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1.
Proc Natl Acad Sci U S A ; 119(16): e2117142119, 2022 04 19.
Article in English | MEDLINE | ID: covidwho-1774040

ABSTRACT

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.


Subject(s)
Coronavirus 3C Proteases , Coronavirus RNA-Dependent RNA Polymerase , SARS-CoV-2 , Antiviral Agents/chemistry , Coronavirus 3C Proteases/chemistry , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Polyproteins/chemistry , Protein Conformation , Proteolysis , SARS-CoV-2/enzymology , Substrate Specificity/genetics
2.
Anal Bioanal Chem ; 414(5): 1773-1785, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1653430

ABSTRACT

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80-8.23% and is stable at 4 °C for 1 week and at -70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Genome, Viral , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Dosage , Gene Expression , Humans , Jurkat Cells , Lentivirus/genetics , Lentivirus/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA, Viral/metabolism , RNA, Viral/standards , Reagent Kits, Diagnostic/supply & distribution , Reference Standards , Reproducibility of Results , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Genome Packaging
3.
Nucleic Acids Res ; 50(3): 1551-1561, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1636373

ABSTRACT

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.


Subject(s)
Evolution, Molecular , Genetic Variation/genetics , Genome, Viral/genetics , Host-Pathogen Interactions/genetics , SARS-CoV-2/genetics , APOBEC-1 Deaminase/genetics , Adenosine Deaminase/genetics , Animals , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/genetics , Databases, Genetic , Immune Evasion/genetics , India/epidemiology , Phylogeny , RNA-Binding Proteins/genetics , SARS-CoV-2/classification , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
4.
Virology ; 567: 1-14, 2022 02.
Article in English | MEDLINE | ID: covidwho-1628759

ABSTRACT

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Intracellular Membranes/metabolism , Viral Replicase Complex Proteins/metabolism , Amino Acid Motifs , Animals , Cell Line , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Mice , Murine hepatitis virus , Mutation , Protein Binding , Protein Domains , RNA, Viral/biosynthesis , Viral Replicase Complex Proteins/chemistry , Viral Replicase Complex Proteins/genetics , Viral Replication Compartments/metabolism
5.
Sci Rep ; 11(1): 24234, 2021 12 20.
Article in English | MEDLINE | ID: covidwho-1585791

ABSTRACT

The main strategy for response and control of COVID-19 demands the use of rapid, accurate diagnostic tests aimed at the first point of health care. During the emergency, an increase in asymptomatic and symptomatic cases results in a great demand for molecular tests, which is promoting the development and application of rapid diagnostic technologies. In this study, we describe the development and evaluation of RT-LAMP to detect SARS-CoV-2 based on three genes (ORF1ab, M and N genes) in monoplex and triplex format. RT-LAMP assays were compared with the gold standard method RT-qPCR. The triplex format (RdRp, M and N genes) allowed obtaining comparable results with de RT-qPCR (RdRp and E genes), presented a sensitivity of 98.9% and a specificity of 97.9%, opening the opportunity to apply this method to detect SARS-CoV-2 at primary health-care centers.


Subject(s)
Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , Coronavirus RNA-Dependent RNA Polymerase/genetics , Humans , Limit of Detection , Nasopharynx/virology , Nucleocapsid Proteins/genetics , Point-of-Care Systems , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Matrix Proteins/genetics
6.
Eur J Med Res ; 26(1): 147, 2021 Dec 17.
Article in English | MEDLINE | ID: covidwho-1582004

