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1.
Biophys J ; 120(14): 2814-2827, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1604124

ABSTRACT

The nucleocapsid (N) protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. The N protein N-terminal domain (N-NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRSs), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of the severe acute respiratory syndrome coronavirus 2 N-NTD to nonspecific (NS) and TRS dsRNAs. We probed dsRNAs' Watson-Crick basepairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, triggering melting initiation. dsRNA destabilization driven by N-NTD was more efficient for dsTRSs than dsNS. N-NTD dynamics, especially a tweezer-like motion of ß2-ß3 and Δ2-ß5 loops, seems to play a key role in Watson-Crick basepairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD/dsRNA), matching MD simulations and raising different possibilities for N-NTD action: 1) two N-NTD arms of dimeric N would bind to two different RNA sites, either closely or spatially spaced in the viral genome, in a cooperative manner; and 2) monomeric N-NTD would be active, opening up the possibility of a regulatory dissociation event.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleocapsid Proteins/genetics , Nucleoproteins , RNA
2.
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
3.
J Korean Med Sci ; 36(48): e328, 2021 Dec 13.
Article in English | MEDLINE | ID: covidwho-1572278

ABSTRACT

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. METHODS: A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. RESULTS: Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. CONCLUSION: The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era.


Subject(s)
COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Nasopharynx/virology , RNA, Viral/analysis , Respiratory Syncytial Virus Infections/diagnosis , COVID-19/virology , Cross Reactions , Humans , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza B virus/genetics , Influenza B virus/isolation & purification , Influenza, Human/virology , Limit of Detection , Nucleocapsid Proteins/genetics , Polyproteins/genetics , RNA, Viral/metabolism , Reagent Kits, Diagnostic , Republic of Korea , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/genetics , Viral Proteins/genetics
4.
J Virol Methods ; 299: 114341, 2022 01.
Article in English | MEDLINE | ID: covidwho-1474839

ABSTRACT

The COVID-19 pandemic has demanded a range of biotechnological products for detection of SARS-CoV-2 variants and evaluation of human seroconversion after infection or vaccination. In this work, we describe an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used for infection of lepidopteran Sf9 cells adapted to high-density suspension. Using Tris-HCl pH 8.0 buffer as mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg N protein could be purified from each 50 mg of total protein from clarified supernatant. Such protein amount allows the manufacturing of serological tests and the development of basic studies on cellular responses to SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Insecta , Nucleocapsid , Nucleocapsid Proteins/genetics , Pandemics
5.
PLoS Biol ; 19(10): e3001425, 2021 10.
Article in English | MEDLINE | ID: covidwho-1463301

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , SARS-CoV-2/metabolism , Viral Genome Packaging/physiology , Animals , COVID-19/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Genome, Viral , Genomics , HEK293 Cells , Humans , Nucleocapsid Proteins/genetics , Phosphoproteins/metabolism , Protein Domains , RNA, Viral/genetics , SARS-CoV-2/genetics , Vero Cells
6.
PLoS One ; 16(9): e0257350, 2021.
Article in English | MEDLINE | ID: covidwho-1435609

ABSTRACT

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Nose/virology , Nucleocapsid Proteins/genetics , Oropharynx/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Adolescent , Brazil , COVID-19/virology , DNA Primers/genetics , Female , Humans , Male , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics , Reference Standards , Sensitivity and Specificity , Specimen Handling/methods
7.
J Clin Microbiol ; 59(10): e0100121, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1430156

