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1.
Virology ; 433(2): 421-30, 2012 Nov 25.
Article in English | MEDLINE | ID: mdl-22995191

ABSTRACT

In previous studies, the Beaudette strain of coronavirus infectious bronchitis virus (IBV) was adapted from chicken embryo to Vero, a monkey kidney cell line, by serial propagation for 65 passages. To characterize the susceptibility of other human and animal cells to IBV, 15 human and animal cell lines were infected with the Vero-adapted IBV and productive infection was observed in four human cell lines: H1299, HepG2, Hep3B and Huh7. In other cell lines, the virus cannot be propagated beyond passage 5. Interestingly, cellular furin abundance in five human cell lines was shown to be strongly correlated with productive IBV infection. Cleavage of IBV spike protein by furin may contribute to the productive IBV infection in these cells. The findings that IBV could productively infect multiple human and animal cells of diverse tissue and organ origins would provide a useful system for studying the pathogenesis of coronavirus.


Subject(s)
Coronavirus Infections/etiology , Furin/metabolism , Infectious bronchitis virus , Animals , Base Sequence , Binding Sites , Cell Line , Chlorocebus aethiops , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus M Proteins , Coronavirus Nucleocapsid Proteins , Furin/antagonists & inhibitors , Furin/genetics , Gene Knockdown Techniques , Hep G2 Cells , Host-Pathogen Interactions , Humans , Infectious bronchitis virus/pathogenicity , Infectious bronchitis virus/physiology , Membrane Glycoproteins/metabolism , Nucleocapsid Proteins/metabolism , RNA, Small Interfering/genetics , Spike Glycoprotein, Coronavirus , Vero Cells , Viral Envelope Proteins/metabolism , Viral Matrix Proteins/metabolism , Virus Cultivation
2.
J Virol ; 84(17): 8571-83, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20573827

ABSTRACT

The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.


Subject(s)
Coronavirus Infections/metabolism , DEAD-box RNA Helicases/metabolism , Infectious bronchitis virus/physiology , Viral Nonstructural Proteins/metabolism , Virus Replication , Animals , Cell Nucleus/metabolism , Chlorocebus aethiops , Coronavirus Infections/genetics , Coronavirus Infections/virology , Cytoplasm/metabolism , DEAD-box RNA Helicases/genetics , HeLa Cells , Humans , Infectious bronchitis virus/genetics , Protein Binding , Protein Transport , Two-Hybrid System Techniques , Vero Cells , Viral Nonstructural Proteins/genetics
3.
J Virol ; 84(14): 7325-36, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20444893

ABSTRACT

Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.


Subject(s)
Cysteine Endopeptidases , Infectious bronchitis virus , Viral Proteins , 3C Viral Proteases , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Cell Line , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Humans , Infectious bronchitis virus/enzymology , Infectious bronchitis virus/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Substrate Specificity/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
4.
PLoS One ; 4(7): e6130, 2009 Jul 02.
Article in English | MEDLINE | ID: mdl-19572016

ABSTRACT

Coronavirus host and cell specificities are determined by specific interactions between the viral spike (S) protein and host cell receptor(s). Avian coronavirus infectious bronchitis (IBV) has been adapted to embryonated chicken eggs, primary chicken kidney (CK) cells, monkey kidney cell line Vero, and other human and animal cells. Here we report that acquisition of the cell-cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. Expression of S protein derived from Vero- and CK-adapted strains showed efficient induction of membrane fusion. However, expression of S protein cloned from the third passage of IBV in chicken embryo (EP3) did not show apparent syncytia formation. By construction of chimeric S constructs and site-directed mutagenesis, a point mutation (L857-F) at amino acid position 857 in the heptad repeat 1 region of S protein was shown to be responsible for its acquisition of the cell-cell fusion activity. Furthermore, a G405-D point mutation in the S1 domain, which was acquired during further propagation of Vero-adapted IBV in Vero cells, could enhance the cell-cell fusion activity of the protein. Re-introduction of L857 back to the S gene of Vero-adapted IBV allowed recovery of variants that contain the introduced L857. However, compensatory mutations in S1 and some distant regions of S2 were required for restoration of the cell-cell fusion activity of S protein carrying L857 and for the infectivity of the recovered variants in cultured cells. This study demonstrates that acquisition of the cell-cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins.