ABSTRACT

BACKGROUND: The outbreak of novel coronavirus disease 2019 (COVID-19) has become a public health emergency of international concern. Quantitative testing of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus is demanded in evaluating the efficacy of antiviral drugs and vaccines and RT-PCR can be widely deployed in the clinical assay of viral loads. Here, we developed a quantitative RT-PCR method for SARS-CoV-2 virus detection in this study. METHODS: RT-PCR kits targeting E (envelope) gene, N (nucleocapsid) gene and RdRP (RNA-dependent RNA polymerase) gene of SARS-CoV-2 from Roche Diagnostics were evaluated and E gene kit was employed for quantitative detection of COVID-19 virus using Cobas Z480. Viral load was calculated according to the standard curve established by series dilution of an E-gene RNA standard provided by Tib-Molbiol (a division of Roche Diagnostics). Assay performance was evaluated. RESULTS: The performance of the assay is acceptable with limit of detection (LOD) below 10E1 copies/µL and lower limit of quantification (LLOQ) as 10E2 copies/µL. CONCLUSION: A quantitative detection of the COVID-19 virus based on RT-PCR was established.


Subject(s)
COVID-19/diagnosis , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Humans , Limit of Detection , Phosphoproteins/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Load/methods
7.
Viruses ; 13(12)2021 12 10.
Article in English | MEDLINE | ID: covidwho-1572657

ABSTRACT

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/isolation & purification , Brazil/epidemiology , COVID-19/epidemiology , Coronavirus RNA-Dependent RNA Polymerase/genetics , DNA Primers , False Negative Reactions , Genome, Viral/genetics , Humans , Mutation , Phosphoproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics
8.
Infect Genet Evol ; 97: 105153, 2022 01.
Article in English | MEDLINE | ID: covidwho-1521407

ABSTRACT

Amid the ongoing COVID-19 pandemic, 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 and the proteins comprising the RNA-Dependent RNA Polymerase (RdRP) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences were downloaded from the GISAID database, aligned, and the variants identified. 437,006 unique viral genomes were analyzed. Polymorphisms in the protein sequence were investigated and examined longitudinally to identify sequence and strain variants appearing between January 5th, 2020 and January 16th, 2021. A structural analysis was also performed to investigate mutations in the receptor binding domain and the N-terminal domain of the spike protein. Within the spike protein, there were 766 unique mutations observed in the N-terminal domain and 360 in the receptor binding domain. Four residues that directly contact ACE2 were mutated in more than 100 sequences, including positions K417, Y453, S494, and N501. Within the furin cleavage site of the spike protein, a high degree of conservation was observed, but the P681H mutation was observed in 10.47% of sequences analyzed. Within the RNA dependent RNA polymerase complex proteins, 327 unique mutations were observed in Nsp8, 166 unique mutations were observed in Nsp7, and 1157 unique mutations were observed in Nsp12. Only 4 sequences analyzed contained mutations in the 9 residues that directly interact with the therapeutic Remdesivir, suggesting limited mutations in drug interacting residues. The identification of new variants emphasizes the need for further study on the effects of the mutations and the implications of increased prevalence, particularly for vaccine or therapeutic efficacy.


Subject(s)
COVID-19/epidemiology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Genome, Viral , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Africa/epidemiology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Asia/epidemiology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Databases, Factual , Epidemiological Monitoring , Europe/epidemiology , Evolution, Molecular , Furin/genetics , Furin/metabolism , Gene Expression , Humans , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , United States/epidemiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
9.
Biologicals ; 75: 29-36, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1517058

ABSTRACT

The RNA dependent RNA polymerase (RdRp) plays crucial role in virus life cycle by replicating the viral genome. The SARS-CoV-2 is an RNA virus that rapidly spread worldwide and acquired mutations. This study was carried out to identify mutations in RdRp as the SARS-CoV-2 spread in India. We compared 50217 RdRp sequences reported from India with the first reported RdRp sequence from Wuhan, China to identify 223 mutations acquired among Indian isolates. Our protein modelling study revealed that several mutants can potentially alter stability and flexibility of RdRp. We predicted the potential B cell epitopes contributed by RdRp and identified thirty-six linear continuous and twenty-five discontinuous epitopes. Among 223 RdRp mutants, 44% of them localises in the B cell epitopes region. Altogether, this study highlights the need to identify and characterize the variations in RdRp to understand the impact of these mutations on SARS-CoV-2.


