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1.
J Med Virol ; 94(7): 3017-3031, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1756619

ABSTRACT

The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus impairs our capacity to set up a toolkit against it. Thus, more studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology are urgently needed. Reverse genetics systems, including viral infectious clones and replicons, are powerful platforms for viral research projects, spanning many aspects such as the rescues of wild-type or mutant viral particles, the investigation of viral replication mechanism, the characterization of viral protein functions, and the studies on viral pathogenesis and antiviral drug development. The operations on viral infectious clones are strictly limited in the Biosafety Level 3 (BSL3) facilities, which are insufficient, especially during the pandemic. In contrast, the operation on the noninfectious replicon can be performed in Biosafety Level 2 (BSL2) facilities, which are widely available. After the outbreak of COVID-19, many reverse genetics systems for SARS-CoV-2, including infectious clones and replicons are developed and given plenty of options for researchers to pick up according to the requirement of their research works. In this review, we summarize the available reverse genetics systems for SARS-CoV-2, by highlighting the features of these systems, and provide a quick guide for researchers, especially those without ample experience in operating viral reverse genetics systems.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Pandemics , Replicon , Reverse Genetics , SARS-CoV-2/genetics
2.
PLoS Pathog ; 18(2): e1010268, 2022 02.
Article in English | MEDLINE | ID: covidwho-1753212

ABSTRACT

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.


Subject(s)
Ebolavirus/genetics , Filoviridae Infections/virology , Filoviridae/genetics , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , Genetic Complementation Test , Genome, Viral , Hemorrhagic Fever, Ebola/virology , Host Microbial Interactions , Humans , Inclusion Bodies/virology , Induced Pluripotent Stem Cells/virology , Macrophages/virology , RNA, Viral , Reverse Genetics , Vero Cells , Virion/genetics
3.
EMBO Rep ; 23(5): e53820, 2022 05 04.
Article in English | MEDLINE | ID: covidwho-1726972

ABSTRACT

Engineering recombinant viruses is a pre-eminent tool for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the large size of coronavirus genomes renders the current reverse genetics methods challenging. Here, we describe a simple method based on "infectious subgenomic amplicons" (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstruction of the complete genomic cDNA and apply this method to SARS-CoV-2 and also to the feline enteric coronavirus. In both cases we rescue wild-type viruses with biological characteristics similar to original strains. Specific mutations and fluorescent red reporter genes can be readily incorporated into the SARS-CoV-2 genome enabling the generation of a genomic variants and fluorescent reporter strains for in vivo experiments, serological diagnosis, and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies, to explore the molecular biological properties of the SARS-CoV-2 variants, and to accelerate the development of effective therapeutic reagents.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antiviral Agents , COVID-19/genetics , Cats , Reverse Genetics , SARS-CoV-2/genetics
4.
Nihon Yakurigaku Zasshi ; 157(2): 134-138, 2022.
Article in Japanese | MEDLINE | ID: covidwho-1714692

ABSTRACT

RNA viruses are responsible for several infectious diseases, including dengue fever, Zika fever, and COVID-19. Reverse genetics is a powerful tool to elucidate which domain or mutations in RNA viruses determine their pathogenicity and ability to evade antiviral drugs and host immune response. Previous reverse genetics systems for flaviviruses and coronaviruses have been technically challenging and time-consuming, thereby hampering the further understanding of events during viral evolution. A novel reverse genetics system-circular polymerase extension reaction (CPER)-has been developed to overcome this limitation. CPER is based on PCR-mediated assembly of DNA fragments that encode the whole genome of these viruses. CPER requires a relatively short time to introduce specific mutations into the viral genome of flaviviruses and SARS-CoV-2. In this review article, we explain the mode of action of this system and discuss the future direction of reverse genetics for RNA viruses.


Subject(s)
COVID-19 , Zika Virus Infection , Zika Virus , Genome, Viral , Humans , RNA, Viral/genetics , Reverse Genetics , SARS-CoV-2 , Zika Virus/genetics , Zika Virus Infection/genetics
5.
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
6.
J Virol ; 96(3): e0156121, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1529876

ABSTRACT

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.


Subject(s)
DNA, Complementary , Genome, Viral , Reverse Genetics , Torovirus/genetics , Animals , Cattle , Cattle Diseases/virology , Cell Line , Cells, Cultured , Chromosomes, Artificial, Bacterial , Cloning, Molecular , Genes, Reporter , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/metabolism , Mutation , Plasmids/genetics , Torovirus/isolation & purification , Torovirus Infections , Transfection
7.
PLoS Biol ; 19(11): e3001284, 2021 11.
Article in English | MEDLINE | ID: covidwho-1502046

ABSTRACT

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2-infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Animals , COVID-19/pathology , COVID-19 Vaccines/therapeutic use , Cell Line , Disease Models, Animal , Female , Humans , Lung/pathology , Mice , Mice, Inbred BALB C , Reverse Genetics , Serial Passage , Virus Replication
8.
Science ; 374(6571): 1099-1106, 2021 Nov 26.
Article in English | MEDLINE | ID: covidwho-1467657

ABSTRACT

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.


