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1.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1356909

ABSTRACT

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


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Interferon-beta , Promoter Regions, Genetic , SARS-CoV-2 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/biosynthesis , Interferon-beta/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
2.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1350002

ABSTRACT

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


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Interferon-beta , Promoter Regions, Genetic , SARS-CoV-2 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/biosynthesis , Interferon-beta/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
3.
J Virol ; 95(14): e0066321, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1291038

ABSTRACT

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.


Subject(s)
COVID-19 , Genome, Viral , Nucleotide Motifs , RNA Folding , RNA, Viral , SARS-CoV-2/physiology , Virus Replication , Animals , COVID-19/genetics , COVID-19/metabolism , Chlorocebus aethiops , HEK293 Cells , Humans , RNA, Viral/genetics , RNA, Viral/metabolism , Vero Cells
4.
RNA ; 27(9): 1025-1045, 2021 09.
Article in English | MEDLINE | ID: covidwho-1269913

ABSTRACT

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.


Subject(s)
Host-Pathogen Interactions/genetics , Protein Biosynthesis , Proteins/chemistry , Proteins/genetics , Viral Nonstructural Proteins/genetics , 5' Untranslated Regions , Autoantigens/genetics , Autoantigens/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Protein Folding , Pyrimidines , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Ribosomes/genetics , Ribosomes/virology , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Viral Nonstructural Proteins/metabolism
5.
J Med Virol ; 93(4): 2301-2306, 2021 04.
Article in English | MEDLINE | ID: covidwho-1217381

ABSTRACT

Assessment of commercial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially available lateral flow immunochromatographic assays (LFIC; Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for coronavirus disease 2019 (COVID-19) diagnosis in hospitalized patients and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS-CoV-2 spike protein) was used. Test line intensity was scored using a 4-level scale (0 to 3+). The overall sensitivity of LFIC assays was 91.1% for the Wondfo SARS-CoV-2 Antibody test, 72.2% for the INNOVITA 2019-nCoV IgG, 85.6% for the INNOVITA 2019-nCoV IgM, and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%, 93.9%, and 93.9%, respectively). Reactivities equal to or more intense than the positive control line (≥2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb50 titers (≥1/160). Our findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID-19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS-CoV-2-S NtAb50 titers.


Subject(s)
Antibodies, Neutralizing/blood , COVID-19 Testing/methods , COVID-19/immunology , Immunoassay/methods , SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/diagnosis , COVID-19/virology , Green Fluorescent Proteins , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Spike Glycoprotein, Coronavirus/immunology
6.
Notf Rett Med ; : 1-9, 2021 Apr 23.
Article in German | MEDLINE | ID: covidwho-1210708

ABSTRACT

BACKGROUND: To contain the coronavirus disease (COVID-19) pandemic, public life was reduced through contact restriction measures (referred to as "lockdown" in the further course for reading simplicity), among other things to make health system resources available for the treatment of COVID-19 patients. In parallel, a decrease in emergency patients was observed in the public health system. METHODS: For two 10-week periods before and during the lockdown, ambulance service deployment rates were analysed in 6 ambulance service areas for 6 tracer diagnoses. Random effects were minimised by comparing the results with the corresponding 2018 and 2019 time periods and a calculated expected value. RESULTS: For emergency ambulance service calls, there was a reduction in call numbers (-16%) during the lockdown. A 20% reduction for the categories cardiac and cerebral ischaemia was found. In the urban area, the reduction in cardiac ischaemia was less pronounced at 14% than in the surrounding area at 23%. The deployment figures for intoxications decreased by 27% and for psychiatric emergencies by 16%. CONCLUSION: The public ambulance service was not overwhelmed by the COVID-19 pandemic; there was a decrease in depolyments during the lockdown. For the reduction in cerebral or cardiac ischaemias, the explanatory models for the influence of the lockdown are missing. Further studies on the utilisation behaviour of the ambulance service during a lockdown appear necessary in order to detect potentially fatal reductions in utilisation for the patient outcome and to be able to counteract them through education.

