Complete Sequence (20 Kilobases) of the Polyprotein-Encoding Gene 1 of Transmissible Gastroenteritis Virus

Éléouët, Jean-François; Rasschaert, Denis; Lambert, Patrick; Lévy, Laurent; Vende, Patrice; Laude, Hubert

doi:10.1006/viro.1995.1004

Cited by 101 publications

(154 citation statements)

References 0 publications

Supporting

Mentioning

145

Contrasting

Order By: Relevance

“…For most of them, the mechanisms used to express the downstream ORFs remain to be determined. On the basis of the available data for other coronaviruses (Brierley et al, 1989;Eleouet et al, 1995;Herold et al, 1993;Kocherhans et al, 2001), it seemed likely that ORF1b expression from the genomic RNA would involve 21 ribosomal frameshifting, a process that essentially depends on two elements, known as the 'slippery' sequence, i.e. the site where the ribosomes shift into the 21 reading frame, and a complex RNA pseudoknot structure (Brierley et al, 1989.…”

Section: Translationmentioning

confidence: 99%

Mechanisms and enzymes involved in SARS coronavirus genome expression

Thiel

Иванов

Putics

et al. 2003

Journal of General Virology

813

960

View full text Add to dashboard Cite

A novel coronavirus is the causative agent of the current epidemic of severe acute respiratory syndrome (SARS). Coronaviruses are exceptionally large RNA viruses and employ complex regulatory mechanisms to express their genomes. Here, we determined the sequence of SARS coronavirus (SARS-CoV), isolate Frankfurt 1, and characterized key RNA elements and protein functions involved in viral genome expression. Important regulatory mechanisms, such as the (discontinuous) synthesis of eight subgenomic mRNAs, ribosomal frameshifting and posttranslational proteolytic processing, were addressed. Activities of three SARS coronavirus enzymes, the helicase and two cysteine proteinases, which are known to be critically involved in replication, transcription and/or post-translational polyprotein processing, were characterized. The availability of recombinant forms of key replicative enzymes of SARS coronavirus should pave the way for high-throughput screening approaches to identify candidate inhibitors in compound libraries. INTRODUCTIONSevere acute respiratory syndrome (SARS) is a lifethreatening form of pneumonia (Peiris et al., 2003a). In the course of a few months in 2003, an epidemic emerged that has spread from its likely origin in Guangdong Province, China, to 32 countries. By 11 June 2003 more than 8400 cases and 789 deaths had been recorded by the World Health Organization. The rapid transmission by aerosols (and probably also the faecal-oral route) and the high mortality rate make SARS a global threat for which no efficacious therapy is available. There is now clear evidence that SARS is caused by a previously unknown coronavirus, provisionally termed SARS coronavirus (SARS-CoV) (Peiris et al., 2003b;Drosten et al., 2003;Ksiazek et al., 2003;Fouchier et al., 2003). Genome sequences of SARS-CoV isolates obtained from a number of index patients have been published recently and provide important information on the organization, phylogeny and variability of the 29?7 kb positive-strand RNA genome of SARS-CoV (Rota et al., 2003;Marra et al., 2003;Ruan et al., 2003). By analogy with other coronaviruses (Lai & Holmes, 2001;Gorbalenya, 2001), SARS-CoV gene expression is expected to involve complex transcriptional, translational and post-translational regulatory mechanisms, whose molecular details remain to be determined. SARS-CoV genome expression starts with the translation of two large replicative polyproteins, pp1a (486 kDa) and pp1ab (790 kDa), which are encoded by the viral replicase gene (21 221 nt) that comprises ORFs 1a and 1b (Fig. 1). Expression of the ORF1b-encoded region of pp1ab is predicted to involve ribosomal frameshifting into the 21 frame just upstream of the ORF1a translation termination codon (Brierley et al., 1989). The pp1a and pp1ab polyproteins are processed by viral proteinases to yield the functional components of the membrane-bound replicase complex (Ziebuhr et al., 2000). In contrast to most other coronaviruses, which use three proteinase activities for replicase polyprotein processing (Ziebuhr et ...

