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1.
Viruses ; 14(2)2022 02 21.
Article in English | MEDLINE | ID: covidwho-1744920

ABSTRACT

Involvement of macrophages in the SARS-CoV-2-associated cytokine storm, the excessive secretion of inflammatory/anti-viral factors leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, is unclear. In this study, we sought to characterize the interplay between the virus and primary human monocyte-derived macrophages (MDM). MDM were stimulated with recombinant IFN-α and/or infected with either live or UV-inactivated SARS-CoV-2 or with two reassortant influenza viruses containing external genes from the H1N1 PR8 strain and heterologous internal genes from a highly pathogenic avian H5N1 or a low pathogenic human seasonal H1N1 strain. Virus replication was monitored by qRT-PCR for the E viral gene for SARS-CoV-2 or M gene for influenza and TCID50 or plaque assay, and cytokine levels were assessed semiquantitatively with qRT-PCR and a proteome cytokine array. We report that MDM are not susceptible to SARS-CoV-2 whereas both influenza viruses replicated in MDM, albeit abortively. We observed a modest cytokine response in SARS-CoV-2 exposed MDM with notable absence of IFN-ß induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-α enhanced proinflammatory cytokine expression upon exposure to virus. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages.


Subject(s)
Inflammation/virology , Macrophages/immunology , Macrophages/virology , SARS-CoV-2/immunology , Virus Replication/genetics , Cell Line , Cell Line, Tumor , Cells, Cultured , Cytokines/analysis , Cytokines/immunology , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/immunology , Interferon-alpha/pharmacology , Macrophages/drug effects , Male , SARS-CoV-2/genetics , SARS-CoV-2/physiology
2.
J Virol ; 96(7): e0151621, 2022 Apr 13.
Article in English | MEDLINE | ID: covidwho-1744134

ABSTRACT

ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.


Subject(s)
Coronavirus , Poly Adenosine Diphosphate Ribose , Adenosine Diphosphate Ribose/metabolism , Animals , Coronavirus/genetics , Mammals/genetics , RNA, Viral/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
3.
J Virol ; 96(4): e0196921, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1702819

ABSTRACT

Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited antiviral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, ∼50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine (Cxcl9, Cxcl10, Ccl2, Ccl5 and Ccl19) and cytokine (Ifn-λ and Tnf-α) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease. IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , Central Nervous System Viral Diseases/immunology , Microglia/immunology , SARS-CoV-2/physiology , Virus Replication/immunology , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , Central Nervous System/immunology , Central Nervous System/virology , Central Nervous System Viral Diseases/genetics , Central Nervous System Viral Diseases/virology , Chemokines/genetics , Chemokines/immunology , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Microglia/virology , Neurons/immunology , Neurons/virology , Virus Replication/genetics
4.
Commun Biol ; 5(1): 154, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1699831

ABSTRACT

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Exonucleases/antagonists & inhibitors , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Sequence , Anilides/pharmacology , Animals , Base Sequence , Benzimidazoles/pharmacology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Drug Synergism , Exonucleases/genetics , Exonucleases/metabolism , Humans , Proline/pharmacology , Pyrrolidines/pharmacology , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Valine/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Virus Replication/genetics
5.
Sci Rep ; 12(1): 2420, 2022 02 14.
Article in English | MEDLINE | ID: covidwho-1684102

ABSTRACT

The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.


Subject(s)
COVID-19/genetics , Dinucleoside Phosphates/genetics , Genome, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Animals , Base Sequence , Binding Sites/genetics , COVID-19/virology , Dinucleoside Phosphates/metabolism , Evolution, Molecular , Host-Pathogen Interactions/genetics , Humans , Nucleotide Motifs/genetics , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/physiology , Virus Replication/genetics
6.
Signal Transduct Target Ther ; 7(1): 29, 2022 01 28.
Article in English | MEDLINE | ID: covidwho-1655546