7.
mBio ; 12(2)2021 04 13.
Article in English | MEDLINE | ID: covidwho-1183285

ABSTRACT

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6's nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection.IMPORTANCE SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6's interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6's disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.


Subject(s)
Cell Nucleus/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Active Transport, Cell Nucleus , Binding Sites , COVID-19/metabolism , COVID-19/virology , Cell Line , Gene Expression Regulation , Humans , Mutation , Nuclear Matrix-Associated Proteins/genetics , Nuclear Pore Complex Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , Protein Binding , RNA, Messenger/metabolism , SARS-CoV-2/genetics , Viral Proteins/chemistry , Viral Proteins/genetics
8.
J Mol Biol ; 433(13): 166983, 2021 06 25.
Article in English | MEDLINE | ID: covidwho-1174385

ABSTRACT

Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases.


Subject(s)
Antibodies, Viral/chemistry , Antigens, Viral/immunology , COVID-19/diagnosis , COVID-19/virology , SARS-CoV-2/isolation & purification , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antigens, Viral/chemistry , Antigens, Viral/isolation & purification , Epitopes/immunology , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Luciferases , Luminescent Measurements/methods , Protein Engineering , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
9.
Nat Med ; 27(5): 917-924, 2021 05.
Article in English | MEDLINE | ID: covidwho-1152868

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 and B.1.351 variants were first identified in the United Kingdom and South Africa, respectively, and have since spread to many countries. These variants harboring diverse mutations in the gene encoding the spike protein raise important concerns about their immune evasion potential. Here, we isolated infectious B.1.1.7 and B.1.351 strains from acutely infected individuals. We examined sensitivity of the two variants to SARS-CoV-2 antibodies present in sera and nasal swabs from individuals infected with previously circulating strains or who were recently vaccinated, in comparison with a D614G reference virus. We utilized a new rapid neutralization assay, based on reporter cells that become positive for GFP after overnight infection. Sera from 58 convalescent individuals collected up to 9 months after symptoms, similarly neutralized B.1.1.7 and D614G. In contrast, after 9 months, convalescent sera had a mean sixfold reduction in neutralizing titers, and 40% of the samples lacked any activity against B.1.351. Sera from 19 individuals vaccinated twice with Pfizer Cominarty, longitudinally tested up to 6 weeks after vaccination, were similarly potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Neutralizing titers increased after the second vaccine dose, but remained 14-fold lower against B.1.351. In contrast, sera from convalescent or vaccinated individuals similarly bound the three spike proteins in a flow cytometry-based serological assay. Neutralizing antibodies were rarely detected in nasal swabs from vaccinees. Thus, faster-spreading SARS-CoV-2 variants acquired a partial resistance to neutralizing antibodies generated by natural infection or vaccination, which was most frequently detected in individuals with low antibody levels. Our results indicate that B1.351, but not B.1.1.7, may increase the risk of infection in immunized individuals.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , COVID-19 Vaccines/immunology , Convalescence , Cross Reactions , Humans , Neutralization Tests , Sensitivity and Specificity , Vaccination
10.
Viruses ; 13(2)2021 02 16.
Article in English | MEDLINE | ID: covidwho-1085036

ABSTRACT

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.


Subject(s)
Antiviral Agents/pharmacology , Enterovirus A, Human/drug effects , Lonicera/chemistry , MicroRNAs/metabolism , Plant Extracts/pharmacology , Virus Replication/drug effects , Animals , Cell Line , Enterovirus A, Human/physiology , Enterovirus Infections/drug therapy , Humans , Mice , Mice, Inbred ICR
11.
Viruses ; 13(2)2021 01 24.
Article in English | MEDLINE | ID: covidwho-1052508

ABSTRACT

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.


Subject(s)
Antiviral Agents/metabolism , Coronavirus 3C Proteases/antagonists & inhibitors , Luciferases/metabolism , Protease Inhibitors/metabolism , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Cell Survival/drug effects , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Lentivirus/genetics , Luciferases/genetics , Protease Inhibitors/pharmacology
12.
Viruses ; 13(2)2021 01 24.
Article in English | MEDLINE | ID: covidwho-1045366

ABSTRACT

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.