ABSTRACT

The purpose of this study was to characterize the diagnostic performance of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of SARS-CoV-2 nucleocapsid protein (NP) in blood. Blood samples were collected during hospitalization of 165 inpatients with PCR-confirmed SARS-CoV-2 infection and from 505 outpatients predominantly with relevant symptoms of COVID-19 simultaneously with PCR testing. For the 143 inpatients who had their first blood sample collected within 2 weeks after PCR-confirmed infection, the diagnostic sensitivity of the ELISA was 91.6%. The mean NP concentration of the 131 ELISA-positive blood samples was 1,734 pg/ml (range, 10 to 3,840 pg/ml). An exponential decline in NP concentration was observed for 368 blood samples collected over the first 4 weeks after PCR-confirmed SARS-CoV-2 infection, and all blood samples taken later had an NP concentration below the 10-pg/ml diagnostic cutoff. The diagnostic sensitivity of the ELISA was 81.4% for the 43 blood samples collected from outpatients with a simultaneous positive PCR test, and the mean NP concentration of the 35 ELISA-positive samples was 157 pg/ml (range, 10 to 1,377 pg/ml). For the 462 outpatients with a simultaneous negative PCR test, the diagnostic specificity of the ELISA was 99.8%. In conclusion, the SARS-CoV-2 NP ELISA is a suitable laboratory diagnostic test for COVID-19, particularly for hospitals, where blood samples are readily available and screening of serum or plasma by ELISA can facilitate prevention of nosocomial infections and reduce the requirement for laborious swab sampling and subsequent PCR analysis to confirmatory tests only.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Clinical Laboratory Techniques , Enzyme-Linked Immunosorbent Assay , Humans , Laboratories , Nucleocapsid Proteins/genetics , Sensitivity and Specificity
8.
Int J Environ Res Public Health ; 18(18)2021 09 13.
Article in English | MEDLINE | ID: covidwho-1409577

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the assay's performance, with the plaque reduction neutralization test and the commercial SARS-CoV-2 spike protein IgG ELISA as gold standards. The SARS-CoV-2 N truncated protein-based ELISA showed similar sensitivity (91.1% vs. 91.9%) and specificity (93.8% vs. 93.8%) between the PRNT and spike IgG ELISA, as well as also higher specificity compared to the full-length N protein (93.8% vs. 89.9%). Our ELISA can be used for the diagnosis and surveillance of COVID-19.


Subject(s)
COVID-19 , Nucleocapsid Proteins , Antibodies, Viral , COVID-19 Testing , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Humans , Immunoglobulin G , Nucleocapsid Proteins/genetics , SARS-CoV-2 , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus
12.
Braz J Microbiol ; 52(4): 2069-2073, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1338313

ABSTRACT

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Seroconversion , Antibodies, Viral/blood , COVID-19/diagnosis , COVID-19/immunology , Humans , Immunity, Humoral , Nucleocapsid Proteins/genetics , Phosphoproteins/immunology , Sensitivity and Specificity
13.
Talanta ; 235: 122691, 2021 Dec 01.
Article in English | MEDLINE | ID: covidwho-1313446

ABSTRACT

The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. This protein has a strong tendency for dimerization and interaction with nucleic acids. For the first time, high titers of NP were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of downstream processing (DSP) steps. An analytical method consisting of native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77% after cell lysis. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The optimized HIC-MALS method enables monitoring of the product purity, and simultaneously access its molecular mass, providing orthogonal information complementary to established SEC-MALS methods. Enhanced resolving power can be achieved over SEC, attributed to the extended variables to tune selectivity in HIC mode.


Subject(s)
COVID-19 , Nucleocapsid Proteins , Chromatography , Escherichia coli/genetics , Humans , Hydrophobic and Hydrophilic Interactions , Nucleocapsid Proteins/genetics , SARS-CoV-2
14.
Viruses ; 12(10)2020 10 18.
Article in English | MEDLINE | ID: covidwho-1305818

ABSTRACT

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.