Subject(s)
COVID-19/immunology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/immunology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Mutation , SARS-CoV-2/enzymology , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Enzyme Stability/genetics , Humans , India , SARS-CoV-2/genetics , SARS-CoV-2/immunology
10.
Virus Res ; 307: 198618, 2022 01 02.
Article in English | MEDLINE | ID: covidwho-1504602

ABSTRACT

The second wave of COVID-19 caused by severe acute respiratory syndrome virus (SARS-CoV-2) is rapidly spreading over the world. Mechanisms behind the flee from current antivirals are still unclear due to the continuous occurrence of SARS-CoV-2 genetic variants. Brazil is the world's second-most COVID-19 affected country. In the present study, we identified the genomic and proteomic variants of Brazilian SARS-CoV-2 isolates. We identified 16 different genotypic variants were found among the 27 isolates. The genotypes of three isolates such as Bra/1236/2021 (G15), Bra/MASP2C844R2/2020 (G11), and Bra/RJ-DCVN5/2020 (G9) have a unique mutant in NSP4 (S184N), 2'O-Mutase (R216N), membrane protein (A2V) and Envelope protein (V5A). A mutation in RdRp of SARS-CoV-2, particularly the change of Pro-to Leu-at 323 resulted in the stabilization of the structure in BRA/CD1739-P4/2020. NSP4, NSP5 protein mutants are more virulent in genotype 15 and 16. A fast protein folding rate changes the structural stability and leads to escape for current antivirals. Thus, our findings help researchers to develop the best potent antivirals based on the new mutant of Brazilian isolates.


Subject(s)
Coronavirus 3C Proteases/genetics , Protein Folding , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Brazil , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Genetic Variation/genetics , Genome, Viral/genetics , Humans , Phosphoproteins/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Virulence/genetics
11.
Cell Res ; 31(12): 1230-1243, 2021 12.
Article in English | MEDLINE | ID: covidwho-1475291

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.


Subject(s)
COVID-19/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/drug therapy , COVID-19/mortality , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cytokines/genetics , Cytokines/metabolism , Down-Regulation/drug effects , ErbB Receptors/metabolism , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Indoles/pharmacology , Indoles/therapeutic use , Lung/pathology , Lung/virology , Mice , Mice, Transgenic , Mutation , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Survival Rate , Transcriptome/drug effects , Viral Load/drug effects , Virus Internalization
12.
Viruses ; 13(10)2021 10 18.
Article in English | MEDLINE | ID: covidwho-1471002

ABSTRACT

West Java Health Laboratory (WJHL) is one of the many institutions in Indonesia that have sequenced SARS-CoV-2 genome. Although having submitted a large number of sequences since September 2020, however, these submitted data lack advanced analyses. Therefore, in this study, we analyze the variant distribution, hotspot mutation, and its impact on protein structure and function of SARS-CoV-2 from the collected samples from WJHL. As many as one hundred sixty-three SARS-CoV-2 genome sequences submitted by West Java Health Laboratory (WJHL), with collection dates between September 2020 and June 2021, were retrieved from GISAID. Subsequently, the frequency and distribution of non-synonymous mutations across different cities and regencies from these samples were analyzed. The effect of the most prevalent mutations from dominant variants on the stability of their corresponding proteins was examined. The samples mostly consisted of people of working-age, and were distributed between female and male equally. All of the sample sequences showed varying levels of diversity, especially samples from West Bandung which carried the highest diversity. Dominant variants are the VOC B.1.617.2 (Delta) variant, B.1.466.2 variant, and B.1.470 variant. The genomic regions with the highest number of mutations are the spike, NSP3, nucleocapsid, NSP12, and ORF3a protein. Mutation analysis showed that mutations in structural protein might increase the stability of the protein. Oppositely, mutations in non-structural protein might lead to a decrease in protein stability. However, further research to study the impact of mutations on the function of SARS-CoV-2 proteins are required.