Subject(s)
Antiviral Agents/metabolism , Coronavirus 3C Proteases/antagonists & inhibitors , Luciferases/metabolism , Protease Inhibitors/metabolism , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Cell Survival/drug effects , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Lentivirus/genetics , Luciferases/genetics , Protease Inhibitors/pharmacology
13.
Cytokine ; 138: 155399, 2021 02.
Article in English | MEDLINE | ID: covidwho-987430

ABSTRACT

BACKGROUND: The overall clinical outcome of inflammatory conditions is the result of the balance between pro-inflammatory and anti-inflammatory mediators. Because nuclear factor kappa B (NF-ĸB) is at the bottom of many inflammatory conditions, methods to evaluate the net effect of inflammation modulators on this master regulator have been conceptualized for years. METHODS: Using an ex vivo NF-ĸB reporter cell line-based assay, plasma samples of patients with rheumatoid arthritis (n = 27), psoriasis (n = 15), or severe coronavirus disease-19 (COVID-19) (n = 21) were investigated for NF-ĸB activation compared to plasma samples from 9 healthy volunteers. RESULTS: When separated by C-reactive protein (CRP) threshold levels, samples of patients exhibiting increased CRP levels (≥5 mg/l) activated NF-ĸB more efficiently than samples from patients with levels below 5 mg/l (P = 0.0001) or healthy controls (P = 0.04). Overall, there was a moderate association of CRP levels with NF-ĸB activation (Spearman r = 0.66; p < 0.0001). Plasma from COVID-19 patients activated NF-ĸB more efficiently (mean 2.4-fold compared to untreated reporter cells) than samples from any other condition (healthy controls, 1.8-fold, P = 0.0025; rheumatoid arthritis, 1.7-fold, P < 0.0001; psoriasis, 1.7-fold, P < 0.0001). In contrast, effects of rheumatoid arthritis, psoriasis, or healthy volunteer samples did not differ. CONCLUSION: This study shows that a NF-ĸB reporter cell line can be used to evaluate the net inflammatory effect of clinical plasma samples. Patients with chronic but stable rheumatoid arthritis or psoriasis do not exhibit increased plasma levels of NF-ĸB-activating compounds as opposed to COVID-19 patients with high inflammatory burden.


Subject(s)
Arthritis, Rheumatoid/pathology , COVID-19/pathology , NF-kappa B/blood , NF-kappa B/metabolism , Psoriasis/pathology , Arthritis, Rheumatoid/blood , C-Reactive Protein/analysis , Cell Line , Enzyme Activation/physiology , Female , HEK293 Cells , Humans , Inflammation/pathology , Inflammation Mediators/metabolism , Male , Middle Aged , Psoriasis/blood , SARS-CoV-2/immunology
14.
J Transl Med ; 18(1): 452, 2020 11 30.
Article in English | MEDLINE | ID: covidwho-948411

ABSTRACT

BACKGROUND: Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-ß, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release. METHODS: ELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1ß relative potency were then employed to confirm the involvement of enriched pathways and TFs. RESULTS: LMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1ß, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1ß release. CONCLUSION: In this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.


Subject(s)
Cytokines/metabolism , Inflammation/prevention & control , Leukocytes, Mononuclear/drug effects , Serum Albumin, Human/pharmacology , Anti-Inflammatory Agents/pharmacology , COVID-19/drug therapy , COVID-19/immunology , COVID-19/pathology , Cells, Cultured , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation Mediators/metabolism , Interferon-Stimulated Gene Factor 3/metabolism , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides , Lymphocyte Activation/drug effects , Molecular Weight , NF-kappa B/metabolism , Serum Albumin, Human/chemistry , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Transcription Factors/metabolism
15.
J Gen Virol ; 102(1)2021 01.
Article in English | MEDLINE | ID: covidwho-910383

ABSTRACT

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.


