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1.
Biophys J ; 120(14): 2890-2901, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1604873

ABSTRACT

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1-1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1-1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.


Subject(s)
COVID-19 , SARS-CoV-2 , Coronavirus Nucleocapsid Proteins , Humans , Nucleocapsid/metabolism , Phosphoproteins , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism
2.
STAR Protoc ; 3(1): 101067, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1595326

ABSTRACT

N 6 -methylation of adenosine (m6A) is the most abundant internal mRNA modification and is an important post-transcriptional regulator of gene expression. Here, we describe a protocol for methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to detect and quantify m6A modifications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. The protocol is optimized for low viral RNA levels and is readily adaptable for other applications. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).


Subject(s)
Adenosine/analogs & derivatives , Immunoprecipitation/methods , Sequence Analysis, RNA/methods , Adenosine/analysis , Adenosine/genetics , Animals , COVID-19/genetics , Caco-2 Cells , Chlorocebus aethiops , Gene Expression/genetics , Gene Expression Regulation/genetics , Genetic Techniques , HEK293 Cells , Humans , Methylation , RNA/chemistry , RNA/genetics , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Vero Cells
3.
Signal Transduct Target Ther ; 6(1): 167, 2021 04 24.
Article in English | MEDLINE | ID: covidwho-1585891

ABSTRACT

The ongoing 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has posed a worldwide pandemic and a major global public health threat. The severity and mortality of COVID-19 are associated with virus-induced dysfunctional inflammatory responses and cytokine storms. However, the interplay between host inflammatory responses and SARS-CoV-2 infection remains largely unknown. Here, we demonstrate that SARS-CoV-2 nucleocapsid (N) protein, the major structural protein of the virion, promotes the virus-triggered activation of NF-κB signaling. After binding to viral RNA, N protein robustly undergoes liquid-liquid phase separation (LLPS), which recruits TAK1 and IKK complex, the key kinases of NF-κB signaling, to enhance NF-κB activation. Moreover, 1,6-hexanediol, the inhibitor of LLPS, can attenuate the phase separation of N protein and restrict its regulatory functions in NF-κB activation. These results suggest that LLPS of N protein provides a platform to induce NF-κB hyper-activation, which could be a potential therapeutic target against COVID-19 severe pneumonia.


Subject(s)
COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , NF-kappa B/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Signal Transduction , A549 Cells , Acrylates/pharmacology , Animals , COVID-19/drug therapy , COVID-19/pathology , Chlorocebus aethiops , HEK293 Cells , HeLa Cells , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Phosphoproteins/metabolism , Vero Cells
4.
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
5.
Sci Rep ; 11(1): 24432, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1585772

ABSTRACT

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/metabolism , Animals , Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Down-Regulation/drug effects , Endoplasmic Reticulum Stress , Humans , Inflammasomes/drug effects , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Janus Kinases/genetics , Janus Kinases/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Poly I-C/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification
6.
Front Immunol ; 12: 774776, 2021.
Article in English | MEDLINE | ID: covidwho-1581334

ABSTRACT

Both RNA N6-methyladenosine (m6A) modification of SARS-CoV-2 and immune characteristics of the human body have been reported to play an important role in COVID-19, but how the m6A methylation modification of leukocytes responds to the virus infection remains unknown. Based on the RNA-seq of 126 samples from the GEO database, we disclosed that there is a remarkably higher m6A modification level of blood leukocytes in patients with COVID-19 compared to patients without COVID-19, and this difference was related to CD4+ T cells. Two clusters were identified by unsupervised clustering, m6A cluster A characterized by T cell activation had a higher prognosis than m6A cluster B. Elevated metabolism level, blockage of the immune checkpoint, and lower level of m6A score were observed in m6A cluster B. A protective model was constructed based on nine selected genes and it exhibited an excellent predictive value in COVID-19. Further analysis revealed that the protective score was positively correlated to HFD45 and ventilator-free days, while negatively correlated to SOFA score, APACHE-II score, and crp. Our works systematically depicted a complicated correlation between m6A methylation modification and host lymphocytes in patients infected with SARS-CoV-2 and provided a well-performing model to predict the patients' outcomes.


