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    Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

    Authors: Florian Kabinger; Carina Stiller; Jana Schmitzová; Christian Dienemann; Hauke S. Hillen; Claudia Höbartner; Patrick Cramer

    doi:10.1101/2021.05.11.443555 Date: 2021-05-11 Source: bioRxiv

    Molnupiravir is an orally available antiviral drug candidate that is in phase III trials for the treatment of COVID-19 MESHD patients1,2. Molnupiravir increases the frequency of viral RNA mutations3,4 and impairs SARS-CoV-2 replication in animal models4-6 and in patients2. Here we establish the molecular mechanisms that underlie molnupiravir-induced RNA mutagenesis by the RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) of the coronavirus SARS-CoV-2. Biochemical assays show that the RdRp PROTEIN readily uses the active form of molnupiravir, {beta}-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of CTP or UTP. Incorporation of NHC monophosphate into nascent RNA does not impair further RdRp PROTEIN progression. When the RdRp PROTEIN uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp PROTEIN-RNA complexes containing mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp PROTEIN active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism likely applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

    Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection MESHD using SYBR Green melting curve analysis

    Authors: Shovon Lal Sarkar; A. S. M. Rubayet Ul Alam; Prosanto Kumar Das; Md. Hasan Ali Pramanik; Hassan M. Al-Emran; Iqbal Kabir Jahid; Md. Anwar Hossain

    doi:10.1101/2021.05.06.21256629 Date: 2021-05-08 Source: medRxiv

    TaqMan probe-based expensive commercial real-time (RT) PCR kits are being used in COVID-19 MESHD diagnosis. The unprecedented scale of SARS-CoV-2 infections MESHD has urgently needed to meet the challenge of testing more persons at a reasonable cost. This study developed a rapid, simple, and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay with a host-specific internal control. A total of 90 randomly selected samples were used for comparing the assay with an available commercial kit to analyse the variation and validity of this in-house developed method. Our customized designed primers specifically detected the virus as similar to commercial kit manufactured by Sansure Biotech Inc. We optimized separately the N, E, S, and RdRp PROTEIN genes by SYBR Green RT-PCR method based on melting curve analysis. Afterwards, a multiplex COVID-19 MESHD diagnosis method targeting N and E genes PROTEIN of the virus along with the { beta}-actin HGNC gene of the host as an internal control has been established. The total run-time of our proposed method was less than 90 minutes. The cost of each sample processing was less than $2. Overall, this one-step and one-tube method can revolutionize the COVID-19 MESHD diagnosis in developing countries.

    Diversity and selection of SARS-CoV-2 minority variants in the early New York City outbreak

    Authors:

    doi:10.1101/2021.05.05.442873 Date: 2021-05-06 Source: bioRxiv

    High error rates of viral RNA-dependent RNA polymerases PROTEIN lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. Here we analyzed minority variants within the SARS-CoV-2 data in 12 samples from the early outbreak in New York City, using replicate sequencing for reliable identification. While most minority variants were unique to a single sample, we found several instances of shared variants. We provide evidence that some higher-frequency minority variants may be transmitted between patients or across short transmission chains, while other lower-frequency, more widely shared variants arise independently. Further, our data indicate that even with a small transmission bottleneck, the heterogeneity of intra-host viral populations is enhanced by minority variants present in transmission samples. Our data suggest that analysis of shared minority variants could help identify regions of the SARS-CoV-2 genome that are under increased selective pressure, as well as inform transmission chains and give insight into variant strain emergence.

    Emerging genetic diversity of SARS-CoV-2 RNA dependent RNA polymerase PROTEIN ( RdRp PROTEIN) alters its B-cell epitopes

    Authors: Sushant Kumar; Gajendra Kumar Azad

    doi:10.1101/2021.05.04.442686 Date: 2021-05-05 Source: bioRxiv

    The RNA dependent RNA polymerase PROTEIN ( RdRp PROTEIN) plays crucial role in virus life cycle by replicating the viral RNA genome. The SARS-CoV-2 is an RNA virus that rapidly spread worldwide and during this process acquired mutations. This study was carried out to identify mutations in RdRp PROTEIN as the SARS-CoV-2 spread in India. We compared the 668 RdRp PROTEIN sequences reported from India with the first reported RdRp PROTEIN sequence from Wuhan, China. Our data revealed that RdRp PROTEIN have acquired sixty mutations among Indian isolates. Our protein modelling study also revealed that several mutants including D833Y, A699S, Y149C and C464F can potentially alter stability and flexibility of RdRp PROTEIN. We also predicted the potential B cell epitopes contributed by RdRp PROTEIN and identified thirty-six linear continuous and twenty-five discontinuous epitopes. Among sixty RdRp PROTEIN mutants identified in this study, 40% of them localizes in the B cell epitopes region. Altogether, this study highlights the need to identify and characterize the variations in RdRp PROTEIN to understand the impact of these mutations on SARS-CoV-2.

    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.

