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    Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase PROTEIN

    Authors: Rupert Beale; Agustina P Bertolin; Berta Canal; John FX Diffley; Lucy S Drury; Michael Howell; Jennifer Milligan; Viktor Posse; Rachel Ulferts; Florian Weissmann; Mary Wu; Jingkun Zeng

    doi:10.1101/2021.04.07.438807 Date: 2021-04-08 Source: bioRxiv

    The coronavirus disease 2019 MESHD ( COVID-19 MESHD) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 MESHD etiologic agent is the severe acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) holoenzyme. The RdRp PROTEIN is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes PROTEIN. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp PROTEIN activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp PROTEIN inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp PROTEIN activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

    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.

    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.

    Characterization of the NiRAN domain from RNA-dependent RNA polymerase PROTEIN provides insights into a potential therapeutic target against SARS-CoV-2

    Authors: Abhisek Dwivedy; Richard Mariadasse; Mohammed Ahmad; Sayan Chakraborty; Deepsikha Kar; Satish Tiwari; Tanmay Majumdar; Jeyaraman Jeyakanthan; Bichitra Biswal

    doi:10.1101/2021.02.03.429510 Date: 2021-02-03 Source: bioRxiv

    Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases PROTEIN ( RdRp PROTEIN) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp PROTEIN associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp PROTEIN from the severe acute respiratory syndrome coronavirus-2 MESHD (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness as well as DFT properties. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp PROTEIN. Interestingly, the proposed kinase inhibitors and a few of the predicted nucleotidyl transferase inhibitors significantly inhibited the aforementioned enzymatic activity. In line with the current global COVID-19 pandemic MESHD urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

    Routine SARS-CoV-2 wastewater surveillance results in Turkey to follow Covid-19 MESHD outbreak

    Authors: Bilge Alpaslan Kocamemi; Halil Kurt; Ahmet Sait; Hamza Kadi; Fahriye Sarac; Ismail Aydin; Ahmet Mete Saatci; Bekir Pakdemirli

    doi:10.1101/2020.12.21.20248586 Date: 2020-12-22 Source: medRxiv

    A global pandemic of Coronavirus Disease 2019 MESHD ( Covid-19 MESHD) caused by severe acute respiratory syndrome coronavirus 2 MESHD (SAR-CoV-2) declared by WHO in March 2019 is still ongoing. As of 13th of December 2020, 70 million people were infected by SARS-CoV-2 and 1.5 million people lost their lives globally (WHO, 2020). Since March 2019, diagnosis of Covid-19 MESHD cases has been done through PCR test of samples from nasopharyngeal and throat swabs. However, in March 2019, it was reported that the faeces [1] and urine [2] of all infected people contain SARS-CoV-2 MESHD. Later, numerous researchers [3-7] detected SARS-CoV-2 in faeces of both symptomatic and asymptomatic patients. Moreover, some studies [1,4,8-12] suggested the possibility of extended duration of viral shedding in faeces after the patients respiratory samples tested negative. In this respect, SARS-CoV-2 wastewater-based epidemiology (WBE), i.e., wastewater surveillance, aiming to estimate the distribution of asymptomatic and symptomatic individuals in a specific region has received worldwide attention. Various research groups worldwide [1, 13-54] have started SARS-CoV-2 detection in wastewater since WBE provides tracking whole population by testing a small number of wastewater samples in a specific region and can predict SARS-CoV-2 RNA in human faeces a few days to a week before onset of symptoms. This makes WBE quite economic tool for continual tracking of decreasing or increasing trend of the Covid-19 MESHD in a particular region. However, up to date, almost all of the WBE studies have been performed with samples from a few treatment plants. There was no reported nationwide wastewater surveillance study that has been integrated into a national Covid-19 MESHD management strategy by decision makers. Nationwide, SARS-CoV-2 surveillance studies have great potential to reflect the actual distribution of Covid-19 MESHD cases in a community by accounting not only symptomatic patients tested but also asymptomatic patients having no or mild symptoms and not been tested. As opposed to clinical surveillance studies, wastewater-based surveillance studies will reflect the number of cases in a community by testing one sample from a treatment plant serving this community instead of performing individual swab tests. Turkey, which is among the few countries that started wastewater based surveillance studies at the early stages of pandemic is a leading country, performing a nationwide surveillance study. The distribution of Covid-19 MESHD cases throughout the country via SARS-CoV-2 measurements in influent, effluent and sludge samples of wastewater treatment plants (WWTPs) located in 81 cities through May 2020- June 2020 was conducted [36, 51, 52]. In June 2020, nationwide routine sampling through 22 regional identified cities has been started. However, from June to September 2020 all samples were detected negative due to problems with RT-pCR primer targeting RdRp PROTEIN gene of SARS-CoV-2 genome. Since September 2020, routine sampling from 22 cities of Turkey with 2 weeks sampling period (weekly for mega city Istanbul) has been continued and regional Covid-19 MESHD distributions have been reported as viral loads on color-scale maps. To the best our knowledge, this is the first routine nationwide surveillance study indicating Covid-19 MESHD distribution regularly using color-scale presentation on a map.