Subject(s)
Genome, Viral/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Papain-Like Proteases/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Disease Hotspot , Female , Humans , Indonesia , Male , Molecular Docking Simulation , Mutation/genetics , Phosphoproteins/genetics , Protein Stability , Spike Glycoprotein, Coronavirus/genetics , Viroporin Proteins/genetics , Whole Genome Sequencing
13.
Sci Rep ; 11(1): 19161, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440480

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2-8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-ß signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online ( http://clip.korea.ac.kr/covid19 ).


Subject(s)
Argonaute Proteins/genetics , COVID-19/prevention & control , MicroRNAs/genetics , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , A549 Cells , Argonaute Proteins/metabolism , Base Sequence , Binding Sites/genetics , COVID-19/virology , Cell Line , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Expression Profiling/methods , HeLa Cells , Humans , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , RNA Interference , RNA-Seq/methods , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid
14.
Sci Rep ; 11(1): 18955, 2021 09 23.
Article in English | MEDLINE | ID: covidwho-1437688

ABSTRACT

The world is facing an exceptional pandemic caused by SARS-CoV-2. To allow the diagnosis of COVID-19 infections, several assays based on the real-time PCR technique have been proposed. The requests for diagnosis are such that it was immediately clear that the choice of the most suitable method for each microbiology laboratory had to be based, on the one hand, on the availability of materials, and on the other hand, on the personnel and training priorities for this activity. Unfortunately, due to high demand, the shortage of commercial diagnostic kits has also become a major problem. To overcome these critical issues, we have developed a new qualitative RT-PCR probe. Our system detects three genes-RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N)-and uses the ß-actin gene as an endogenous internal control. The results from our assay are in complete agreement with the results obtained using a commercially available kit, except for two samples that did not pass the endogenous internal control. The coincidence rate was 0.96. The LoD of our assay was 140 cp/reaction for N and 14 cp/reaction for RdRp and E. Our kit was designed to be open, either for the nucleic acid extraction step or for the RT-PCR assay, and to be carried out on several instruments. Therefore, it is free from the industrial production logics of closed systems, and conversely, it is hypothetically available for distribution in large quantities to any microbiological laboratory. The kit is currently distributed worldwide (called MOLgen-COVID-19; Adaltis). A new version of the kit for detecting the S gene is also available.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , COVID-19/genetics , COVID-19 Testing/methods , Clinical Laboratory Techniques/methods , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Humans , Pandemics , Phosphoproteins/genetics , Qualitative Research , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
15.
PLoS Pathog ; 17(9): e1009929, 2021 09.
Article in English | MEDLINE | ID: covidwho-1430555

ABSTRACT

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Drug Resistance, Microbial/genetics , SARS-CoV-2/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Biological Evolution , COVID-19/drug therapy , Chlorocebus aethiops , Humans , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
16.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1387102

ABSTRACT

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Coronavirus RNA-Dependent RNA Polymerase/genetics , Ebolavirus/drug effects , SARS-CoV-2/drug effects , Virus Replication/drug effects , Adenosine Monophosphate/genetics , Adenosine Monophosphate/metabolism , Alanine/genetics , Alanine/metabolism , Antiviral Agents/metabolism , Base Pairing , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/metabolism , Models, Molecular , Protein Biosynthesis/drug effects , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
17.
J Med Virol ; 93(9): 5339-5349, 2021 09.
Article in English | MEDLINE | ID: covidwho-1363673

