A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

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.

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Construction of a synthetic messenger RNA encoding a membrane protein.

The Journal of Cell Biology ◽

10.1083/jcb.96.5.1464 ◽

1983 ◽

Vol 96 (5) ◽

pp. 1464-1469 ◽

Cited By ~ 4

Author(s):

J L Rubenstein ◽

T G Chappell

Keyword(s):

G Protein ◽

Messenger Rna ◽

In Vitro Transcription ◽

Membrane Insertion ◽

Wheat Germ Extract ◽

E Coli ◽

Infected Cells ◽

Vesicular Stomatitis ◽

Synthetic Mrna

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.

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Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations

Journal of Virology ◽

10.1128/jvi.72.8.6283-6290.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6283-6290 ◽

Cited By ~ 39

Author(s):

Ervin Fodor ◽

Peter Palese ◽

George G. Brownlee ◽

Adolfo García-Sastre

Keyword(s):

Influenza Virus ◽

Transcription Initiation ◽

Influenza A ◽

Reverse Genetics ◽

Mrna Levels ◽

Double Mutation ◽

Ribonucleoprotein Complexes ◽

Virus Rna ◽

Infected Cells

ABSTRACT We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G→A-U double mutation at positions 11 and 12′ at the 3′ and 5′ ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.

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Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA

Journal of Virology ◽

10.1128/jvi.72.11.8913-8920.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8913-8920 ◽

Cited By ~ 44

Author(s):

Kun Yao ◽

Vikram N. Vakharia

Keyword(s):

Rna Polymerase ◽

Reverse Genetics ◽

In Vitro Transcription ◽

Infectious Pancreatic Necrosis Virus ◽

Rna Dependent Rna Polymerase ◽

Rna Transcripts ◽

Recovered Virus ◽

Cloned Cdna ◽

Pancreatic Necrosis Virus

ABSTRACT We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of theBirnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3′-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3′ end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

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Recovery of Infectious Bluetongue Virus from RNA

Journal of Virology ◽

10.1128/jvi.01819-06 ◽

2006 ◽

Vol 81 (5) ◽

pp. 2179-2186 ◽

Cited By ~ 53

Author(s):

Mark Boyce ◽

Polly Roy

Keyword(s):

Bluetongue Virus ◽

Reverse Genetics ◽

Infectious Virus ◽

Helper Virus ◽

Rnase A ◽

Active Components ◽

Viral Protein Synthesis ◽

Transfected Cells ◽

Infected Cells

ABSTRACT Bluetongue virus (BTV) is an insect-vectored emerging pathogen of ruminants with the potential for devastating economic impact on European agriculture. BTV and many other members of the Reoviridae have remained stubbornly refractory to the development of methods for the rescue of infectious virus from cloned nucleic acid (reverse genetics). Partially disassembled virus particles are transcriptionally active, synthesizing viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. With the goal of generating a reverse-genetics system for BTV, we examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (single-stranded RNA [ssRNA]) synthesized in vitro using BTV core particles. Following transfection, viral-protein synthesis was detected by immunoblotting, and confocal examination of the cells showed a punctate cytoplasmic distribution of inclusion bodies similar to that seen in infected cells. Viral double-stranded RNA (dsRNA) was isolated from ssRNA-transfected cells, demonstrating that replication of the ssRNA had occurred. Additionally, infectious virus was present in the medium of transfected cells, as demonstrated by the passage of infectivity in BSR cells. Infectivity was sensitive to single-strand-specific RNase A, and cotransfection of genomic BTV dsRNA with transcribed ssRNA demonstrated that the ssRNA species, rather than dsRNA, were the active components. We conclude that it is possible to recover infectious BTV wholly from ssRNA, which suggests a means for establishing helper virus-independent reverse-genetics systems for members of the Reoviridae.

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Targeted mutations in a highly conserved motif of the nsp1β protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus

Journal of General Virology ◽

10.1099/vir.0.051748-0 ◽

2013 ◽

Vol 94 (9) ◽

pp. 1972-1983 ◽

Cited By ~ 25

Author(s):

Yanhua Li ◽

Longchao Zhu ◽

Steven R. Lawson ◽

Ying Fang

Keyword(s):