    A recombinant fragment of Human surfactant protein D HGNC binds Spike protein PROTEIN and inhibits infectivity and replication of SARS-CoV-2 in clinical samples

    Authors: Taruna Madan; Barnali Biswas; Praveen Varghese; Rambhadur Subedi; Hrishikesh Pandit; Susan Idicula-Thomas; Indra Kundu; Sheetalnath Babasaheb Rooge; Reshu Aggarwal; Dinesh Tripathi; Savneet Kaur; Ekta Gupta; Sanjeev Gupta

    doi:10.1101/2020.12.18.423415 Date: 2020-12-18 Source: bioRxiv

    Rationale COVID-19 MESHD is an acute infectious disease MESHD caused by the Severe Acute Respiratory Syndrome Coronavirus 2 MESHD (SARS-CoV-2). Human surfactant protein D HGNC ( SP-D HGNC) is known to interact with spike protein PROTEIN of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known. ObjectiveThis study aimed to examine the potential of a recombinant fragment of human SP-D HGNC (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2 MESHD. MethodsrfhSP-D interaction with spike protein PROTEIN of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein PROTEIN and ACE-2 HGNC by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp PROTEIN gene of the virus using qPCR. Measurements and Main ResultsIn-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 PROTEIN were commonly involved in interacting with rfhSP-D and ACE-2 HGNC. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5M rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 M). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5M rfhSP-D. ConclusionsThese results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike PROTEIN protein and human ACE-2 HGNC, its host cell receptor, and a significant reduction in SARS-CoV-2 infection MESHD and replication in-vitro.

    Consistent and High-Frequency Identification of an Intra-Sample Genetic Variant of SARS-CoV-2 with Elevated Fusogenic Properties

    Authors: Lynda Rocheleau; Geneviève Laroche; Kathy Fu; Marceline Côté; Patrick M Giguère; Marc-André Langlois; Martin Pelchat; Peter Mohr; Remo Gamboni; Thanos D. Halazonetis; Kai-Thomas Schneider; Kristian Daniel Ralph Roth; Philipp Kuhn; Peggy Riese; Dorina Schäckermann; Janin Korn; Allan Koch; Susanne Zock-Emmenthal; Marlies Becker; Margitta Scholz; Gustavo Marçal Schmidt Garcia Moreira; Esther Veronika Wenzel; Giulio Russo; Hendrikus S.P. Garritsen; Sebastian Casu; Andreas Gerstner; Günter Roth; Andreas Hermann; Thomas Schirrmann; Stefan Dübel; André Frenzel; Joop Van den Heuvel; Luka Cicin-Sain; Maren Schubert; Michael Hust