Subject(s)
Adenosine/analogs & derivatives , COVID-19/immunology , COVID-19/virology , Host-Pathogen Interactions/immunology , Leukocytes/immunology , RNA, Viral/genetics , SARS-CoV-2/physiology , Adenosine/metabolism , Cluster Analysis , Computational Biology/methods , Disease Susceptibility/immunology , Gene Expression Profiling , Humans , Leukocytes/metabolism , RNA, Viral/metabolism , ROC Curve
7.
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
8.
Proc Natl Acad Sci U S A ; 118(52)2021 12 28.
Article in English | MEDLINE | ID: covidwho-1569356

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19), continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2-infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named CoV2-miR-O7a (for SARS-CoV-2 miRNA-like ORF7a-derived small RNA). Its abundance ranges from low to moderate as compared to host miRNAs and it associates with Argonaute proteins-core components of the RNA interference pathway. We identify putative targets for CoV2-miR-O7a, including Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which participates in interferon signaling. We demonstrate that CoV2-miR-O7a production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence.


Subject(s)
Gene Expression Regulation, Viral , RNA, Small Untranslated/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Proteins/genetics , COVID-19/metabolism , COVID-19/virology , Host-Pathogen Interactions , Humans , RNA, Small Untranslated/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics
9.
ACS Appl Mater Interfaces ; 13(50): 60612-60624, 2021 Dec 22.
Article in English | MEDLINE | ID: covidwho-1569206

ABSTRACT

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Nasal Mucosa/virology , Polymers/chemistry , RNA, Viral/metabolism , SARS-CoV-2 , Biofouling , Biological Assay , Biosensing Techniques , Humans , Ions , Limit of Detection , Mass Spectrometry , Nasopharynx/virology , Phosphoproteins/chemistry , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Specimen Handling
10.
Eur Rev Med Pharmacol Sci ; 25(21): 6745-6766, 2021 11.
Article in English | MEDLINE | ID: covidwho-1524863

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has created the current pandemic, has caused a worldwide worry. Different countries have since enforced varying levels of lockdowns and guidelines for their populations to follow in a serious effort to mitigate the spread. Up until recently, the majority of these regulations and policies were established on the assumption that the dominant routes of transmission of this virus are through droplets and fomite contact. However, there is now a substantial amount of research pointing towards the strong possibility that SARS-CoV-2 can spread through airborne means. The World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) have recently recognized this, which poses the question of whether our collective methods of lessening transmission risk and keeping people safe have been sufficient. This paper is a comprehensive review of the evidence on SARS-CoV-2 being an airborne disease, through different epidemiological, experimental, and animal-model based published research. Studies opposing this evidence have also been discussed. The majority of these studies are favoring the high plausibility of SARS-CoV-2 aerosol transmission, and therefore the many implications of aerosol transmission have been discussed in this paper to suggest effective mitigation and control strategies.


Subject(s)
Aerosols , COVID-19/transmission , SARS-CoV-2/physiology , Animals , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Cricetinae , Disease Models, Animal , Equipment and Supplies, Hospital/virology , Feces/virology , Humans , Masks , Pandemics , Particulate Matter , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Ventilation/standards , Virus Inactivation
11.
Int J Mol Sci ; 22(21)2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1518611

ABSTRACT

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Interferon-alpha/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/genetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Exoribonucleases/genetics , Genetic Vectors , HeLa Cells , Humans , Interferon-alpha/administration & dosage , Luciferases/genetics , Luciferases/metabolism , Naphthyridines/administration & dosage , Naphthyridines/pharmacology , Oxadiazoles/administration & dosage , Oxadiazoles/pharmacology , RNA, Viral/drug effects , Replicon
12.
Chem Commun (Camb) ; 57(92): 12270-12272, 2021 Nov 19.
Article in English | MEDLINE | ID: covidwho-1506302

ABSTRACT

An automated Cas12a-microfluidic system was constructed to distinguish the B.1.617.2 (delta) variant of SARS-CoV-2 from the wild-type virus rapidly and was validated using 30 clinical samples, showing 100% consistency with next-generation sequencing. It will be a potential tool for the rapid differential diagnosis of the delta variant of SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Microfluidics/methods , SARS-CoV-2/genetics , Automation , COVID-19/virology , High-Throughput Nucleotide Sequencing , Humans , Mutation , Polymerase Chain Reaction , RNA, Guide/genetics , RNA, Guide/metabolism , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification
13.
J Virol ; 95(12)2021 05 24.
Article in English | MEDLINE | ID: covidwho-1501541