    TMPRSS2 HGNC and RNA-dependent RNA polymerase PROTEIN are effective targets of therapeutic intervention for treatment of COVID-19 MESHD caused by SARS-CoV-2 variants (B.1.1.7 and B.1.351)

    Authors: Jihye Lee; JinAh Lee; Hyeon Ju Kim; Meehyun Ko; Youngmee Jee; Seungtaek Kim

    doi:10.1101/2021.04.06.438540 Date: 2021-04-08 Source: bioRxiv

    SARS-CoV-2 is a causative agent of COVID-19 pandemic MESHD and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development and this effort was partially successful. Since the beginning of COVID-19 pandemic MESHD, the emergence of SARS-CoV-2 variants has been reported in many parts of the world and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. The viral entry and viral RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) are the main targets of current drug development, thus the inhibitory effects of TMPRSS2 HGNC and RdRp PROTEIN inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the UK and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 HGNC and RdRp PROTEIN are equally effective against the two variants of concern.

    Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease PROTEIN ExoN

    Authors: Nicholas H Moeller; Ke Shi; Özlem Demir; Surajit Banerjee; Lulu Yin; Christopher Belica; Cameron Durfee; Rommie E Amaro; Hideki Aihara

    doi:10.1101/2021.04.02.438274 Date: 2021-04-04 Source: bioRxiv

    High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease PROTEIN (ExoN) in non-structural protein 14 PROTEIN (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-[A] resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease PROTEIN activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

    Screening of HLA-A HGNC restricted T cell epitopes of SARS-CoV-2 and induction of CD8+ T cell responses in HLA-A HGNC transgenic mice

    Authors: Xiaoxiao Jin; Yan Ding; Shihui Sun; Xinyi Wang; Zining Zhou; Xiaotao Liu; Miaomiao Li; Xian Chen; Anran Shen; Yandan Wu; Bicheng Liu; Jianqiong Zhang; Jian Li; Yi Yang; Haibo Qiu; Chuanlai Shen; Yuxian He; Guangyu Zhao

    doi:10.1101/2021.04.01.438020 Date: 2021-04-01 Source: bioRxiv

    While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 MESHD patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8 T cell epitopes from E, M, N, S and RdRp PROTEIN proteins of SARS-CoV-2 were validated by on-silicon prediction, DC-peptide-PBL costimulation with PBMCs of healthy donors and HLA-A HGNC molecule competitive binding experiments. Among them, 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV MESHD, OC43, NL63, HKU1, and 229E, respectively. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8 T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. These data have provided a library of SARS-CoV-2 CD8 T cell epitopes which restricted by a series of high-frequency HLA-A HGNC allotypes and covered broad population in Asia, and initially confirmed the feasibility of human MHC class I molecule-restricted SARS-CoV2 epitope peptide cocktail vaccines, thus will facilitate the development of T cell epitope vaccines and specific cellular function detection kits.

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

    Authors: Subhas Chandra Bera; Mona Seifert; Robert Kirchdoerfer; Pauline van Nies; Yibulayin Wubulikasimu; Salina Quack; Flavia S. Papini; Jamie J. Arnold; Bruno Canard; Craig E. Cameron; Martin Depken; David Dulin

    doi:10.1101/2021.03.27.437309 Date: 2021-03-27 Source: bioRxiv

    Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase PROTEIN 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 PROTEIN uses a thermal ratchet instead of a power stroke MESHD 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.

    SARS-CoV-2 N gene PROTEIN dropout and N gene PROTEIN Ct value shift as indicator for the presence of B.1.1.7 lineage in a widely used commercial multiplex PCR assay

    Authors: Paul Wollschlaeger; Nadja Gerlitz; Daniel Todt; Stephanie Pfaender; Thomas Bollinger; Andreas Sing; Alexandra Dangel; Nikolaus Ackermann; Klaus Korn; Armin Ensser; Eike Steinmann; Michael Buhl; Joerg Steinmann

    doi:10.1101/2021.03.23.21254171 Date: 2021-03-26 Source: medRxiv

    Objectives. Increased importance in detection and surveillance of SARS-CoV-2 has been demonstrated due to the emergence of variants of concern (VOCs). In this study we evaluated if a commercially available real-time SARS-CoV-2 PCR assay can identify B.1.1.7 lineage samples by a specific N gene PROTEIN dropout or Ct value shift compared to the S or RdRP PROTEIN gene. Methods. Patients samples with confirmed B.1.1.7 variant by whole-genome sequencing and variant-specific PCR (n=48) and non-B.1.1.7 samples (n=53) were tested by the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay for presence of S, RdRP PROTEIN and N gene PROTEIN of SARS CoV-2. The N gene PROTEIN coding sequence of SARS-CoV-2 with and without D3L mutation (specific for B.1.1.7) were cloned into pCR-TOPO vectors and Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay was performed. Results. All studied B.1.1.7 patient samples showed significantly higher Ct values (delta 6-10, N-gene PROTEIN dropout on Ct values >29) in the N gene PROTEIN compared to the respective values of S and RdRP PROTEIN gene. Receiver operating characteristic (ROC) curve analysis resulted in 100% sensitivity and specificity for delta Ct N/ RdRP PROTEIN and delta Ct N/S. As a result of the reversed genetic experiments we found also the shift in Ct values for the 3L variant N-gene PROTEIN. Conclusions. N gene PROTEIN dropout or Ct value shift is specific for B.1.1.7 positive samples using the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins


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