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted on mink farms between minks and humans in many countries. However, the systemic pathological features of SARS-CoV-2-infected minks are mostly unknown. Here, we demonstrated that minks were largely permissive to SARS-CoV-2, characterized by severe and diffuse alveolar damage, and lasted at least 14 days post inoculation (dpi). We first reported that infected minks displayed multiple organ-system lesions accompanied by an increased inflammatory response and widespread viral distribution in the cardiovascular, hepatobiliary, urinary, endocrine, digestive, and immune systems. The viral protein partially co-localized with activated Mac-2+ macrophages throughout the body. Moreover, we first found that the alterations in lipids and metabolites were correlated with the histological lesions in infected minks, especially at 6 dpi, and were similar to that of patients with severe and fatal COVID-19. Particularly, altered metabolic pathways, abnormal digestion, and absorption of vitamins, lipids, cholesterol, steroids, amino acids, and proteins, consistent with hepatic dysfunction, highlight metabolic and immune dysregulation. Enriched kynurenine in infected minks contributed to significant activation of the kynurenine pathway and was related to macrophage activation. Melatonin, which has significant anti-inflammatory and immunomodulating effects, was significantly downregulated at 6 dpi and displayed potential as a targeted medicine. Our data first illustrate systematic analyses of infected minks to recapitulate those observations in severe and fetal COVID-19 patients, delineating a useful animal model to mimic SARS-CoV-2-induced systematic and severe pathophysiological features and provide a reliable tool for the development of effective and targeted treatment strategies, vaccine research, and potential biomarkers.


Subject(s)
COVID-19/metabolism , Lung/metabolism , Macrophages, Alveolar/metabolism , Metabolome , Mink/virology , SARS-CoV-2/metabolism , Amino Acids/metabolism , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/genetics , COVID-19/pathology , Disease Models, Animal , Female , Humans , Lung/pathology , Lung/virology , Macrophages, Alveolar/pathology , Macrophages, Alveolar/virology , Melatonin/metabolism , Metabolic Networks and Pathways/genetics , Molecular Targeted Therapy/methods , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Sterols/metabolism , Virulence , Virus Replication/genetics
7.
Nat Commun ; 13(1): 460, 2022 01 24.
Article in English | MEDLINE | ID: covidwho-1651070

ABSTRACT

The SARS-CoV-2 Delta variant has spread rapidly worldwide. To provide data on its virological profile, we here report the first local transmission of Delta in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of quarantined individuals indicated that the viral loads of Delta infections, when they first become PCR-positive, were on average ~1000 times greater compared to lineage A/B infections during the first epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. The estimated transmission bottleneck size of the Delta variant was generally narrow, with 1-3 virions in 29 donor-recipient transmission pairs. However, the transmission of minor iSNVs resulted in at least 3 of the 34 substitutions that were identified in the outbreak, highlighting the contribution of intra-host variants to population-level viral diversity during rapid spread.


Subject(s)
COVID-19/transmission , Contact Tracing/methods , Disease Outbreaks/prevention & control , SARS-CoV-2/isolation & purification , Animals , COVID-19/epidemiology , COVID-19/virology , Chlorocebus aethiops , Humans , RNA-Seq/methods , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Time Factors , Vero Cells , Viral Load/genetics , Viral Load/physiology , Virus Replication/genetics , Virus Replication/physiology , Virus Shedding/genetics , Virus Shedding/physiology
8.
Viruses ; 14(2)2022 01 18.
Article in English | MEDLINE | ID: covidwho-1625168

ABSTRACT

The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.


Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral/genetics , Reverse Genetics/methods , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Virus Replication/genetics , Amino Acid Substitution , Animals , Chlorocebus aethiops , Chromosomes, Artificial, Bacterial/genetics , Humans , Polymorphism, Genetic , Replicon/drug effects , Replicon/genetics , Vero Cells
9.
Nucleic Acids Res ; 50(3): 1484-1500, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1624985

ABSTRACT

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/metabolism , Genome, Viral/genetics , Genomic Instability , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Exoribonucleases/antagonists & inhibitors , Genome, Viral/drug effects , Genomic Instability/drug effects , Genomic Instability/genetics , HIV Integrase Inhibitors/pharmacology , Isoindoles/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Organoselenium Compounds/pharmacology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Raltegravir Potassium/pharmacology , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Virus Replication/drug effects , Virus Replication/genetics
10.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1614505

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.


