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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1350002

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
13.
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
14.
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
15.
Sci Rep ; 11(1): 13705, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1294480

ABSTRACT

The D614G mutation in the Spike protein of the SARS-CoV-2 has effectively replaced the early pandemic-causing variant. Using pseudotyped lentivectors, we confirmed that the aspartate replacement by glycine in position 614 is markedly more infectious. Molecular modelling suggests that the G614 mutation facilitates transition towards an open state of the Spike protein. To explain the epidemiological success of D614G, we analysed the evolution of 27,086 high-quality SARS-CoV-2 genome sequences from GISAID. We observed striking coevolution of D614G with the P323L mutation in the viral polymerase. Importantly, the exclusive presence of G614 or L323 did not become epidemiologically relevant. In contrast, the combination of the two mutations gave rise to a viral G/L variant that has all but replaced the initial D/P variant. Our results suggest that the P323L mutation, located in the interface domain of the RNA-dependent RNA polymerase, is a necessary alteration that led to the epidemiological success of the present variant of SARS-CoV-2. However, we did not observe a significant correlation between reported COVID-19 mortality in different countries and the prevalence of the Wuhan versus G/L variant. Nevertheless, when comparing the speed of emergence and the ultimate predominance in individual countries, it is clear that the G/L variant displays major epidemiological supremacy over the original variant.


Subject(s)
COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Point Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , COVID-19/epidemiology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Humans , Models, Molecular , Protein Conformation , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry
16.
Biomolecules ; 11(7)2021 06 22.
Article in English | MEDLINE | ID: covidwho-1282440

ABSTRACT

SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) protein is the target for the antiviral drug Remdesivir (RDV). With RDV clinical trials on COVID-19 patients showing a reduced hospitalisation time. During the spread of the virus, the RdRp has developed several mutations, with the most frequent being A97V and P323L. The current study sought to investigate whether A97V and P323L mutations influence the binding of RDV to the RdRp of SARS-CoV-2 compared to wild-type (WT). The interaction of RDV with WT-, A97V-, and P323L-RdRp were measured using molecular dynamic (MD) simulations, and the free binding energies were extracted. Results showed that RDV that bound to WT- and A97V-RdRp had a similar dynamic motion and internal residue fluctuations, whereas RDV interaction with P323L-RdRp exhibited a tighter molecular conformation, with a high internal motion near the active site. This was further corroborated with RDV showing a higher binding affinity to P323L-RdRp (-24.1 kcal/mol) in comparison to WT-RdRp (-17.3 kcal/mol). This study provides insight into the potential significance of administering RDV to patients carrying the SARS-CoV-2 P323L-RdRp mutation, which may have a more favourable chance of alleviating the SARS-CoV-2 illness in comparison to WT-RdRp carriers, thereby suggesting further scientific consensus for the usage of Remdesivir as clinical candidate against COVID-19.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus RNA-Dependent RNA Polymerase/genetics , Point Mutation , SARS-CoV-2/genetics , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Binding Sites/drug effects , COVID-19/virology , Catalytic Domain/drug effects , Humans , Molecular Dynamics Simulation , Point Mutation/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
17.
mBio ; 12(3): e0142321, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1280400

ABSTRACT

The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCE In vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Guanosine Tetraphosphate/pharmacology , SARS-CoV-2/drug effects , Adenosine Triphosphate/pharmacology , COVID-19/drug therapy , Catalytic Domain/physiology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Humans , Ribosomes/metabolism
18.
Comput Biol Chem ; 93: 107532, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1275230

ABSTRACT

Zoonotic Novel coronavirus disease 2019 (COVID-19) is highly pathogenic and transmissible considered as emerging pandemic disease. The virus belongs from a large virus Coronaviridae family affect respiratory tract of animal and human likely originated from bat and homology to SARA-CoV and MERS-CoV. The virus consists of single-stranded positive genomic RNA coated by nucleocapsid protein. The rate of mutation in any virulence gene may influence the phenomenon of host radiation. We have studied the molecular evolution of selected virulence genes (HA, N, RdRP and S) of novel COVID-19. We used a site-specific comparison of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions. Maximum Likelihood genealogies based on differential gamma distribution rates were used for the analysis of null and alternate hypothesis. The null hypothesis was found more suitable for the analysis using Likelihood Ratio Test (LRT) method, confirming higher rate of substitution. The analysis revealed that RdRP gene had the fastest rate evolution followed by HA gene. We have also reported the new motifs for different virulence genes, which are further useful to design new detection and diagnosis kit for COVID -19.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Hemagglutinins/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virulence/genetics , Amino Acid Substitution , Base Sequence , Evolution, Molecular , Genes, Viral , Phosphoproteins/genetics , SARS-CoV-2/pathogenicity
19.
J Am Soc Mass Spectrom ; 32(7): 1618-1630, 2021 Jul 07.
Article in English | MEDLINE | ID: covidwho-1267989

ABSTRACT

Coronavirus (CoV) nonstructural 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 and other nsps. To date, no structure of the full-length SARS-CoV-2 nsp7:nsp8 complex has been published. The current understanding of this complex is based on structures from truncated constructs, with missing electron densities, or from related CoV species where SARS-CoV-2 nsp7 and nsp8 share upward of 90% sequence identity. Despite available structures solved using crystallography and cryo-EM representing detailed static 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 individual proteins and how they complex to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and cross-linking mass spectrometry (XL-MS), illuminate the dynamics of SARS-CoV-2 full-length nsp7 and nsp8 proteins and the nsp7:nsp8 protein complex. Results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of the 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 significantly altering nsp7-nsp8 interactions.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Proteomics/methods , Viral Nonstructural Proteins/chemistry , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Hydrogen Deuterium Exchange-Mass Spectrometry , Models, Molecular , Protein Conformation , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
20.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1263454

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
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