Subject(s)
Copper/metabolism , Nucleocapsid Proteins/metabolism , Nucleocapsid/metabolism , Prions/metabolism , Zinc/metabolism , Computational Biology , Meta-Analysis as Topic , Molecular Dynamics Simulation , Neurodegenerative Diseases/virology , Nucleocapsid/genetics , Nucleocapsid Proteins/genetics , Prions/genetics , Protein Domains , Viral Proteins/genetics , Viral Proteins/metabolism
15.
Nat Cell Biol ; 23(7): 718-732, 2021 07.
Article in English | MEDLINE | ID: covidwho-1303773

ABSTRACT

Patients with Coronavirus disease 2019 exhibit low expression of interferon-stimulated genes, contributing to a limited antiviral response. Uncovering the underlying mechanism of innate immune suppression and rescuing the innate antiviral response remain urgent issues in the current pandemic. Here we identified that the dimerization domain of the SARS-CoV-2 nucleocapsid protein (SARS2-NP) is required for SARS2-NP to undergo liquid-liquid phase separation with RNA, which inhibits Lys63-linked poly-ubiquitination and aggregation of MAVS and thereby suppresses the innate antiviral immune response. Mice infected with an RNA virus carrying SARS2-NP exhibited reduced innate immunity, an increased viral load and high morbidity. Notably, we identified SARS2-NP acetylation at Lys375 by host acetyltransferase and reported frequently occurring acetylation-mimicking mutations of Lys375, all of which impaired SARS2-NP liquid-liquid phase separation with RNA. Importantly, a peptide targeting the dimerization domain was screened out to disrupt the SARS2-NP liquid-liquid phase separation and demonstrated to inhibit SARS-CoV-2 replication and rescue innate antiviral immunity both in vitro and in vivo.


Subject(s)
Nucleocapsid Proteins/immunology , Nucleocapsid Proteins/metabolism , SARS-CoV-2/genetics , Animals , Immunity, Innate/immunology , Immunity, Innate/physiology , Mice , Nucleocapsid Proteins/genetics , RNA Viruses/genetics , SARS-CoV-2/physiology
16.
Sci Rep ; 11(1): 13804, 2021 07 05.
Article in English | MEDLINE | ID: covidwho-1297315

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a pandemic threat worldwide and causes severe health and economic burdens. Contaminated environments, such as personal items and room surfaces, are considered to have virus transmission potential. Ultraviolet C (UVC) light has demonstrated germicidal ability and removes environmental contamination. UVC has inactivated SARS-CoV-2; however, the underlying mechanisms are not clear. It was confirmed here that UVC 253.7 nm, with a dose of 500 µW/cm2, completely inactivated SARS-CoV-2 in a time-dependent manner and reduced virus infectivity by 10-4.9-fold within 30 s. Immunoblotting analysis for viral spike and nucleocapsid proteins showed that UVC treatment did not damage viral proteins. The viral particle morphology remained intact even when the virus completely lost infectivity after UVC irradiation, as observed by transmission electronic microscopy. In contrast, UVC irradiation-induced genome damage was identified using the newly developed long reverse-transcription quantitative-polymerase chain reaction (RT-qPCR) assay, but not conventional RT-qPCR. The six developed long RT-PCR assays that covered the full-length viral genome clearly indicated a negative correlation between virus infectivity and UVC irradiation-induced genome damage (R2 ranging from 0.75 to 0.96). Altogether, these results provide evidence that UVC inactivates SARS-CoV-2 through the induction of viral genome damage.


Subject(s)
Disinfection , RNA, Viral/radiation effects , SARS-CoV-2 , Ultraviolet Rays , Virus Inactivation/radiation effects , Animals , COVID-19/prevention & control , Chlorocebus aethiops , Disinfection/methods , Genome, Viral/genetics , Nucleocapsid Proteins/genetics , RNA, Viral/analysis , SARS-CoV-2/pathogenicity , SARS-CoV-2/radiation effects , Vero Cells
17.
Brief Bioinform ; 22(6)2021 11 05.
Article in English | MEDLINE | ID: covidwho-1276146