Subject(s)
Amino Acid Substitution , Cell Fusion , Coronavirus/pathogenicity , Membrane Glycoproteins/physiology , Viral Envelope Proteins/physiology , Amino Acid Sequence , Animals , Blotting, Western , Cells, Cultured , Electroporation , Flow Cytometry , Fluorescent Antibody Technique , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics
5.
J Virol Methods ; 160(1-2): 48-56, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19409420

ABSTRACT

Manipulation of the coronavirus genome to accommodate and express foreign genes is an attractive approach for gene delivery and vaccine development. By using an infectious cloning system developed recently for the avian coronavirus infectious bronchitis virus (IBV), the enhanced green fluorescent protein (EGFP) gene, the firefly luciferase gene and several host and viral genes (eIF3f, SARS ORF6, Dengue virus 1 core protein gene) were inserted into various positions of the IBV genome, and the effects on gene expression, virus recovery, and stability in cell culture were studied. Selected viruses were also inoculated into chicken embryos for studies of foreign gene expression at different tissue level. The results demonstrated the stability of recombinant viruses depends on the intrinsic properties of the foreign gene itself as well as the position at which the foreign genes were inserted. For unstable viruses, the loss of expression of the inserted genes was found to result from a large deletion of the inserted gene and even IBV backbone sequences. This represents a promising system for development of coronavirus-based gene delivery vectors and vaccines against coronavirus and other viral infections in chicken.


Subject(s)
Genetic Therapy , Genetic Vectors , Infectious bronchitis virus/genetics , Viral Vaccines/genetics , Animals , Chick Embryo , Chickens , Dengue Virus/genetics , Genes, Reporter , Genomic Instability , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Severe acute respiratory syndrome-related coronavirus/genetics , Sequence Deletion , Viral Proteins/genetics , Viral Vaccines/immunology
6.
Virology ; 379(2): 175-80, 2008 Sep 30.
Article in English | MEDLINE | ID: mdl-18678384

ABSTRACT

Coronavirus 3C-like proteinase (3CLpro) plays important roles in viral life cycle through extensive processing of the polyproteins 1a and 1ab into 12 mature, non-structural proteins (nsp5-nsp16). Structural and biochemical studies have revealed that all confirmed 3CLpro cleavage sites have a conserved Gln residue at the P1 position, which is thought to be absolutely required for efficient cleavage. Recent studies on murine hepatitis virus (MHV) showed that processing of the 1a polyprotein at the position between nsp10-nsp11 is essential for viral replication. In this report, we investigated the requirement of processing at the equivalent position for replication of avian coronavirus infectious bronchitis virus (IBV), using an infectious cloning system. The results showed that mutation of the P1 Gln to Pro or deletion of the Gln residue in the nsp10-nsp11/12 site completely abolished the 3CLpro-mediated processing, but allowed production of infectious recombinant viruses with variable degrees of growth defect, suggesting that cleavage at the nsp10-nsp11/12 site of IBV is dispensable for viral replication in cultured cells. This study would pave a way for potential vaccine development by generation of attenuated IBV from field isolates through manipulation of the nsp10-nsp11/12 cleavage site. Similar approaches would be also applicable to other human and animal coronaviruses.


Subject(s)
Infectious bronchitis virus/metabolism , Viral Nonstructural Proteins/metabolism , Viral Proteins/metabolism , Amino Acid Substitution , Animals , Catalytic Domain/genetics , Chlorocebus aethiops , Coronavirus 3C Proteases , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Humans , Infectious bronchitis virus/genetics , Infectious bronchitis virus/physiology , Mutagenesis, Site-Directed , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Vero Cells , Virus Replication
7.
FEBS J ; 274(16): 4211-22, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17645546

ABSTRACT

The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). To understand the functions of these proteins, we cloned cDNA fragments covering these ORFs into expression plasmids, and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein, 8a; no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site-directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N-linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region, protein 8b undergoes rapid degradation by proteasomes, and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b-induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation could also stabilize protein 8ab. More interestingly, the two proteins could bind to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of severe acute respiratory syndrome coronavirus.