show abstract

Section: Translationmentioning

confidence: 99%

Mechanisms and enzymes involved in SARS coronavirus genome expression

Thiel

Иванов

Putics

et al. 2003

Journal of General Virology

813

960

View full text Add to dashboard Cite

show abstract

“…Transmissible gastroenteritis virus (TGEV), a group I coronavirus, contains a ϳ28.5-kb genomic RNA that is packaged into a helical nucleocapsid structure and surrounded by an envelope that contains three virus-specific glycoprotein spikes, including the S glycoprotein, membrane glycoprotein (M), and a small envelope glycoprotein (E) (17,18,33,36). The TGEV genome is polycistronic and encodes eight large open reading frames (ORFs), which are expressed from full-length or subgenome-length mRNAs during infection (17,42,43). The 5Ј-most ϳ20 kb encode the RNA replicase genes, which are encoded in two large ORFs, designated 1a and 1b, the latter of which is expressed by ribosomal frameshifting (3,17).…”

mentioning

confidence: 99%

“…In addition to the viral mRNAs, full-length and subgenome-length negativestrand RNAs are implicated in mRNA synthesis (4,26,40,42,43). Another unique feature of coronavirus replication is the high RNA recombination frequencies associated with infection (6,25,26).The large size of the coronavirus genome, coupled with the inability to clone portions of the polymerase gene in microbial vectors, has hampered the ability to perform precise manipulations and reverse genetics in members of the Coronaviridae (17,18,26,44). Recently these problems were overcome when a full-length cDNA of TGEV was stably cloned in bacterial artificial chromosome (BAC) vectors (3).…”

mentioning

confidence: 99%

“…The TGEV genome is polycistronic and encodes eight large open reading frames (ORFs), which are expressed from full-length or subgenome-length mRNAs during infection (17,42,43). The 5Ј-most ϳ20 kb encode the RNA replicase genes, which are encoded in two large ORFs, designated 1a and 1b, the latter of which is expressed by ribosomal frameshifting (3,17). ORF1a encodes at least two viral proteases and several other nonstructural proteins, while ORF1b contains polymerase, helicase, and metal-binding motifs typical of an RNA polymerase (3,17,19).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model

Yount

Curtis

Baric

2000

J Virol

244

284

View full text Add to dashboard Cite

A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The ϳ30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of ϳ28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.Molecular genetic analysis of the structure and function of RNA virus genomes has been profoundly advanced by the availability of full-length cDNA clones, the source of infectious RNA transcripts that replicate efficiently when introduced into permissive cell lines (2, 9). Recombinant DNA technology has allowed the isolation of infectious cDNA clones from a number of positive-stranded RNA viruses, including picornaviruses, caliciviruses, alphaviruses, flaviviruses, and arteriviruses, whose RNA genomes range in size from ϳ7 to 15 kb in length (1,13,32,34,35,47,48,54). The availability of these clones has enhanced our understanding of the molecular mechanisms of viral replication and pathogenesis and resulted in new approaches for heterologous gene expression and vaccine development.The order Nidovirales includes mammalian positive-polarity single-stranded RNA viruses in the arterivirus and coronavirus families (10, 16). The Coronaviridae family includes the Coronavirus and Torovirus genera (10, 46). Despite significant size differences (ϳ13 to 32 kb), the polycistronic genome organization and regulation of gene expression from a nested set of subgenomic mRNAs are similar for all members of the order (16, 4...

show abstract

“…This enteropathogenic virus causes acute and fatal diarrhea in newborn piglets. TGEV replicates in enterocytes and provokes villous atrophy, is closely related to the human respiratory coronavirus HCoV-229E (9), and can also infect the respiratory tract. Moreover, some variant strains of TGEV, such as the porcine respiratory coronavirus (PRCoV), have lost their intestinal tropism (11,18).…”

mentioning

confidence: 99%

The Viral Nucleocapsid Protein of Transmissible Gastroenteritis Coronavirus (TGEV) Is Cleaved by Caspase-6 and -7 during TGEV-Induced Apoptosis

Éléouët

Slee

Saurini

et al. 2000

J Virol

View full text Add to dashboard Cite

The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and ␣-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD 359 2. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.

show abstract

Complete Sequence (20 Kilobases) of the Polyprotein-Encoding Gene 1 of Transmissible Gastroenteritis Virus

Cited by 101 publications

References 0 publications

Mechanisms and enzymes involved in SARS coronavirus genome expression

Mechanisms and enzymes involved in SARS coronavirus genome expression

Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model

The Viral Nucleocapsid Protein of Transmissible Gastroenteritis Coronavirus (TGEV) Is Cleaved by Caspase-6 and -7 during TGEV-Induced Apoptosis

Contact Info

Product

Resources

About