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of the coronavirus disease (COVID-19), is a part of the $\beta $-Coronaviridae family. The virus contains five major protein classes viz., four structural proteins [nucleocapsid (N), membrane (M), envelop (E) and spike glycoprotein (S)] and replicase polyproteins (R), synthesized as two polyproteins (ORF1a and ORF1ab). Due to the severity of the pandemic, most of the SARS-CoV-2-related research are focused on finding therapeutic solutions. However, studies on the sequences and structure space throughout the evolutionary time frame of viral proteins are limited. Besides, the structural malleability of viral proteins can be directly or indirectly associated with the dysfunctionality of the host cell proteins. This dysfunctionality may lead to comorbidities during the infection and may continue at the post-infection stage. In this regard, we conduct the evolutionary sequence-structure analysis of the viral proteins to evaluate their malleability. Subsequently, intrinsic disorder propensities of these viral proteins have been studied to confirm that the short intrinsically disordered regions play an important role in enhancing the likelihood of the host proteins interacting with the viral proteins. These interactions may result in molecular dysfunctionality, finally leading to different diseases. Based on the host cell proteins, the diseases are divided in two distinct classes: (i) proteins, directly associated with the set of diseases while showing similar activities, and (ii) cytokine storm-mediated pro-inflammation (e.g. acute respiratory distress syndrome, malignancies) and neuroinflammation (e.g. neurodegenerative and neuropsychiatric diseases). Finally, the study unveils that males and postmenopausal females can be more vulnerable to SARS-CoV-2 infection due to the androgen-mediated protein transmembrane serine protease 2.


Subject(s)
COVID-19/genetics , Genome, Viral/genetics , Protein Conformation , SARS-CoV-2/ultrastructure , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/ultrastructure , Humans , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/ultrastructure , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/ultrastructure , Viral Replicase Complex Proteins/genetics , Viral Replicase Complex Proteins/ultrastructure , Viral Structural Proteins/genetics , Viral Structural Proteins/ultrastructure
18.
J Biol Chem ; 297(1): 100821, 2021 07.
Article in English | MEDLINE | ID: covidwho-1240418

ABSTRACT

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein-mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.


Subject(s)
Arginine/metabolism , COVID-19/metabolism , COVID-19/virology , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/physiology , Amino Acid Motifs , COVID-19/genetics , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , HEK293 Cells , Humans , Methylation , Nucleocapsid Proteins/genetics , RNA Stability , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Virus Replication
19.
Anal Chem ; 93(19): 7283-7291, 2021 05 18.
Article in English | MEDLINE | ID: covidwho-1217666

ABSTRACT

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.


Subject(s)
COVID-19 , Camelids, New World , Single-Domain Antibodies , Animals , Humans , Nucleocapsid Proteins/genetics , SARS-CoV-2
20.
Nat Commun ; 12(1): 2114, 2021 04 09.
Article in English | MEDLINE | ID: covidwho-1174670

ABSTRACT

Lack of detailed knowledge of SARS-CoV-2 infection has been hampering the development of treatments for coronavirus disease 2019 (COVID-19). Here, we report that RNA triggers the liquid-liquid phase separation (LLPS) of the SARS-CoV-2 nucleocapsid protein, N. By analyzing all 29 proteins of SARS-CoV-2, we find that only N is predicted as an LLPS protein. We further confirm the LLPS of N during SARS-CoV-2 infection. Among the 100,849 genome variants of SARS-CoV-2 in the GISAID database, we identify that ~37% (36,941) of the genomes contain a specific trio-nucleotide polymorphism (GGG-to-AAC) in the coding sequence of N, which leads to the amino acid substitutions, R203K/G204R. Interestingly, NR203K/G204R exhibits a higher propensity to undergo LLPS and a greater effect on IFN inhibition. By screening the chemicals known to interfere with N-RNA binding in other viruses, we find that (-)-gallocatechin gallate (GCG), a polyphenol from green tea, disrupts the LLPS of N and inhibits SARS-CoV-2 replication. Thus, our study reveals that targeting N-RNA condensation with GCG could be a potential treatment for COVID-19.


Subject(s)
Amino Acid Substitution/drug effects , COVID-19/prevention & control , Catechin/analogs & derivatives , Nucleocapsid Proteins/genetics , SARS-CoV-2/drug effects , Virus Replication/drug effects , COVID-19/virology , Catechin/pharmacology , Genome, Viral/genetics , Humans , Liquid-Liquid Extraction , Nucleocapsid Proteins/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , Virus Replication/genetics
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