Subject(s)
COVID-19/diagnosis , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Caco-2 Cells , Cell Line, Tumor , Chlorocebus aethiops , Humans , In Situ Hybridization/methods , Microscopy, Electron/methods , RNA, Viral/ultrastructure , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sensitivity and Specificity , Vero Cells , Virus Release/drug effects , Virus Release/genetics , Virus Release/physiology , Virus Replication/drug effects , Virus Replication/physiology
11.
PLoS Biol ; 19(12): e3001065, 2021 12.
Article in English | MEDLINE | ID: covidwho-1594053

ABSTRACT

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air-liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.


Subject(s)
Epithelial Cells/immunology , Hot Temperature , Immunity, Innate/immunology , Interferons/immunology , Respiratory Mucosa/immunology , SARS-CoV-2/immunology , Virus Replication/immunology , Adolescent , Animals , COVID-19/genetics , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Epithelial Cells/metabolism , Epithelial Cells/virology , Female , Gene Expression Profiling/methods , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate/genetics , Interferons/genetics , Interferons/metabolism , Male , Middle Aged , Models, Biological , RNA-Seq/methods , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Tissue Culture Techniques , Vero Cells , Virus Replication/genetics , Virus Replication/physiology
12.
Cells ; 11(1)2021 12 24.
Article in English | MEDLINE | ID: covidwho-1580994

ABSTRACT

The ongoing pandemic of coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), needs better treatment options both at antiviral and anti-inflammatory levels. It has been demonstrated that the aminothiol cysteamine, an already human applied drug, and its disulfide product of oxidation, cystamine, have anti-infective properties targeting viruses, bacteria, and parasites. To determine whether these compounds exert antiviral effects against SARS-CoV-2, we used different in vitro viral infected cell-based assays. Moreover, since cysteamine has also immune-modulatory activity, we investigated its ability to modulate SARS-CoV-2-specific immune response in vitro in blood samples from COVID-19 patients. We found that cysteamine and cystamine decreased SARS-CoV-2-induced cytopathic effects (CPE) in Vero E6 cells. Interestingly, the antiviral action was independent of the treatment time respect to SARS-CoV-2 infection. Moreover, cysteamine and cystamine significantly decreased viral production in Vero E6 and Calu-3 cells. Finally, cysteamine and cystamine have an anti-inflammatory effect, as they significantly decrease the SARS-CoV-2 specific IFN-γ production in vitro in blood samples from COVID-19 patients. Overall, our findings suggest that cysteamine and cystamine exert direct antiviral actions against SARS-CoV-2 and have in vitro immunomodulatory effects, thus providing a rational to test these compounds as a novel therapy for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Cysteamine/pharmacology , Drug Repositioning/methods , SARS-CoV-2/drug effects , Aged , Animals , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Cystamine/pharmacology , Cystine Depleting Agents/pharmacology , Female , Humans , Male , Middle Aged , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Vero Cells , Virus Replication/drug effects , Virus Replication/genetics
13.
Virol J ; 18(1): 244, 2021 12 07.
Article in English | MEDLINE | ID: covidwho-1559217

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a huge challenge worldwide. Although previous studies have suggested that type I interferon (IFN-I) could inhibit the virus replication, the expression characteristics of IFN-I signaling-related miRNAs (ISR-miRNAs) during acute severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and its relationship with receptor-binding domain (RBD) IgG antibody response at the recovery phase remain unclear. METHODS: Expression profiles of 12 plasma ISR-miRNAs in COVID-19 patients and healthy controls were analyzed using RT-qPCR. The level of RBD-IgG antibody was determined using the competitive ELISA. Spearman correlation was done to measure the associations of plasma ISR-miRNAs with clinical characteristics during acute SARS-CoV-2 infection and RBD-IgG antibody response at the recovery phase. RESULTS: Compared with the healthy controls, COVID-19 patients exhibited higher levels of miR-29b-3p (Z = 3.15, P = 0.002) and miR-1246 (Z = 4.98, P < 0.001). However, the expression of miR-186-5p and miR-15a-5p were significantly decreased. As the results shown, miR-30b-5p was negatively correlated with CD4 + T cell counts (r = - 0.41, P = 0.027) and marginally positively correlated with fasting plasma glucose in COVID-19 patients (r = 0.37, P = 0.052). The competitive ELISA analysis showed the plasma level of miR-497-5p at the acute phase was positively correlated with RBD-IgG antibody response (r = 0.48, P = 0.038). CONCLUSIONS: Our present results suggested that the expression level of ISR-miRNAs was not only associated with acute SARS-CoV-2 infection but also with RBD-IgG antibody response at the recovery phase of COVID-19. Future studies should be performed to explore the biological significance of ISR-miRNAs in SARS-CoV-2 infection.