    doi:10.1101/2020.12.03.409714 Date: 2020-12-03 Source: bioRxiv

    The severe acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2) has a genome comprised of a ~30K nucleotides non-segmented, positive single-stranded RNA. Although its RNA-dependent RNA polymerase PROTEIN exhibits exonuclease proofreading activity, viral sequence diversity can be induced by replication errors and host factors. These variations can be observed in the population of viral sequences isolated from infected host cells and are not necessarily reflected in the genome of transmitted founder viruses. We profiled intra-sample genetic diversity of SARS-CoV-2 variants using 15,289 high-throughput sequencing datasets from infected individuals and infected cell lines. Most of the genetic variations observed, including C->U and G->U, were consistent with errors due to heat-induced DNA damage during sample processing, and/or sequencing protocols. Despite high mutational background, we confidently identified intra-variable positions recurrent in the samples analyzed, including several positions at the end of the gene encoding the viral S protein PROTEIN. Notably, most of the samples possesses a C->A missense mutation resulting in the S protein PROTEIN lacking the last 20 amino acids (S{Delta}20). Here we demonstrate that S{Delta}20 exhibits increased cell-to-cell fusion and syncytia formations MESHD. Our findings are suggestive of the consistent emergence of high-frequency viral quasispecies that are not horizontally transmitted but involved in intra-host infection MESHD and spread. Author summaryThe severe acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2) and its associated disease, COVID-19 MESHD, has caused significant worldwide mortality and unprecedented economic burden. Here we studied the intra-host genetic diversity of SARS-CoV-2 genomes and identified a high-frequency and recurrent non-sense mutation yielding a truncated form of the viral spike protein PROTEIN, in both human COVID-19 MESHD samples and in cell culture experiments. Through the use of a functional assay, we observed that this truncated spike protein PROTEIN displays an elevated fusogenic potential and forms syncytia. Given the high frequency at which this mutation independently arises across various samples, it can be hypothesized that this deletion mutation provides a selective advantage to viral replication and may also have a role in pathogenesis in humans.

    Long-chain polyphosphates impair SARS-CoV-2 infection MESHD and replication: a route for therapy in man

    Authors: Veronica Ferrucci; Young Kong Dae; Fatemeh Asadzadeh; Laura Marrone; Roberto Siciliano; Pellegrino Cerino; Giuseppina Criscuolo; Ida Pisano; Fabrizio Quarantelli; Barbara Izzo; Giovanna Fusco; Marika Comegna; Angelo Boccia; Maurizio Viscardi; Giorgia Borriello; Sergio Brandi; Bianca Maria Pierri; Claudia Tiberio; Luigi Atripaldi; Giovanni Paolella; Giuseppe Castaldo; Stefano Pascarella; Martina Bianchi; Rosa Della Monica; Lorenzo Chiariotti; Kyong Seop Yun; Jae Ho Cheong; Hong Yeoul Kim; Massimo Zollo; Katie Jeffery; David W Eyre; Talat Mokhtari-Azad; Reza Najafipour; Reza Malekzadeh; Kimia Kahrizi; Seyed Mohammad Jazayeri; Hossein Najmabadi

    doi:10.1101/2020.11.18.388413 Date: 2020-11-18 Source: bioRxiv

    Anti-viral activities of long-chain inorganic polyphosphates (PolyPs) against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection MESHD were investigated. In molecular docking analyses, PolyPs interacted with several conserved angiotensin-converting enzyme ( ACE)2 HGNC and RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) amino acids. We thus tested PolyPs for functional interactions in vitro in SARS-CoV-2-infected MESHD Vero E6, Caco2 and human primary nasal epithelial cells. Immunofluorescence, qPCR, direct RNA sequencing, FISH and Immunoblotting were used to determine virus loads and transcription levels of genomic(g)RNAs and sub-genomic(sg)RNAs. We show that PolyP120 binds to ACE2 HGNC and enhances its proteasomal degradation. PolyP120 shows steric hindrance of the genomic Sars-CoV-2-RNA/ RdRP complex PROTEIN, to impair synthesis of positive-sense gRNAs, viral subgenomic transcripts and structural proteins needed for viral replication. Thus, PolyP120 impairs infection MESHD and replication of Korean and European (containing non-synonymous variants) SARS-CoV-2 strains. As PolyPs have no toxic activities, we envision their use as a nebulised formula for oropharyngeal delivery to prevent infections of SARS-CoV-2 and during early phases of antiviral therapy.