ABSTRACT

Long disregarded as junk DNA or genomic dark matter, endogenous retroviruses (ERVs) have turned out to represent important components of the antiviral immune response. These remnants of once-infectious retroviruses not only regulate cellular immune activation, but may even directly target invading viral pathogens. In this Gem, we summarize mechanisms by which retroviral fossils protect us from viral infections. One focus will be on recent advances in the role of ERVs as regulators of antiviral gene expression.


Subject(s)
Endogenous Retroviruses/physiology , Retroelements , Virus Diseases/immunology , Animals , Endogenous Retroviruses/genetics , Enhancer Elements, Genetic , Gene Expression Regulation , Humans , Immunity, Cellular , Promoter Regions, Genetic , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Receptors, Pattern Recognition/metabolism , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/metabolism , Viral Proteins/metabolism , Virion/metabolism , Virus Diseases/genetics , Virus Diseases/virology
14.
Sci Rep ; 11(1): 21462, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1500517

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 (COVID-19). More than 143 million cases of COVID-19 have been reported to date, with the global death rate at 2.13%. Currently, there are no licensed therapeutics for controlling SARS-CoV-2 infection. The antiviral effects of heme oxygenase-1 (HO-1), a cytoprotective enzyme that inhibits the inflammatory response and reduces oxidative stress, have been investigated in several viral infections. To confirm whether HO-1 suppresses SARS-CoV-2 infection, we assessed the antiviral activity of hemin, an effective and safe HO-1 inducer, in SARS-CoV-2 infection. We found that treatment with hemin efficiently suppressed SARS-CoV-2 replication (selectivity index: 249.7012). Besides, the transient expression of HO-1 using an expression vector also suppressed the growth of the virus in cells. Free iron and biliverdin, which are metabolic byproducts of heme catalysis by HO-1, also suppressed the viral infection. Additionally, hemin indirectly increased the expression of interferon-stimulated proteins known to restrict SARS-CoV-2 replication. Overall, the findings suggested that HO-1, induced by hemin, effectively suppressed SARS-CoV-2 in vitro. Therefore, HO-1 could be potential therapeutic candidate for COVID-19.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Heme Oxygenase-1/metabolism , Hemin/therapeutic use , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/virology , Cell Survival/drug effects , Chlorocebus aethiops , Heme Oxygenase-1/antagonists & inhibitors , Heme Oxygenase-1/genetics , Hemin/chemistry , Hemin/pharmacology , Humans , RNA Interference , RNA, Small Interfering/metabolism , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Up-Regulation/drug effects , Vero Cells , Virus Replication/drug effects
15.
Int J Mol Sci ; 22(21)2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1488612

ABSTRACT

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Interferon-alpha/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/genetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Exoribonucleases/genetics , Genetic Vectors , HeLa Cells , Humans , Interferon-alpha/administration & dosage , Luciferases/genetics , Luciferases/metabolism , Naphthyridines/administration & dosage , Naphthyridines/pharmacology , Oxadiazoles/administration & dosage , Oxadiazoles/pharmacology , RNA, Viral/drug effects , Replicon
16.
PLoS Pathog ; 17(10): e1009726, 2021 10.
Article in English | MEDLINE | ID: covidwho-1484867

ABSTRACT

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.


Subject(s)
Prenylation/physiology , RNA Viruses/drug effects , RNA-Binding Proteins/pharmacology , Virus Replication/physiology , CpG Islands/physiology , HEK293 Cells , HIV-1/physiology , HeLa Cells , Humans , RNA Viruses/physiology , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Binding Motifs/physiology , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , SARS-CoV-2/physiology , Transfection , Virus Replication/drug effects
17.
Genome Biol Evol ; 13(11)2021 11 05.
Article in English | MEDLINE | ID: covidwho-1483441