Subject(s)
Antibodies, Viral/immunology , COVID-19/diagnosis , Immunoglobulin G/immunology , Interferon Type I/genetics , MicroRNAs , Virus Replication/genetics , COVID-19/blood , COVID-19 Nucleic Acid Testing , Case-Control Studies , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin G/blood , Interferon Type I/blood , Male , MicroRNAs/blood , MicroRNAs/genetics , MicroRNAs/metabolism , Middle Aged , Pregnancy , SARS-CoV-2
14.
Int J Mol Sci ; 22(24)2021 Dec 07.
Article in English | MEDLINE | ID: covidwho-1554804

ABSTRACT

In the last few years, microRNA-mediated regulation has been shown to be important in viral infections. In fact, viral microRNAs can alter cell physiology and act on the immune system; moreover, cellular microRNAs can regulate the virus cycle, influencing positively or negatively viral replication. Accordingly, microRNAs can represent diagnostic and prognostic biomarkers of infectious processes and a promising approach for designing targeted therapies. In the past 18 months, the COVID-19 infection from SARS-CoV-2 has engaged many researchers in the search for diagnostic and prognostic markers and the development of therapies. Although some research suggests that the SARS-CoV-2 genome can produce microRNAs and that host microRNAs may be involved in the cellular response to the virus, to date, not enough evidence has been provided. In this paper, using a focused bioinformatic approach exploring the SARS-CoV-2 genome, we propose that SARS-CoV-2 is able to produce microRNAs sharing a strong sequence homology with the human ones and also that human microRNAs may target viral RNA regulating the virus life cycle inside human cells. Interestingly, all viral miRNA sequences and some human miRNA target sites are conserved in more recent SARS-CoV-2 variants of concern (VOCs). Even if experimental evidence will be needed, in silico analysis represents a valuable source of information useful to understand the sophisticated molecular mechanisms of disease and to sustain biomedical applications.


Subject(s)
MicroRNAs/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , COVID-19/genetics , Computational Biology/methods , DNA Viruses/genetics , Gene Expression/genetics , Gene Expression Regulation, Viral/genetics , Genome, Viral/genetics , Host-Pathogen Interactions/genetics , RNA, Viral/genetics , Sequence Homology
15.
Nucleic Acids Res ; 49(21): 12502-12516, 2021 12 02.
Article in English | MEDLINE | ID: covidwho-1546005

ABSTRACT

Circular RNAs (circRNAs) are noncoding RNAs that exist in all eukaryotes investigated and are derived from back-splicing of certain pre-mRNA exons. Here, we report the application of artificial circRNAs designed to act as antisense-RNAs. We systematically tested a series of antisense-circRNAs targeted to the SARS-CoV-2 genome RNA, in particular its structurally conserved 5'-untranslated region. Functional assays with both reporter transfections as well as with SARS-CoV-2 infections revealed that specific segments of the SARS-CoV-2 5'-untranslated region can be efficiently accessed by specific antisense-circRNAs, resulting in up to 90% reduction of virus proliferation in cell culture, and with a durability of at least 48 h. Presenting the antisense sequence within a circRNA clearly proved more efficient than in the corresponding linear configuration and is superior to modified antisense oligonucleotides. The activity of the antisense-circRNA is surprisingly robust towards point mutations in the target sequence. This strategy opens up novel applications for designer circRNAs and promising therapeutic strategies in molecular medicine.


Subject(s)
Genome, Viral/genetics , RNA, Antisense/genetics , RNA, Circular/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , 5' Untranslated Regions/genetics , Animals , Antiviral Agents/metabolism , Base Sequence , COVID-19/prevention & control , COVID-19/virology , Cell Proliferation/genetics , Chlorocebus aethiops , Drug Design , HeLa Cells , Host-Pathogen Interactions/genetics , Humans , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA-Seq/methods , SARS-CoV-2/physiology , Vero Cells
16.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Article in English | MEDLINE | ID: covidwho-1541316

ABSTRACT

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.