ABSTRACT

The present study was conducted from July 1, 2020 to September 25, 2020 in a dedicated coronavirus disease 2019 (COVID-19) hospital in Delhi, India to provide evidence for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in atmospheric air and surfaces of the hospital wards. Swabs from hospital surfaces (patient's bed, ward floor, and nursing stations area) and suspended particulate matter in ambient air were collected by a portable air sampler from the medicine ward, intensive care unit, and emergency ward admitting COVID-19 patients. By performing reverse-transcriptase polymerase chain reaction (RT-PCR) for E-gene and RdRp gene, SARS-CoV-2 virus was detected from hospital surfaces and particulate matters from the ambient air of various wards collected at 1 and 3-m distance from active COVID-19 patients. The presence of the virus in the air beyond a 1-m distance from the patients and surfaces of the hospital indicates that the SARS-CoV-2 virus has the potential to be transmitted by airborne and surface routes from COVID-19 patients to health-care workers working in COVID-19 dedicated hospital. This warrants that precautions against airborne and surface transmission of COVID-19 in the community should be taken when markets, industries, educational institutions, and so on, reopen for normal activities.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/epidemiology , COVID-19/transmission , Fomites/virology , RNA, Viral/genetics , SARS-CoV-2/genetics , Air/analysis , COVID-19/prevention & control , Coronavirus Envelope Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Hospitals , Humans , India/epidemiology , Intensive Care Units , Particulate Matter/analysis
18.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1356909

ABSTRACT

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Interferon-beta , Promoter Regions, Genetic , SARS-CoV-2 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/biosynthesis , Interferon-beta/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
19.
J Med Virol ; 93(10): 6016-6026, 2021 10.
Article in English | MEDLINE | ID: covidwho-1303275

ABSTRACT

Novel mutations have been emerging in the genome of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2); consequently, the evolving of more virulent and treatment resistance strains have the potential to increase transmissibility and mortality rates. The characterization of full-length SARS-CoV-2 genomes is critical for understanding the origin and transmission pathways of the virus, as well as identifying mutations that affect the transmissibility and pathogenicity of the virus. We present an analysis of the mutation pattern and clade distribution of full-length SARS-CoV-2 genome sequences obtained from specimens tested at Gazi University Medical Virology Laboratory. Viral RNA was extracted from nasopharyngeal specimens. Next-generation sequencing libraries were prepared and sequenced on Illumina iSeq 100 platform. Raw sequencing data were processed to obtain full-length genome sequences and variant calling was performed to analyze amino acid changes. Clade distribution was determined to understand the phylogenetic background in relation to global data. A total of 293 distinct mutations were identified, of which 152 missense, 124 synonymous, 12 noncoding, and 5 deletions. The most frequent mutations were P323L (nsp12), D614G (ORF2/S), and 2421C>T (5'-untranslated region) found simultaneously in all sequences. Novel mutations were found in nsp12 (V111A, H133R, Y453C, M626K) and ORF2/S (R995G, V1068L). Nine different Pangolin lineages were detected. The most frequently assigned lineage was B.1.1 (17 sequences), followed by B.1 (7 sequences) and B.1.1.36 (3 sequences). Sequence information is essential for revealing genomic diversity. Mutations might have significant functional implications and analysis of these mutations provides valuable information for therapeutic and vaccine development studies. Our findings point to the introduction of the virus into Turkey through various sources and the subsequent spread of several key variants.


Subject(s)
COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Adult , COVID-19/epidemiology , COVID-19/transmission , Female , Genome, Viral/genetics , Humans , Male , Mutation , Mutation Rate , Phylogeny , RNA, Viral/genetics , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , Turkey/epidemiology
20.
Sci Signal ; 14(690)2021 07 06.
Article in English | MEDLINE | ID: covidwho-1299215

ABSTRACT

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Polyphosphates/pharmacology , SARS-CoV-2/drug effects , Administration, Inhalation , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , COVID-19/metabolism , COVID-19/virology , Caco-2 Cells , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cytokines/metabolism , HEK293 Cells , Host Microbial Interactions/drug effects , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , In Vitro Techniques , Models, Biological , Molecular Docking Simulation , Nebulizers and Vaporizers , Polyphosphates/administration & dosage , Polyphosphates/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Domains and Motifs , Proteolysis/drug effects , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Signal Transduction/drug effects , Vero Cells , Virus Replication/drug effects
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