Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products.
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The coronavirus Avian infectious bronchitis virus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly(673)-Gly(674) dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly(2265)-Gly(2266) dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn(2313) residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q(2583)-G(2584)) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product. (+info)
Leader switching occurs during the rescue of defective RNAs by heterologous strains of the coronavirus infectious bronchitis virus.
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A defective RNA (D-RNA), CD-61, derived from the Beaudette strain of the avian coronavirus infectious bronchitis virus (IBV), was rescued (replicated and packaged) using four heterologous strains of IBV as helper virus. Sequence analysis of the genomic RNA from the four heterologous IBV strains (M41, H120, HV10 and D207) identified nucleotide differences of up to 17% within the leader sequence and up to 4.3% within the whole of the adjacent 5' untranslated region (UTR). Analysis of the 5' ends of the rescued D-RNAs showed that the Beaudette leader sequence, present on the initial CD-61, had been replaced with the corresponding leader sequence from the helper IBV strain but the adjacent 5' UTR sequence of the rescued D-RNAs corresponded to the original CD-61 Beaudette sequence. These results demonstrated that the phenomenon of leader switching previously identified for the coronaviruses murine hepatitis virus and bovine coronavirus (BCoV) also occurred during the replication of IBV D-RNAs. Three predicted stem-loop structures were identified within the 5' UTR of IBV. Stem-loop I showed a high degree of covariance amongst the IBV strains providing phylogenetic evidence that this structure exists and is potentially involved in replication, supporting previous observations that a BCoV stem-loop homologue was essential for replication of BCoV defective interfering RNAs. (+info)
Epithelial cell kinetics in the inflammatory process of chicken trachea infected with infectious bronchitis virus.
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All stages of degeneration and regeneration in chicken tracheal epithelium were studied morphologically following an intratracheal inoculation of infectious bronchitis virus (IBV). Viral antigen was detected in the cytoplasm of tracheal epithelium from 1 to 7 days post-inoculation (d.p.i.) with a peak on 3 d.p.i. At 1 d.p.i., almost all epithelial cells were involved in the degeneration. At this time, labelling index of bromodeoxyuridine (BrdU) in the basal cells showed significantly high value compared with control. At 2 and 3 d.p.i., a great number of basal cells were recognized, but the BrdU labelling index tended to decrease. At 4 and 5 d.p.i., the BrdU labelling index of basal cells significantly decreased than 1 d.p.i., and a few number of regenerated immature ciliated epithelia appeared. At 6 to 11 d.p.i., the ciliated columnar epithelia increased rapidly in number, and returned to the normal appearance except for non-ciliated patch by 13 d.p.i. These results suggested that the tracheal epithelial cells infected with IBV degenerated within 24 hours and proliferating activity of basal cells functioned immediately, and 3 to 4 days later, these basal cells were differentiated to the ciliated epithelia. (+info)
Adoptive transfer of infectious bronchitis virus primed alphabeta T cells bearing CD8 antigen protects chicks from acute infection.
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Infectious bronchitis virus (IBV) infection and associated illness may be dramatically modified by passive transfer of immune T lymphocytes. Lymphocytes collected 10 days postinfection were transferred to naive chicks before challenge with virus. As determined by respiratory illness and viral load, transfer of syngeneic immune T lymphocytes protected chicks from challenge infection, whereas no protection was observed in the chicks receiving the MHC compatible lymphocytes from uninfected chicks. Protection following administration of T lymphocytes could be observed in chicks with three distinct MHC haplotypes: B(8)/B(8), B(12)/B(12), and B(19)/B(19). Nearly complete elimination of viral infection and illness was observed in chicks receiving cells enriched in alphabeta lymphocytes. In contrast, removal of gammadelta T lymphocytes had only a small effect on their potential to protect chicks. The adoptive transfer of enriched CD8(+) or CD4(+) T lymphocytes indicated that protection was also a function primarily of CD8-bearing cells. These results indicated that alphabeta T lymphocytes bearing CD8(+) antigens are critical in protecting chicks from IBV infection. (+info)
Infectious bronchitis virus E protein is targeted to the Golgi complex and directs release of virus-like particles.
