vivo studies.

    Synergistic Inhibition of SARS-CoV-2 Replication using Disulfiram/Ebselen and Remdesivir

    Authors: Ting Chen; Cheng-Yin Fei; Yi-Ping Chen; Karen Sargsyan; Chun Ping Chang; Hanna S. Yuan; Carmay Lim

    doi:10.26434/chemrxiv.13604015.v2 Date: 2021-03-11 Source: ChemRxiv

    The SARS-CoV-2 replication and transcription complex (RTC) comprising nonstructural protein (nsp) 2-16 plays crucial roles in viral replication, reducing the efficacy of broad-spectrum nucleoside analog drugs such as remdesivir and in evading innate immune responses. Most studies target a specific viral component of the RTC such as the main protease PROTEIN or the RNA-dependent RNA polymerase PROTEIN. In contrast, our strategy is to target multiple conserved domains of the RTC to prevent SARS-CoV-2 genome replication and to create a high barrier to viral resistance and/or evasion of antiviral drugs. We show that clinically-safe Zn-ejector drugs, disulfiram/ebselen, can target conserved Zn2+-sites in SARS-CoV-2 nsp13 and nsp14 and inhibit nsp13 ATPase and nsp14 exoribonuclease PROTEIN activities. As the SARS-CoV-2 nsp14 domain targeted by disulfiram/ebselen is involved in RNA fidelity control, our strategy allows coupling of the Zn-ejector drug with a broad-spectrum nucleoside analog that would otherwise be excised by the nsp14 proofreading domain. As proof-of-concept, we show that disulfiram/ebselen, when combined with remdesivir, can synergistically inhibit SARS-CoV-2 replication in Vero E6 cells. We present a mechanism of action and the advantages of our multi-targeting strategy, which can be applied to any type of coronavirus with conserved Zn2+-sites.

    Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N2, RdRP PROTEIN and human RP genes

    Authors: Huseyin Tombuloglu; Hussein Sabit; Ebtesam Al-Suhaimi; Hamoud Al-Khallaf; Juma Kabanja; Najat Al-Saleh

    doi:10.21203/rs.3.rs-308136/v1 Date: 2021-03-07 Source: ResearchSquare

    Corona Virus Disease MESHD 2019 ( COVID-19 MESHD) is a disease caused by severe acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative accuracy, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, some of these assays have the disadvantages of taking time to show the result and might produce false-negative and false-positive ones. Therefore, designing rapid and accurate PCR-based testing assay is of paramount importance for early detection of this virus and for more efficient control of the spread of this disease. We, here, describe a fast, reliable, easy-to- use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N2 and RdRP PROTEIN) and a human gene (RP) simultaneously. The performance and the accuracy of the assay was tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.25 copies/µL or 5 copies/reaction. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

    Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

    Authors: Valentine Courouble; Sanjay Dey; Ruchi Yadav; Jennifer Timm; Jerry Harrison; Francesc X Ruiz; Eddy Arnold; Patrick R Griffin

    doi:10.1101/2021.03.06.434214 Date: 2021-03-06 Source: bioRxiv

    Coronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

    Analysis of SARS-CoV-2 Mutations Over Time Reveals Increasing Prevalence of Variants in the Spike Protein PROTEIN and RNA-Dependent RNA Polymerase PROTEIN

    Authors: William M Showers; Sonia M Leach; Katerina Kechris; Michael Strong

    doi:10.1101/2021.03.05.433666 Date: 2021-03-05 Source: bioRxiv

    Amid the ongoing COVID-19 pandemic MESHD, it has become increasingly important to monitor the mutations that arise in the SARS-CoV-2 virus, to prepare public health strategies and guide the further development of vaccines and therapeutics. The spike (S) protein PROTEIN and the proteins comprising the RNA-Dependent RNA Polymerase PROTEIN ( RdRP PROTEIN) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences for the spike proteins PROTEIN and RNA-dependent RNA polymerase PROTEIN proteins were downloaded from the GISAID database, aligned, and the variants identified. Polymorphisms in the protein sequence were investigated at the protein structural level and examined longitudinally in order to identify sequence and strain variants that are emerging over time. Our analysis revealed a group of variants in the spike protein PROTEIN and the polymerase complex that appeared in August, and account for around five percent of the genomes analyzed up to the last week of October. A structural analysis also facilitated investigation of several unique variants in the receptor binding domain and the N-terminal domain of the spike protein PROTEIN, with high-frequency mutations occurring more commonly in these regions. The identification of new variants emphasizes the need for further study on the effects of these mutations and the implications of their increased prevalence, particularly as these mutations may impact vaccine or therapeutic efficacy.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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