ABSTRACT

Adenosine Deaminases that Act on RNA (ADARs) are RNA editing enzymes that play a dynamic and nuanced role in regulating transcriptome and proteome diversity. This editing can be highly selective, affecting a specific site within a transcript, or nonselective, resulting in hyperediting. ADAR editing is important for regulating neural functions and autoimmunity, and has a key role in the innate immune response to viral infections, where editing can have a range of pro- or antiviral effects and can contribute to viral evolution. Here we examine the role of ADAR editing across a broad range of viral groups. We propose that the effect of ADAR editing on viral replication, whether pro- or antiviral, is better viewed as an axis rather than a binary, and that the specific position of a given virus on this axis is highly dependent on virus- and host-specific factors, and can change over the course of infection. However, more research needs to be devoted to understanding these dynamic factors and how they affect virus-ADAR interactions and viral evolution. Another area that warrants significant attention is the effect of virus-ADAR interactions on host-ADAR interactions, particularly in light of the crucial role of ADAR in regulating neural functions. Answering these questions will be essential to developing our understanding of the relationship between ADAR editing and viral infection. In turn, this will further our understanding of the effects of viruses such as SARS-CoV-2, as well as many others, and thereby influence our approach to treating these deadly diseases.


Subject(s)
Adenosine Deaminase/metabolism , RNA Editing , RNA Viruses/genetics , Adenosine Deaminase/genetics , Animals , Evolution, Molecular , Host-Pathogen Interactions/immunology , Humans , Immunity , RNA Viruses/classification , RNA Viruses/physiology , RNA, Viral/genetics , RNA, Viral/metabolism , Virus Replication/genetics
18.
Commun Biol ; 4(1): 1215, 2021 10 22.
Article in English | MEDLINE | ID: covidwho-1479821

ABSTRACT

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/genetics , COVID-19/metabolism , Coronavirus Nucleocapsid Proteins , Polymerase Chain Reaction/methods , RNA, Viral/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Transcriptome , Computational Biology , Humans , Limit of Detection , Open Reading Frames , Phosphoproteins , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reproducibility of Results
19.
Biosensors (Basel) ; 11(10)2021 Oct 13.
Article in English | MEDLINE | ID: covidwho-1470794

ABSTRACT

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Humans , Microelectrodes , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Phosphoproteins/genetics , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/metabolism , Reaction Time , SARS-CoV-2/isolation & purification , Saliva/virology
20.
PLoS Pathog ; 17(9): e1009947, 2021 09.
Article in English | MEDLINE | ID: covidwho-1470670

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) preferentially infects and causes Kaposi's sarcoma (KS) in male patients. However, the biological mechanisms are largely unknown. This study was novel in confirming the extensive nuclear distribution of the androgen receptor (AR) and its co-localization with viral oncoprotein of latency-associated nuclear antigen in KS lesions, indicating a transcription way of AR in KS pathogenesis. The endogenous AR was also remarkably higher in KSHV-positive B cells than in KSHV-negative cells and responded to the ligand treatment of 5α-dihydrotestosterone (DHT), the agonist of AR. Then, the anti-AR antibody-based chromatin immunoprecipitation (ChIP)-associated sequencing was used to identify the target viral genes of AR, revealing that the AR bound to multiple regions of lytic genes in the KSHV genome. The highest peak was enriched in the core promoter sequence of polyadenylated nuclear RNA (PAN), and the physical interaction was verified by ChIP-polymerase chain reaction (PCR) and the electrophoretic mobility shift assay (EMSA). Consistently, male steroid treatment significantly transactivated the promoter activity of PAN in luciferase reporter assay, consequently leading to extensive lytic gene expression and KSHV production as determined by real-time quantitative PCR, and the deletion of nuclear localization signals of AR resulted in the loss of nuclear transport and transcriptional activity in the presence of androgen and thus impaired the expression of PAN RNA. Oncogenically, this study identified that the AR was a functional prerequisite for cell invasion, especially under the context of KSHV reactivation, through hijacking the PAN as a critical effector. Taken together, a novel mechanism from male sex steroids to viral noncoding RNA was identified, which might provide a clue to understanding the male propensity in KS.


Subject(s)
RNA, Messenger/metabolism , RNA, Viral/metabolism , Receptors, Androgen/metabolism , Sarcoma, Kaposi/metabolism , Sex Characteristics , Carcinogenesis/metabolism , Female , Herpesvirus 8, Human , Humans , Male , RNA, Untranslated/metabolism
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