Subject(s)
Genome, Viral , RNA, Messenger/genetics , Severe acute respiratory syndrome-related coronavirus/metabolism , Viral Proteins/metabolism , Animals , Blotting, Northern , Blotting, Western , COS Cells , Chlorocebus aethiops , Cloning, Molecular , Gene Expression Regulation, Viral/drug effects , Glycosylation/drug effects , Humans , Protease Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus/genetics , Severe Acute Respiratory Syndrome/virology , Ubiquitin/metabolism , Vero Cells , Viral Proteins/genetics
8.
Virology ; 358(1): 136-47, 2007 Feb 05.
Article in English | MEDLINE | ID: mdl-16979681

ABSTRACT

Genetic manipulation of the RNA genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of RNA viruses. During construction of an infectious clone from a Vero cell-adapted coronavirus infectious bronchitis virus (IBV), we found that a G-C point mutation at nucleotide position 15526, causing Arg-to-Pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of IBV on Vero cells. When the in vitro-synthesized full-length transcripts containing this mutation were introduced into Vero cells, no infectious virus was rescued. Upon correction of the mutation, infectious virus was recovered. Further characterization of the in vitro-synthesized full-length transcripts containing the G15526C mutation demonstrated that this mutation may block the transcription of subgenomic RNAs. Substitution mutation of the Arg132 residue to a positively charged amino acid Lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. However, mutation of the Arg132 residue to Leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. The recovered mutant virus showed much smaller-sized plaques. On the contrary, a G-C and a G-A point mutations at nucleotide positions 4330 and 9230, respectively, causing Glu-Gln and Gly-Glu mutations in or near the catalytic centers of the papain-like (Nsp3) and 3C-like (Nsp5) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses.


Subject(s)
Infectious bronchitis virus/growth & development , Infectious bronchitis virus/genetics , Point Mutation , RNA Helicases/genetics , Viral Proteins/genetics , Virus Replication/genetics , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Chlorocebus aethiops , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Hydrolases/genetics , Peptide Hydrolases/physiology , RNA Helicases/physiology , RNA, Viral/metabolism , Sequence Homology, Amino Acid , Transcription, Genetic , Vero Cells , Viral Plaque Assay , Viral Proteins/physiology
9.
Biochem Biophys Res Commun ; 336(2): 417-23, 2005 Oct 21.
Article in English | MEDLINE | ID: mdl-16137658

ABSTRACT

An interesting question posed by the current evidence that severe acute respiratory syndrome coronavirus may be originated from an animal coronavirus is how such an animal coronavirus breaks the host species barrier and becomes zoonotic. In this report, we study the chronological order of genotypic changes in the spike protein of avian coronavirus infectious bronchitis virus (IBV) during its adaptation to a primate cell line. Adaptation of the Beaudette strain of IBV from chicken embryo to Vero cells showed the accumulation of 49 amino acid mutations. Among them, 26 (53.06%) substitutions were located in the S protein. Sequencing analysis and comparison of the S gene demonstrated that the majority of the mutations were accumulated and fixed at passage 7 on Vero cells and minor variants were isolated in several passages. Evidence present suggests that the dominant Vero cell-adapted IBV strain may be derived from the chicken embryo passages by selection of and potential recombination between the minor variants. This may explain why adaptation is a rapid process and the dominant strain, once adapted to a new host cell, becomes relatively stable.


Subject(s)
Adaptation, Physiological/genetics , Chickens/virology , Chlorocebus aethiops/virology , Infectious bronchitis virus/genetics , Membrane Glycoproteins/genetics , Recombination, Genetic/genetics , Selection, Genetic , Viral Envelope Proteins/genetics , Amino Acid Sequence , Animals , Gene Transfer, Horizontal/genetics , Genetic Variation/genetics , Membrane Glycoproteins/chemistry , Molecular Sequence Data , Primates/virology , Spike Glycoprotein, Coronavirus , Vero Cells , Viral Envelope Proteins/chemistry
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