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    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.

    Investigation of Anti-Coronavirus, Anti-HCV, Nucleotide Inhibitors, and Bioactive Molecules efficacy Against RNA-directed RNA polymerase of Nipah Virus: Molecular Docking Study

    Authors: Peter T. Habib

    doi:10.21203/rs.3.rs-294115/v2 Date: 2021-03-03 Source: ResearchSquare

    The infections with the Nipah virus (NiV) are highly infectious and may lead to severe febrile encephalitis MESHD. High rates of mortality in southeastern Asia including Bengal, Malaysia, Papua New Guinea, Vietnam, Cambodia, Indonesia, Madagascar, the Philippines, Thailand, and India have been reported in NiV outbreaks. Considering the high risk of an epidemic, NiV was declared a priority pathogen by the World Health Organization (WHO). However, for the treatment of this infection, there is no effective therapy or approved FDA medicines. RNA-dependent polymerase RNA PROTEIN ( RdRp PROTEIN) plays an important role in viral replication among the nine well-known proteins of NiV. Therefore, fourteen antiviral molecules have been computerized for NiV RNA-dependent RNA polymerase PROTEIN and demonstrated a potential inhibition effect against coronavirus (NiV- RdRp PROTEIN). A multi-step molecular docking process, followed by extensive analyzes of molecular binding interactions, binding energy estimates, synthetic accessibility assessments, and toxicity MESHD tests. Analysis reveals that Uprifosbuvir is the most suitable inhibitor for RdRp PROTEIN of Nipah Virus regarding the binding affinity and binding in the target cavity. Although, such studies need clinical confirmation.

    Atorvastatin effectively inhibits late replicative cycle steps of SARS-CoV-2 in vitro

    Authors: Maria Isabel Zapata-Cardona; Lizdany Flórez-Álvarez; Wildeman Zapata-Builes; Ariadna Guerra-Sandoval; Carlos Guerra-Almonacid; Jaime Hincapié-García; Maria Teresa Rugeles; Juan Carlos Hernández

    doi:10.1101/2021.03.01.433498 Date: 2021-03-03 Source: bioRxiv

    Introduction: SARS-CoV-2 has caused a pandemic of historic proportions and continues to spread worldwide. Currently, there is no effective therapy against this virus. This article evaluated the in vitro antiviral effect of Atorvastatin against SARS-CoV-2 and also identified the interaction affinity between Atorvastatin and three SARS-CoV-2 proteins MESHD, using in silico structure-based molecular docking approach. Materials and methods: The antiviral activity of Atorvastatin against SARS-CoV-2 was evaluated by three different treatment strategies using a clinical isolate of SARS-CoV-2. The interaction of Atorvastatin with Spike, RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) and 3C-like protease ( 3CLpro PROTEIN) was evaluated by molecular docking. Results: Atorvastatin showed anti-SARS-CoV-2 activity of 79%, 54.8%, 22.6% and 25% at 31.2, 15.6, 7.9, and 3.9 {micro}M, respectively, by pre-post-treatment strategy. In addition, atorvastatin demonstrated an antiviral effect of 26.9% at 31.2 {micro}M by pre-infection treatment. This compound also inhibited SARS-CoV-2 in 66.9%, 75%, 27.9% and 29.2% at concentrations of 31.2, 15.6, 7.9, and 3.9 {micro}M, respectively, by post-infection treatment. The interaction of atorvastatin with SARS-CoV-2 Spike PROTEIN SARS-CoV-2 Spike MESHD, RdRp PROTEIN and 3CL protease PROTEIN yielded a binding affinity of -8.5 Kcal/mol, -6.2 Kcal/mol, and -7.5 Kcal/mol, respectively. Conclusion: Our study demonstrated the in vitro anti-SARS-CoV-2 activity of Atorvastatin, mainly against the late steps of the viral replicative cycle. A favorable binding affinity with viral proteins by bioinformatics methods was also shown. Due to its low cost, availability, well-established safety and tolerability, and the extensive clinical experience of atorvastatin, it could prove valuable in reducing morbidity and mortality from COVID-19 MESHD.

    Impact of COVID-19 MESHD pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results

    Authors: Jonathan B Gubbay; Heather Rilkoff; Heather L. Kristjanson; Jessica D. Forbes; Michelle Murti; AliReza Eshaghi; George Broukhanski; Antoine Corbeil; Nahuel Fittipaldi; Jessica Hopkins; Erik Kristjanson; Julianne V Kus; Liane Macdonald; Anna Majury; Gustavo V Mallo; Tony Mazzulli; Roberto G Melano; Romy Olsha; Stephen J Perusini; Vanessa Tran; Vanessa Gray Allen; Samir N Patel

    doi:10.1101/2021.03.02.21252768 Date: 2021-03-03 Source: medRxiv

    Background: Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent resolved infection with persistent RNA shedding, presymptomatic or asymptomatic infection, or a false positive test. This study assessed clinical specificity of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Materials and Methods: A total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) gene followed by Sanger sequencing. Results: Significantly less positive results in the lowest pre-test probability group (facilities with institution-wide screening having [≤]3 positive asymptomatic cases) were reproduced with the nested RdRp PROTEIN gene RT-PCR assay than in all other groups combined (5/32, 15.6% vs 61/90, 68%; p <0.0001), and in each subgroup with higher pre-test probability (individual subgroup range 50.0% to 85.0%). Conclusions: A higher proportion of false-positive test results are likely with lower pre-test probability. Positive SARS-CoV-2 PCR results should be interpreted within the context of patient history, clinical setting, known exposure, and estimated community disease prevalence. Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

    The impact of mutations on the structural and functional properties of SARS-CoV-2 proteins: A comprehensive bioinformatics analysis

    Authors: Aqsa Ikram; Anam Naz; Faryal Awan; Bisma Rauff; Ayesha Obaid; Mohamad S. Hakim; Arif Malik

    doi:10.1101/2021.03.01.433340 Date: 2021-03-01 Source: bioRxiv

    An in-depth analysis of first wave SARS-CoV-2 genome is required to identify various mutations that significantly affect viral fitness MESHD. In the present study, we have performed comprehensive in-silico mutational analysis of 3C-like protease ( 3CLpro PROTEIN), RNA dependent RNA polymerase PROTEIN ( RdRp PROTEIN), and spike (S) proteins PROTEIN with the aim of gaining important insights into first wave virus mutations and their functional and structural impact on SARS-CoV-2 proteins. Our integrated analysis gathered 3465 SARS-CoV-2 sequences and identified 92 mutations in S, 37 in RdRp PROTEIN, and 11 in 3CLpro PROTEIN regions. The impact of those mutations was also investigated using various in silico approaches. Among these 32 mutations in S, 15 in RdRp PROTEIN, and 3 in 3CLpro PROTEIN proteins are found to be deleterious in nature and could alter the structural and functional behavior of the encoded proteins. D614G mutation in spike and P323L in RdRp PROTEIN are the globally dominant variants with a high frequency. Most of them have also been found in the binding moiety of the viral proteins which determine their critical involvement in the host-pathogen interactions and drug targets. The findings of the current study may facilitate better understanding of COVID-19 MESHD diagnostics, vaccines, and therapeutics.

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SARS-CoV-2 Proteins


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