Subject(s)
RNA, Viral/genetics , Replicon/physiology , SARS-CoV-2/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antiviral Agents/pharmacology , Cell Line , Humans , Interferons/pharmacology , Microbial Sensitivity Tests , Mutation , Plasmids , RNA, Viral/metabolism , Replicon/genetics , Reverse Genetics , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virion/genetics , Virion/physiology , Virus Replication
9.
Vet Microbiol ; 254: 109014, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1107294

ABSTRACT

TW-like infectious bronchitis virus (IBV) with high pathogenicity is becoming the predominant IBV type circulating in China. To develop vaccines against TW-like IBV strains and investigate the critical genes associated with their virulence, GD strain was attenuated by 140 serial passages in specific-pathogen-free embryonated eggs and the safety and efficacy of the attenuated GD strain (aGD) were examined. The genome sequences of GD and aGD were also compared and the effects of mutations in the S gene were observed. The results revealed that aGD strain showed no obvious pathogenicity with superior protective efficacy against TW-like and QX-like virulent IBV strains. The genomes of strains aGD and GD shared high similarity (99.87 %) and most of the mutations occurred in S gene. Recombinant IBV strain rGDaGD-S, in which the S gene was replaced with the corresponding regions from aGD, showed decreased pathogenicity compared with its parental strain. In conclusion, attenuated TW-like IBV strain aGD is a potential vaccine candidate and the S gene is responsible for its attenuation. Our research has laid the foundation for future exploration of the attenuating molecular mechanism of IBV.


Subject(s)
Chickens/virology , Infectious bronchitis virus/genetics , Infectious bronchitis virus/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Viral Vaccines/genetics , Virulence Factors/genetics , Animals , Chick Embryo , Coronavirus Infections/prevention & control , Infectious bronchitis virus/immunology , Poultry Diseases/prevention & control , Poultry Diseases/virology , Reverse Genetics/methods , Serial Passage , Specific Pathogen-Free Organisms , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Attenuated/immunology , Viral Vaccines/immunology
10.
Nat Commun ; 12(1): 3431, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1262001

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.


Subject(s)
Reverse Genetics , SARS-CoV-2/genetics , Amino Acid Sequence , Animals , Base Sequence , Chlorocebus aethiops , Culicidae/virology , Furin/metabolism , Genome, Viral , HEK293 Cells , Humans , Mice , Mutation/genetics , NIH 3T3 Cells , Polymerase Chain Reaction , RAW 264.7 Cells , Receptors, Virus/metabolism , Vero Cells , Viral Proteins/chemistry , Virus Replication
11.
Viruses ; 13(6)2021 05 28.
Article in English | MEDLINE | ID: covidwho-1256665

ABSTRACT

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. In this review, we discuss the application of this technique to RNA viruses including members of the Flaviviridae family, Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2. We also briefly discuss the reverse genetics systems which allow for analysis of viral replication cycles, next-generation sequencing technologies and the bioinformatics tools that facilitate this research.


Subject(s)
High-Throughput Nucleotide Sequencing , Mutation/genetics , RNA Viruses/genetics , Sequence Analysis, RNA , Computational Biology , Gene Library , Genome, Viral/genetics , RNA Viruses/classification , RNA Viruses/physiology , Reverse Genetics , Viral Proteins/genetics
12.
mBio ; 12(2)2021 04 20.
Article in English | MEDLINE | ID: covidwho-1195827

