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1.
Cell Rep ; 37(3): 109841, 2021 10 19.
Article in English | MEDLINE | ID: covidwho-1439922

ABSTRACT

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.


Subject(s)
COVID-19/genetics , Gene Expression Regulation, Viral , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Anisotropy , COVID-19/immunology , DNA Mutational Analysis , Female , Gene Expression Profiling , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Kinetics , Mutation , Phenotype , Point Mutation , Protein Biosynthesis , Protein Domains , RNA Stability , Ribosome Subunits, Small, Eukaryotic/metabolism , Ribosomes/metabolism
2.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Article in English | MEDLINE | ID: covidwho-1387160

ABSTRACT

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Subject(s)
Fluorescence , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Molecular Imaging , RNA, Messenger/analysis , RNA, Messenger/metabolism , COVID-19/drug therapy , Cell Line, Tumor , Cytosine/analogs & derivatives , Cytosine/analysis , Cytosine/chemical synthesis , Cytosine/chemistry , Fluorescent Dyes/chemical synthesis , Green Fluorescent Proteins/metabolism , Histones/metabolism , Humans , Molecular Structure , RNA, Messenger/chemistry , RNA, Messenger/therapeutic use , Spectrometry, Fluorescence
3.
J Virol ; 95(18): e0068721, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1373942

ABSTRACT

The emerging coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide, resulting in global public health emergencies and economic crises. In the present study, a noninfectious and biosafety level 2 (BSL2)-compatible SARS-CoV-2 replicon expressing a nano luciferase (nLuc) reporter was constructed in a bacterial artificial chromosomal (BAC) vector by reverse genetics. The nLuc reporter is highly sensitive, easily quantifiable, and high throughput adaptable. Upon transfecting the SARS-CoV-2 replicon BAC plasmid DNA into Vero E6 cells, we could detect high levels of nLuc reporter activity and viral RNA transcript, suggesting the replication of the replicon. The replicon replication was further demonstrated by the findings that deleting nonstructural protein 15 or mutating its catalytic sites significantly reduced replicon replication, whereas providing the nucleocapsid protein in trans enhanced replicon replication in a dose-dependent manner. Finally, we showed that remdesivir, a U.S. Food and Drug Administration-approved antiviral drug, significantly inhibited the replication of the replicon, providing proof of principle for the application of our replicon as a useful tool for developing antivirals. Taken together, this study established a sensitive and BSL2-compatible reporter system in a single BAC plasmid for investigating the functions of SARS-CoV-2 proteins in viral replication and evaluating antiviral compounds. This should contribute to the global effort to combat this deadly viral pathogen. IMPORTANCE The COVID-19 pandemic caused by SARS-CoV-2 is having a catastrophic impact on human lives. Combatting the pandemic requires effective vaccines and antiviral drugs. In the present study, we developed a SARS-CoV-2 replicon system with a sensitive and easily quantifiable reporter. Unlike studies involving infectious SARS-CoV-2 virus that must be performed in a biosafety level 3 (BSL3) facility, the replicon is noninfectious and thus can be safely used in BSL2 laboratories. The replicon will provide a valuable tool for testing antiviral drugs and studying SARS-CoV-2 biology.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Evaluation, Preclinical , Green Fluorescent Proteins/metabolism , Replicon , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , COVID-19/virology , Chlorocebus aethiops , Green Fluorescent Proteins/genetics , HEK293 Cells , High-Throughput Screening Assays , Humans
4.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Article in English | MEDLINE | ID: covidwho-1373615

ABSTRACT

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Subject(s)
Antibodies, Neutralizing/pharmacology , Containment of Biohazards/methods , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Viral Matrix Proteins/antagonists & inhibitors , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Embryo, Mammalian , Fibroblasts/drug effects , Fibroblasts/virology , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , MCF-7 Cells , Mice , Molecular Mimicry , Plasmids/chemistry , Plasmids/metabolism , Primary Cell Culture , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Transfection , Vero Cells , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism
5.
Commun Biol ; 4(1): 366, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1351981

ABSTRACT

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.