Subject(s)
Host-Pathogen Interactions/genetics , Lysine/genetics , Ribosomal Proteins/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Amino Acid Sequence , Amino Acid Substitution , Computational Biology/methods , G1 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Lysine/metabolism , Mutation , Ribosomal Proteins/antagonists & inhibitors , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/virology , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Viral Nonstructural Proteins/metabolism
16.
J Virol ; 94(22)2020 10 27.
Article in English | MEDLINE | ID: covidwho-901265

ABSTRACT

In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro 3CLpro is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CLpro Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CLpro This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread.IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/chemistry , Coronavirus Infections/virology , Drug Discovery , High-Throughput Screening Assays/methods , Pneumonia, Viral/virology , Protease Inhibitors/pharmacology , Animals , COVID-19 , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus Infections/drug therapy , Cysteine Endopeptidases , Humans , Microscopy, Fluorescence/methods , Pandemics , Pneumonia, Viral/drug therapy , SARS-CoV-2 , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors
17.
Dtsch Arztebl Int ; 117(33-34): 545-552, 2020 08 17.
Article in English | MEDLINE | ID: covidwho-846177

ABSTRACT

BACKGROUND: In this study, we investigate the number of emergency room consultations during the COVID-19 pandemic of 2020 in Germany compared to figures from the previous year. METHODS: Case numbers from calendar weeks 1 through 22 of the two consecutive years 2019 and 2020 were obtained from 29 university hospitals and 7 non-university hospitals in Germany. Information was also obtained on the patients' age, sex, and urgency, along with the type of case (outpatient/inpatient), admitting ward, and a small number of tracer diagnoses (I21, myocardial infarction; J44, COPD; and I61, I63, I64, G45, stroke /TIA), as well as on the number of COVID-19 cases and of tests performed for SARS-CoV-2, as a measure of the number of cases in which COVID-19 was suspected or at least included in the differential diagnoses. RESULTS: A total of 1 022 007 emergency room consultations were analyzed, of which 546 940 took place in 2019 and 475 067 in 2020. The number of consultations with a positive test for the COVID-19 pathogen was 3122. The total number of emergency room consultations in the observation period was 13% lower in 2020 than in 2019, with a maximum drop by 38% coinciding with the highest number of COVID-19 cases (calendar week 14; 572 cases). After the initiation of interpersonal contact restrictions in 2020, there was a marked drop in COVID-19 case numbers, by a mean of -240 cases per week per emergency room (95% confidence interval [-284; -128]). There was a rise in case numbers thereafter, by a mean of 17 patients per week [14; 19], and the number of cases of myocardial infarction returned fully to the level seen in 2019. CONCLUSION: In Germany, the COVID-19 pandemic led to a significant drop in medical emergencies of all kinds presenting to the nation's emergency departments. A recovery effect began to be seen as early as calendar week 15, but the levels seen in 2019 were not yet reached overall by calendar week 22; only the prevalence of myocardial infarction had renormalized by then. The reasons for this require further investigation.


Subject(s)
Coronavirus Infections/epidemiology , Emergencies/epidemiology , Emergency Service, Hospital/statistics & numerical data , Pandemics , Pneumonia, Viral/epidemiology , COVID-19 , Germany/epidemiology , Humans
18.
Sci Rep ; 10(1): 16615, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-834915

ABSTRACT

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Subject(s)
Antibodies, Viral/analysis , Antibody-Dependent Cell Cytotoxicity/immunology , Coronavirus Infections/immunology , Cytotoxicity Tests, Immunologic/methods , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/virology , Genes, Reporter , HEK293 Cells , Humans , Jurkat Cells , Luciferases/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , NFATC Transcription Factors/genetics , Receptors, IgG/genetics , Receptors, IgG/immunology , Response Elements , Spike Glycoprotein, Coronavirus/metabolism , Transfection
19.
Balkan Med J ; 37(1): 208-214, 2020 06 01.
Article in English | MEDLINE | ID: covidwho-826668