ABSTRACT

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has caused extensive mortality and morbidity and wreaked havoc on socioeconomic structures. The urgent need to better understand SARS-CoV-2 biology and enable continued development of effective countermeasures is aided by the production of laboratory tools that facilitate SARS-CoV-2 research. We previously created a directly accessible SARS-CoV-2 toolkit containing user-friendly reverse genetic (RG) infectious clones of SARS-CoV-2. Here, using K18-human ACE2 (hACE2) mice, we confirmed the validity of RG-rescued SARS-CoV-2 viruses to reproduce the infection profile, clinical disease, and pathogenesis already established in mice infected with natural SARS-CoV-2 isolates, often patient derived. RG-rescued SARS-CoV-2-infected K18-hACE2 mice developed substantial clinical disease and weight loss by day 6 postinfection. RG-rescued SARS-CoV-2 was recovered from the lungs and brains of infected K18-hACE2 mice, and infection resulted in viral pneumonia with considerable changes in lung pathology, as seen previously with natural SARS-CoV-2 infection. In mice infected with RG-rescued SARS-CoV-2-mCherry, mCherry was detected in areas of lung consolidation and colocalized with clinically relevant SARS-CoV-2-assocated immunopathology. RG-rescued SARS-CoV-2 viruses successfully recapitulated many of the features of severe COVID-19 associated with the K18-hACE2 model of SARS-CoV-2 infection. With utility in vivo, the RG-rescued SARS-CoV-2 viruses will be valuable resources to advance numerous areas of SARS-CoV-2 basic research and COVID-19 vaccine development.IMPORTANCE To develop COVID-19 countermeasures, powerful research tools are essential. We produced a SARS-COV-2 reverse genetic (RG) infectious clone toolkit that will benefit a variety of investigations. In this study, we further prove the toolkit's value by demonstrating the in vivo utility of RG-rescued SARS-CoV-2 isolates. RG-rescued SARS-CoV-2 isolates reproduce disease signs and pathology characteristic of the K18-hACE2 mouse model of severe COVID-19 in infected mice. Having been validated as a model of severe COVID-19 previously using only natural SARS-CoV-2 isolated from patients, this is the first investigation of RG-rescued SARS-CoV-2 viruses in K18-hACE2 mice. The RG-rescued SARS-CoV-2 viruses will facilitate basic understanding of SARS-CoV-2 and the preclinical development of COVID-19 therapeutics.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/etiology , SARS-CoV-2/pathogenicity , Animals , COVID-19/pathology , COVID-19/virology , Cytokine Release Syndrome/etiology , Disease Models, Animal , Female , Host Microbial Interactions , Humans , Inflammation Mediators/metabolism , Lung/immunology , Lung/pathology , Lung/virology , Male , Mice , Mice, Transgenic , Pandemics , Pneumonia, Viral/etiology , Pneumonia, Viral/virology , Reverse Genetics/methods , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Viral Tropism , Virus Replication
13.
Cell Rep ; 35(3): 109014, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1163485

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.


Subject(s)
COVID-19/genetics , Reverse Genetics , SARS-CoV-2/genetics , Animals , Cricetinae , HEK293 Cells , Humans
15.
Lancet Microbe ; 2(5): e210-e218, 2021 05.
Article in English | MEDLINE | ID: covidwho-1117258

ABSTRACT

BACKGROUND: The COVID-19 agent, SARS-CoV-2, is conspecific with SARS-CoV, the causal agent of the severe acute respiratory syndrome epidemic in 2002-03. Although the viruses share a completely homologous repertoire of proteins and use the same cellular entry receptor, their transmission efficiencies and pathogenetic traits differ. We aimed to compare interferon antagonism by SARS-CoV and SARS-CoV-2. METHODS: For this functional study, we infected Vero E6 and Calu-3 cells with strains of SARS-CoV and SARS-CoV-2. We studied differences in cell line-specific replication (Vero E6 vs Calu-3 cells) and analysed these differences in relation to TMPRSS2-dependent cell entry based on inhibition with the drug camostat mesilate. We evaluated viral sensitivity towards type I interferon treatment and assessed cytokine induction and type I interferon signalling in the host cells by RT-PCR and analysis of transcription factor activation and nuclear translocation. Based on reverse genetic engineering of SARS-CoV, we investigated the contribution of open reading frame 6 (ORF6) to the observed phenotypic differences in interferon signalling, because ORF6 encodes an interferon signalling antagonist. We did a luciferase-based interferon-stimulated response element promotor activation assay to evaluate the antagonistic capacity of SARS-CoV-2 wild-type ORF6 constructs and three mutants (Gln51Glu, Gln56Glu, or both) that represent amino acid substitutions between SARS-CoV and SARS-CoV-2 protein 6 in the carboxy-terminal domain. FINDINGS: Overall, replication was higher for SARS-CoV in Vero E6 cells and for SARS-CoV-2 in Calu-3 cells. SARS-CoV-2 was reliant on TMPRSS2, found only in Calu-3 cells, for more efficient entry. SARS-CoV-2 was more sensitive to interferon treatment, less efficient in suppressing cytokine induction via IRF3 nuclear translocation, and permissive of a higher level of induction of interferon-stimulated genes MX1 and ISG56. SARS-CoV-2 ORF6 expressed in the context of a fully replicating SARS-CoV backbone suppressed MX1 gene induction, but this suppression was less efficient than that by SARS-CoV ORF6. Mutagenesis showed that charged amino acids in residues 51 and 56 shift the phenotype towards more efficient interferon antagonism, as seen in SARS-CoV. INTERPRETATION: SARS-CoV-2 ORF6 interferes less efficiently with human interferon induction and interferon signalling than SARS-CoV ORF6. Because of the homology of the genes, onward selection for fitness could involve functional optimisation of interferon antagonism. Charged amino acids at positions 51 and 56 in ORF6 should be monitored for potential adaptive changes. FUNDING: Bundesministerium für Bildung und Forschung, EU RECOVER project.