Gene Expression ◽

Reverse Genetics ◽

Innate Immune ◽

Respiratory Syndrome Virus ◽

Structural Protein ◽

Virus Growth ◽

Conserved Motif ◽

Immune Genes ◽

Infected Cells

Non-structural protein 1β (nsp1β) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a papain-like cysteine protease (PLPβ) domain and has been identified as the main viral protein antagonizing the host innate immune response. In this study, nsp1β was determined to suppress the expression of reporter genes as well as to suppress ‘self-expression’ in transfected cells, and this activity appeared to be associated with its interferon (IFN) antagonist function. To knock down the effect of nsp1β on IFN activity, a panel of site-specific mutations in nsp1β was analysed. Double mutations K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV) targeting a highly conserved motif of nsp1β, GKYLQRRLQ (in bold), impaired the ability of nsp1β to suppress IFN-β and reporter gene expression, as well as to suppress ‘self-expression’ in vitro. Subsequently, viable recombinant viruses vSD01-08-K130A/R134A and vSD95-21-K124A/R128A, containing double mutations in the GKYLQRRLQ motif were generated using reverse genetics. In comparison with WT viruses, these nsp1β mutants showed impaired growth ability in infected cells, but the PLPβ cleavage function was not directly affected. The expression of selected innate immune genes was determined in vSD95-21-K124A/R128A mutant-infected cells. The results consistently showed that gene expression levels of IFN-α, IFN-β and IFN-stimulated gene 15 were upregulated in cells that were infected with the vSD95-21-K124A/R128A compared with that of WT virus. These data suggest that PRRSV nsp1β may selectively suppress cellular gene expression, including expression of genes involved in the host innate immune function. Modifying the key residues in the highly conserved GKYLQRRLQ motif could attenuate virus growth and improve the cellular innate immune responses.

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Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe

Journal of General Virology ◽

10.1099/vir.0.049981-0 ◽

2013 ◽

Vol 94 (4) ◽

pp. 851-859 ◽

Cited By ~ 41

Author(s):

Richard M. Elliott ◽

Gjon Blakqori ◽

Ingeborg C. van Knippenberg ◽

Elina Koudriakova ◽

Ping Li ◽

...

Keyword(s):

Host Cell ◽

Recombinant Virus ◽

Reverse Genetics ◽

Cell Metabolism ◽

Schmallenberg Virus ◽

Wild Type Virus ◽

Cell Protein ◽

Virus Recombinant ◽

Rescue System

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.

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Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells

eLife ◽

10.7554/elife.01535 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 72

Author(s):

Safia Deddouche ◽

Delphine Goubau ◽

Jan Rehwinkel ◽

Probir Chakravarty ◽

Sharmin Begum ◽

...

Keyword(s):

Antisense Rna ◽

Rna Synthesis ◽

In Vitro Transcription ◽

Encephalomyocarditis Virus ◽

Type I Interferons ◽

Type I ◽

Specific Sequence ◽

Infected Cells ◽

Rna Complexes

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells.

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Phenotypic Diversity of Infectious Red Sea Bream Iridovirus Isolates from Cultured Fish in Japan

Applied and Environmental Microbiology ◽

10.1128/aem.02255-08 ◽

2009 ◽

Vol 75 (11) ◽

pp. 3535-3541 ◽

Cited By ~ 31

Author(s):

Hajime Shinmoto ◽

Ken Taniguchi ◽

Takuya Ikawa ◽

Kenji Kawai ◽

Syun-ichirou Oshima

Keyword(s):

Red Sea ◽

Phenotypic Diversity ◽

Sea Bream ◽

Cell Culture Supernatant ◽

Red Sea Bream ◽

Virus Family ◽

Recent Emergence ◽

Infected Cells

ABSTRACT Megalocytivirus is causing economically serious mass mortality by infecting fish in and around the Pacific region of Asia. The recent emergence of many new iridoviruses has drawn attention to the marked taxonomic variation within this virus family. Most studies of these viruses have not included extensive study of these emergent species. We explored the emergence of red sea bream iridovirus (RSIV) on a fish farm in Japan, and we specifically endeavored to quantify genetic and phenotypic differences between RSIV isolates using in vitro and in vivo methods. The three isolates had identical major capsid protein sequences, and they were closely related to Korean RSIV isolates. In vitro studies revealed that the isolates differed in replication rate, which was determined by real-time quantitative PCR of viral genomes in infected cells and cell culture supernatant, and in cell viability, estimated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay for infected cells. In vivo studies showed that the isolates exhibit different virulence characteristics: infected red sea bream showed either acute death or subacute death according to infection with different isolates. Significant differences were seen in the antigenicity of isolates by a formalin-inactivated vaccine test. These results revealed that variant characteristics exist in the same phylogenetic location in emergent iridoviruses. We suggest that this strain variation would expand the host range in iridoviral epidemics.

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