ABSTRACT

Background: Brain metastasis is a major cause of cancer death in patients with lung cancer. Sirtuin 1 and hsa-miR-217 have been identified to mediate the development of non-small cell lung cancer. Aims: To investigate the roles of hsa-miR-217, its target sirtuin 1, and the P53/KAI1 axis in the brain metastasis from non-small cell lung cancer. Study Design: Cell culture study. Methods: Human pulmonary adenocarcinoma brain metastasis cell line PC-14/B were incubated and treated with constructed lentiviral plasmids expressing miR-217 and/or sirtuin 1. BEAS-2B cell line was used as a control. The targeted regulation of miR-217 to sirtuin 1was examined using a dual-luciferase reporter assay. Cell proliferation, migration, invasion, and related protein expression were detected to examine the effect of the miR-217/sirtuin 1 expression on metastasis. Results: PC-14/B cells expressed higher sirtuin 1 and lower P53 and KAI1 compared with BEAS-2B control cells (p<0.05). Sirtuin 1 was a direct target of miR-217. MiR-217 expression suppressed PC-14/B cell invasion (p=0.004), migration (p=0.001), and proliferation (p<0.05), whereas sirtuin 1 overexpression reversed all processes. sirtuin 1 expression inhibited P53, KAI1/CD82, matrix metalloproteinase-9, and ß-catenin but upregulated E-cadherin protein. MiR-217 overexpression induced reverse changes. Conclusion: Hsa-miR-217 and its target sirtuin 1 acted as metastasis suppressor and promoter gene in non-small cell lung cancer, respectively. The hsa-miR-217/sirtuin 1/P53/KAI1 metastasis regulatory pathway showed novel and crucial roles in brain metastasis from non-small cell lung cancer. This axis might be a potential target for the treatment of brain metastasis of lung cancer.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Kangai-1 Protein/drug effects , MicroRNAs/pharmacology , Sirtuin 1/drug effects , Tumor Suppressor Protein p53/drug effects , Brain Neoplasms/etiology , Brain Neoplasms/physiopathology , Brain Neoplasms/prevention & control , Carcinoma, Non-Small-Cell Lung/physiopathology , Cell Culture Techniques/methods , Cell Proliferation/drug effects , Humans , MicroRNAs/therapeutic use , Signal Transduction/drug effects
20.
Nucleic Acids Res ; 48(19): 10890-10908, 2020 11 04.
Article in English | MEDLINE | ID: covidwho-817440

ABSTRACT

Although endogenous retroviruses (ERVs) are known to harbor cis-regulatory elements, their role in modulating cellular immune responses remains poorly understood. Using an RNA-seq approach, we show that several members of the ERV9 lineage, particularly LTR12C elements, are activated upon HIV-1 infection of primary CD4+ T cells. Intriguingly, HIV-1-induced ERVs harboring transcription start sites are primarily found in the vicinity of immunity genes. For example, HIV-1 infection activates LTR12C elements upstream of the interferon-inducible genes GBP2 and GBP5 that encode for broad-spectrum antiviral factors. Reporter assays demonstrated that these LTR12C elements drive gene expression in primary CD4+ T cells. In line with this, HIV-1 infection triggered the expression of a unique GBP2 transcript variant by activating a cryptic transcription start site within LTR12C. Furthermore, stimulation with HIV-1-induced cytokines increased GBP2 and GBP5 expression in human cells, but not in macaque cells that naturally lack the GBP5 gene and the LTR12C element upstream of GBP2. Finally, our findings suggest that GBP2 and GBP5 have already been active against ancient viral pathogens as they suppress the maturation of the extinct retrovirus HERV-K (HML-2). In summary, our findings uncover how human cells can exploit remnants of once-infectious retroviruses to regulate antiviral gene expression.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Endogenous Retroviruses/genetics , Gene Expression Regulation/immunology , HIV Infections/genetics , Promoter Regions, Genetic , T-Lymphocyte Subsets/immunology , Animals , CD4-Positive T-Lymphocytes/cytology , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , HEK293 Cells , HIV Infections/immunology , HIV-1 , Humans , Macaca mulatta , T-Lymphocyte Subsets/cytology
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