Subject(s)
COVID-19 , Interferon Type I , SARS Virus , Amino Acids/genetics , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Interferon Type I/genetics , Reverse Genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Viral Proteins/chemistry
16.
PLoS Biol ; 19(2): e3001091, 2021 02.
Article in English | MEDLINE | ID: covidwho-1102372

ABSTRACT

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.


Subject(s)
COVID-19 Vaccines , COVID-19/diagnosis , COVID-19/virology , Reverse Genetics , SARS-CoV-2/genetics , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Codon , Humans , Hydrazones/pharmacology , Mice , Morpholines/pharmacology , Open Reading Frames , Plasmids/genetics , Pyrimidines/pharmacology , Serine Endopeptidases/metabolism , Vero Cells , Viral Proteins/metabolism
17.
Cell Host Microbe ; 29(3): 489-502.e8, 2021 03 10.
Article in English | MEDLINE | ID: covidwho-1064930

ABSTRACT

The SARS-CoV-2 virus, the causative agent of COVID-19, is undergoing constant mutation. Here, we utilized an integrative approach combining epidemiology, virus genome sequencing, clinical phenotyping, and experimental validation to locate mutations of clinical importance. We identified 35 recurrent variants, some of which are associated with clinical phenotypes related to severity. One variant, containing a deletion in the Nsp1-coding region (Δ500-532), was found in more than 20% of our sequenced samples and associates with higher RT-PCR cycle thresholds and lower serum IFN-ß levels of infected patients. Deletion variants in this locus were found in 37 countries worldwide, and viruses isolated from clinical samples or engineered by reverse genetics with related deletions in Nsp1 also induce lower IFN-ß responses in infected Calu-3 cells. Taken together, our virologic surveillance characterizes recurrent genetic diversity and identified mutations in Nsp1 of biological and clinical importance, which collectively may aid molecular diagnostics and drug design.


Subject(s)
COVID-19/immunology , COVID-19/virology , Interferon Type I/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Viral Nonstructural Proteins/genetics , A549 Cells , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Base Sequence , COVID-19/blood , Cell Line , Child , Child, Preschool , Chlorocebus aethiops , Female , Gene Deletion , Genomics , HEK293 Cells , Humans , Infant , Interferon Type I/blood , Interferon-beta/blood , Interferon-beta/metabolism , Male , Middle Aged , Molecular Epidemiology , Reverse Genetics , Vero Cells , Viral Nonstructural Proteins/immunology , Young Adult
18.
Nat Protoc ; 16(3): 1761-1784, 2021 03.
Article in English | MEDLINE | ID: covidwho-1054034

ABSTRACT

Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA (cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.


Subject(s)
Genetic Engineering/methods , Reverse Genetics/methods , SARS-CoV-2/genetics , DNA, Viral/genetics , Genome, Viral/genetics
19.
Viruses ; 12(12)2020 12 21.
Article in English | MEDLINE | ID: covidwho-1000348

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.


Subject(s)
3' Untranslated Regions/genetics , COVID-19/virology , Nucleic Acid Conformation , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , Base Sequence , Cell Line , Conserved Sequence , Cricetinae , High-Throughput Nucleotide Sequencing , Humans , Mesocricetus , Models, Molecular , Plasmids , Point Mutation , Reverse Genetics/methods , SARS-CoV-2/physiology , Sequence Alignment , Sequence Homology, Nucleic Acid , Sulfuric Acid Esters , Transcription, Genetic , Virion/genetics , Virion/physiology
20.
mBio ; 11(5)2020 09 25.
Article in English | MEDLINE | ID: covidwho-797116

ABSTRACT

Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.


Subject(s)
Betacoronavirus/genetics , Chromosomes, Artificial, Bacterial/genetics , Animals , Betacoronavirus/pathogenicity , Betacoronavirus/physiology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/virology , Cricetinae , DNA, Complementary/genetics , Genome, Viral/genetics , Pandemics , Pneumonia, Viral/virology , RNA, Viral/genetics , Reverse Genetics , SARS-CoV-2 , Vero Cells , Virus Replication
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