Subject(s)
Chromatography, Gel , Green Fluorescent Proteins/metabolism , Membrane Proteins/metabolism , Nanotechnology , Peptides/metabolism , Single-Domain Antibodies/immunology , Animals , Cryoelectron Microscopy , Cysteine Loop Ligand-Gated Ion Channel Receptors/genetics , Cysteine Loop Ligand-Gated Ion Channel Receptors/immunology , Cysteine Loop Ligand-Gated Ion Channel Receptors/metabolism , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/immunology , Oryzias/genetics , Oryzias/metabolism , Peptides/genetics , Peptides/immunology , Protein Stability , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spectrometry, Fluorescence , Temperature , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism
6.
J Trace Elem Med Biol ; 68: 126818, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1300926

ABSTRACT

CONTEXT: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SARS-CoV-2. METHODS: Vero E6 cells were cultured with or without copper gluconate 18-24 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were incubated in fresh medium containing varying concentration of copper gluconate (supplemented with bovine serum albumin or not) for an additional 48 -h period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT and propidium iodide assays. RESULTS: The viability of Vero E6 cells exposed to copper gluconate up to 200 µM was found to be similar to that of unexposed cells, but it dropped below 70 % with 400 µM of this agent after 72 h of continuous exposure. The infection rate was 23.8 %, 18.9 %, 20.6 %, 6.9 %, 5.3 % and 5.2 % in cells treated prior infection with 0, 2, 10, 25, 50 and 100 µM of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71 %, 77 %, and 78 % with 25, 50, and 100 µM of copper gluconate respectively (p < 0.05). In cells treated only post-infection, the rate of infection dropped by 73 % with 100 µM of copper gluconate (p < 0.05). However, the antiviral activity of copper gluconate was abolished by the addition of bovine serum albumin. CONCLUSION: Copper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells but this effect was abolished by albumin, which suggests that copper will not retain its activity in serum. Furthers studies are needed to investigate whether copper gluconate could be of benefit in mucosal administration such as mouthwash, nasal spray or aerosols.


Subject(s)
Gluconates/pharmacology , Microscopy, Confocal , SARS-CoV-2/drug effects , Animals , Antiviral Agents/pharmacology , COVID-19/pathology , COVID-19/virology , Cell Survival/drug effects , Chlorocebus aethiops , Green Fluorescent Proteins/metabolism , Vero Cells
7.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1294968

ABSTRACT

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Subject(s)
COVID-19/virology , Peptide Library , SARS-CoV-2/metabolism , Viral Proteins/metabolism , A549 Cells , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Host Microbial Interactions/physiology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Time-Lapse Imaging , Viral Proteins/genetics
8.
J Virol Methods ; 295: 114221, 2021 09.
Article in English | MEDLINE | ID: covidwho-1284316

ABSTRACT

SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.


Subject(s)
Neutralization Tests/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/pharmacology , Animals , Antibodies, Neutralizing/pharmacology , Antiviral Agents/pharmacology , Cell Line , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Lentivirus/genetics , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virion , Virus Internalization/drug effects
9.
Sci Rep ; 11(1): 9136, 2021 04 28.
Article in English | MEDLINE | ID: covidwho-1207152

ABSTRACT

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism of viral spike protein-mediated fusion or screening for viral inhibitors under biosafety level 1 conditions.


Subject(s)
Host-Pathogen Interactions/physiology , Peptides/chemistry , Peptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cell Fusion , Circular Dichroism , Giant Cells/virology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Membrane Fusion , Peptides/genetics , Protein Engineering/methods , Protein Multimerization , Protein Stability , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transcription, Genetic
10.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Article in English | MEDLINE | ID: covidwho-1164792

ABSTRACT

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Subject(s)
Fluorescence , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Molecular Imaging , RNA, Messenger/analysis , RNA, Messenger/metabolism , COVID-19/drug therapy , Cell Line, Tumor , Cytosine/analogs & derivatives , Cytosine/analysis , Cytosine/chemical synthesis , Cytosine/chemistry , Fluorescent Dyes/chemical synthesis , Green Fluorescent Proteins/metabolism , Histones/metabolism , Humans , Molecular Structure , RNA, Messenger/chemistry , RNA, Messenger/therapeutic use , Spectrometry, Fluorescence
11.
J Biosci Bioeng ; 131(6): 696-702, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1141952

ABSTRACT

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.


Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Hybridomas/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/analysis , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/isolation & purification , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , Green Fluorescent Proteins/metabolism , Humans , Hybridomas/cytology , Immunoglobulin Isotypes , Immunoprecipitation , Mice , Time Factors
12.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1119222

ABSTRACT

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Subject(s)
COVID-19/virology , Peptide Library , SARS-CoV-2/metabolism , Viral Proteins/metabolism , A549 Cells , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Host Microbial Interactions/physiology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Time-Lapse Imaging , Viral Proteins/genetics
13.
J Virol ; 95(4)2021 01 28.
Article in English | MEDLINE | ID: covidwho-1117221

ABSTRACT

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.


Subject(s)
COVID-19/virology , Dengue Virus/isolation & purification , Dengue/virology , SARS-CoV-2/isolation & purification , A549 Cells , Animals , COVID-19/diagnosis , COVID-19/metabolism , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Dengue/diagnosis , Dengue/metabolism , Dengue/pathology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Nuclear Localization Signals/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication
14.
Mol Biotechnol ; 63(3): 240-248, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1037255

ABSTRACT

The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.


Subject(s)
Antiviral Agents , Coronavirus 3C Proteases/antagonists & inhibitors , High-Throughput Screening Assays/methods , SARS-CoV-2/drug effects , COVID-19/virology , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Drug Development , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Small Molecule Libraries
15.
J Virol ; 95(4)2021 01 28.
Article in English | MEDLINE | ID: covidwho-952556

ABSTRACT

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.


Subject(s)
COVID-19/virology , Dengue Virus/isolation & purification , Dengue/virology , SARS-CoV-2/isolation & purification , A549 Cells , Animals , COVID-19/diagnosis , COVID-19/metabolism , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Dengue/diagnosis , Dengue/metabolism , Dengue/pathology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Nuclear Localization Signals/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication
16.
J Biol Chem ; 296: 100103, 2021.
Article in English | MEDLINE | ID: covidwho-936211

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses, is restricted to BSL-3 facilities. Such BSL-3 classification makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.


Subject(s)
Coronavirus Envelope Proteins/genetics , Nucleocapsid Proteins/genetics , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Virion/growth & development , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Containment of Biohazards/classification , Coronavirus Envelope Proteins/metabolism , Gene Expression , Genes, Reporter , Government Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Electron , Nucleocapsid Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/metabolism , Virion/genetics , Virion/metabolism , Virion/ultrastructure , Virus Assembly/physiology , Virus Internalization , Virus Release/physiology
17.
J Gen Virol ; 102(1)2021 01.
Article in English | MEDLINE | ID: covidwho-873186

ABSTRACT

Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.


Subject(s)
Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , COVID-19/diagnosis , Henipavirus Infections/diagnosis , High-Throughput Screening Assays , Respiratory Syncytial Virus Infections/diagnosis , Viral Fusion Proteins/antagonists & inhibitors , Animals , Antibodies, Neutralizing/isolation & purification , Antibodies, Neutralizing/metabolism , Antibodies, Viral/isolation & purification , Antibodies, Viral/metabolism , COVID-19/immunology , COVID-19/virology , Cell Fusion , Convalescence , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Henipavirus Infections/immunology , Henipavirus Infections/virology , Humans , Immune Sera/chemistry , Luciferases/genetics , Luciferases/metabolism , Models, Molecular , Nipah Virus/immunology , Nipah Virus/pathogenicity , Protein Conformation , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/immunology , Respiratory Syncytial Virus, Human/pathogenicity , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Swine , Viral Fusion Protein Inhibitors/chemistry , Viral Fusion Protein Inhibitors/metabolism , Viral Fusion Protein Inhibitors/pharmacology , Viral Fusion Proteins/genetics , Viral Fusion Proteins/immunology
18.
Nucleic Acids Res ; 48(17): 9694-9709, 2020 09 25.
Article in English | MEDLINE | ID: covidwho-745778

ABSTRACT

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.


Subject(s)
DNA Damage , DNA Repair/drug effects , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Binding Sites , Catalytic Domain , Cell Line, Tumor , DNA Repair/physiology , Fluorescence Resonance Energy Transfer , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Indazoles/pharmacology , Kinetics , Molecular Imaging , NAD/metabolism , Piperidines/pharmacology , Poly(ADP-ribose) Polymerases/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , X-ray Repair Cross Complementing Protein 1/genetics , X-ray Repair Cross Complementing Protein 1/metabolism
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