    Methyltransferase-like 3 HGNC modulates severe acute respiratory syndrome coronavirus-2 RNA N6-methyladenosine modification and replication

    Authors: Xueyan Zhang Sr.; Haojie Hao Sr.; Li Ma Sr.; Yecheng Zhang Sr.; Xiao Hu Sr.; Zhen Chen Sr.; Di Liu Sr.; Jianhui Yuan Sr.; Zhangli Hu Sr.; Wuxiang Guan Sr.

    doi:10.1101/2020.10.14.338558 Date: 2020-10-14 Source: bioRxiv

    The coronavirus disease 2019 MESHD pandemic caused by severe acute respiratory syndrome coronavirus-2 MESHD (SARS-CoV-2) is an ongoing global public crisis. Although viral RNA modification has been reported based on the transcriptome architecture, the types and functions of RNA modification are still unknown. In this study, we evaluated the roles of RNA N6-methyladenosine (m6A) modification in SARS-CoV-2. Our methylated RNA immunoprecipitation sequencing (MeRIP-Seq) analysis showed that SARS-CoV-2 RNA contained m6A modification. Moreover, SARS-CoV-2 infection MESHD not only increased the expression of methyltransferase-like 3 HGNC ( METTL3 HGNC) but also altered its distribution. Modification of METTL3 HGNC expression by short hairpin RNA or plasmid transfection for knockdown or overexpression, respectively, affected viral replication. Furthermore, the viral key protein RdRp PROTEIN interacted with METTL3 HGNC, and METTL3 HGNC was distributed in both the nucleus and cytoplasm in the presence of RdRp PROTEIN. RdRp PROTEIN appeared to modulate the sumoylation and ubiquitination of METTL3 HGNC via an unknown mechanism. Taken together, our findings demonstrated that the host m6A modification complex interacted with viral proteins to modulate SARS-CoV-2 replication.

    Extensive Genetic Diversity and Host Range of Rodent-borne Coronaviruses

    Authors: Wen Wang; Xian-Dan Lin; Hai-Lin Zhang; Miao-Ruo Wang; Xiao-Qing Guan; Edward C Holmes; Yong-Zhen Zhang

    doi:10.1101/2020.08.11.245415 Date: 2020-08-14 Source: bioRxiv

    To better understand the genetic diversity, host association and evolution of coronaviruses (CoVs) in China we analyzed a total of 696 rodents encompassing 16 different species sampled from Zhejiang and Yunnan provinces. Based on the reverse transcriptase PCR-based CoV screening CoVs of fecal samples and subsequent sequence analysis of the RdRp PROTEIN gene, we identified CoVs in diverse rodent species, comprising Apodemus agrarius, Apodemus latronum, Bandicota indica, Eothenomys miletus, E. eleusis, Rattus andamanesis, Rattus norvegicus, and R. tanezumi. Apodemus chevrieri was a particularly rich host, harboring 25 rodent CoVs. Genetic and phylogenetic analysis revealed the presence of three groups of CoVs carried by a range of rodents that were closely related to the Lucheng Rn rat coronavirus (LRNV), China Rattus coronavirus HKU24 MESHD (ChRCoV_HKU24) and Longquan Rl rat coronavirus (LRLV) identified previously. One newly identified A. chevrieri-associated virus closely related to LRNV lacked an NS2 gene. This virus had a similar genetic organization to AcCoV-JC34, recently discovered in the same rodent species in Yunnan, suggesting that it represents a new viral subtype. Notably, additional variants of LRNV were identified that contained putative nonstructural NS2b genes located downstream of the NS2 gene that were likely derived from the host genome. Recombination events were also identified in the ORF1a PROTEIN gene of Lijiang-71. In sum, these data reveal the substantial genetic diversity and genomic complexity of rodent-borne CoVs, and greatly extend our knowledge of these major wildlife virus reservoirs.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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