Subject(s)
Exoribonucleases/chemistry , Models, Molecular , Protein Conformation , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Catalytic Domain , Conserved Sequence , Exoribonucleases/genetics , Exoribonucleases/metabolism , Microbial Viability , Nucleotide Motifs , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Proteins , Structure-Activity Relationship , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
17.
Sci Immunol ; 6(66): eabm3131, 2021 Dec 17.
Article in English | MEDLINE | ID: covidwho-1483985

ABSTRACT

SARS-CoV-2 has caused morbidity and mortality across the globe. As the virus spreads, new variants are arising that show enhanced capacity to bypass preexisting immunity. To understand the memory response to SARS-CoV-2, here, we monitored SARS-CoV-2­specific T and B cells in a longitudinal study of infected and recovered golden hamsters (Mesocricetus auratus). We demonstrated that engagement of the innate immune system after SARS-CoV-2 infection was delayed but was followed by a pronounced adaptive response. Moreover, T cell adoptive transfer conferred a reduction in virus levels and rapid induction of SARS-CoV-2­specific B cells, demonstrating that both lymphocyte populations contributed to the overall response. Reinfection of recovered animals with a SARS-CoV-2 variant of concern showed that SARS-CoV-2­specific T and B cells could effectively control the infection that associated with the rapid induction of neutralizing antibodies but failed to block transmission to both naïve and seroconverted animals. These data suggest that the adaptive immune response to SARS-CoV-2 is sufficient to provide protection to the host, independent of the emergence of variants.


Subject(s)
COVID-19/immunology , Disease Models, Animal , Immunologic Memory/immunology , SARS-CoV-2/immunology , Virus Replication/immunology , Adaptive Immunity/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , B-Lymphocytes/virology , COVID-19/virology , Cricetinae , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate/immunology , Mesocricetus , SARS-CoV-2/genetics , SARS-CoV-2/physiology , T-Lymphocytes/immunology , T-Lymphocytes/virology , Virus Replication/genetics
18.
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
19.
Viruses ; 13(10)2021 10 08.
Article in English | MEDLINE | ID: covidwho-1463841

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide and led to approximately 4 million deaths as of August 2021. Despite successful vaccine development, treatment options are limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed eight small interfering RNAs (siRNAs) targeting the highly conserved 5'-untranslated region (5'-UTR) of SARS-CoV-2. The most promising candidate identified in initial reporter assays, termed siCoV6, targets the leader sequence of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS-CoV-2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS-CoV-2 alpha variant and has perfect homology against all sequences of the delta variant that were analyzed by bioinformatic means. Interestingly, the siRNA was even highly active in virus replication assays with the SARS-CoV-1 family member. This work thus identified a very potent siRNA with a broad activity against various SARS-CoV viruses that represents a promising candidate for the development of new treatment options.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/therapy , RNA Interference , RNA, Small Interfering/pharmacology , SARS-CoV-2/growth & development , Virus Replication/drug effects , 5' Untranslated Regions/genetics , Animals , Cell Line, Tumor , Chlorocebus aethiops , Drug Evaluation, Preclinical , HeLa Cells , Humans , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , Vero Cells , Virus Replication/genetics
20.
Viruses ; 13(10)2021 10 04.
Article in English | MEDLINE | ID: covidwho-1463827

ABSTRACT

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.


Subject(s)
Coronavirus Infections/veterinary , Deltacoronavirus/genetics , Deltacoronavirus/metabolism , Genes, Reporter/genetics , Luciferases/genetics , A549 Cells , Animals , Cell Line , Chlorocebus aethiops , Chromosomes, Artificial, Bacterial/genetics , Coronavirus Infections/pathology , Deltacoronavirus/growth & development , Dogs , Genome, Viral/genetics , Humans , Luciferases/biosynthesis , Madin Darby Canine Kidney Cells , Nanostructures , Swine , Swine Diseases/virology , Vero Cells , Virus Replication/genetics
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