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The coronavirus E protein is a poorly characterized small envelope protein present in low levels in virions. We are interested in the role of E in the intracellular targeting of infectious bronchitis virus (IBV) membrane proteins. We generated a cDNA clone of IBV E and antibodies to the E protein to study its cell biological properties in the absence of virus infection. We show that IBV E is an integral membrane protein when expressed in cells from cDNA. Epitope-specific antibodies revealed that the C terminus of IBV E is cytoplasmic and the N terminus is translocated. The short luminal N terminus of IBV E contains a consensus site for N-linked glycosylation, but the site is not used. When expressed using recombinant vaccinia virus, the IBV E protein is released from cells at low levels in sedimentable particles that have a density similar to that of coronavirus virions. The IBV M protein is incorporated into these particles when present. Indirect immunofluorescence microscopy showed that E is localized to the Golgi complex in cells transiently expressing IBV E. When coexpressed with IBV M, both from cDNA and in IBV infection, the two proteins are colocalized in Golgi membranes, near the coronavirus budding site. Thus, even though IBV E is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly. (+info)
Kinetics of lymphocytic subsets in chicken tracheal lesions infected with infectious bronchitis virus.
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The kinetics of T-cells (CD3 positive (+), CD4+ and CD8+ cells) and B-cells (IgG+, IgM+ and IgA+ cells) in chicken trachea were studied immunohistochemically and histopathologically following an intratracheal inoculation of infectious bronchitis virus (IBV). Viral antigen was detected in the cytoplasm of tracheal epithelium from 16 hr to 6 days post-inoculation (p.i.) with a peak on 4 days p.i. A few IgG+, IgM+ and IgA+ cells were detected in the submucosa from 8 hr p.i. Thereafter IgG+ and IgM+ cells were gradually increased in number, and dramatically increased from 3 days p.i., peaked on 4 days p.i., and gradually decreased after 5 days p.i. IgA+ cells were detected in a small number than IgG+ and IgM+ cells during the all experimental period. These B cells mainly existed in the lamina propria, and some cells were recognized in the interepithelial space. After 14 days p.i., small number of IgG+ and IgM+ cells were detected in the germinal center of lymph follicles in the lamina propria. From 24 to 60 hr p.i., a few number of CD3+, CD4+ and CD8+ cells were detected at the perivascular area in the lamina propria. After 3 or 4 days p.i., each positive T-cells increased rapidly in number, and reached on the peak at 5 days p.i. CD3+, CD4+ and CD8+ cells tend to distribute diffusely, perivascular area, and surrounding area of CD4+ cells, respectively. CD4+ cells were dramatically decreased from 7 days p.i., and CD3+ and CD8+ cells were decreased from 14 days p.i. No T-cells were detected in the lymph follicles in the lamina propria. (+info)
Expression of reporter genes from the defective RNA CD-61 of the coronavirus infectious bronchitis virus.
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The defective RNA (D-RNA) CD-61, derived from the Beaudette strain of the avian coronavirus infectious bronchitis virus (IBV), was used as an RNA vector for the expression of two reporter genes, luciferase and chloramphenicol acetyltransferase (CAT). D-RNAs expressing the CAT gene were demonstrated to be capable of producing CAT protein in a helper-dependent expression system to about 1.6 microgram per 10(6) cells. The reporter genes were expressed from two different sites within the CD-61 sequence and expression was not affected by interruption of the CD-61-specific ORF. Expression of the reporter genes was under the control of a transcription-associated sequence (TAS) derived from the Beaudette gene 5, normally used for the transcription of IBV subgenomic mRNA 5. The Beaudette gene 5 TAS is composed of two tandem repeats of the IBV canonical consensus sequence involved in the acquisition of a leader sequence during the discontinuous transcription of IBV subgenomic mRNAs. It is demonstrated that only one canonical sequence is required for expression of mRNA 5 or for the expression of an mRNA from a D-RNA and that either sequence can function as an acceptor site for acquisition of the leader sequence. (+info)
Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site.
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Coronavirus infectious bronchitis virus (IBV) encodes a trypsin-like proteinase (3C-like proteinase) by ORF 1a, which has been demonstrated to play a pivotal role in proteolytic processing of gene 1-encoded polyproteins. In our previous studies, the proteinase was identified as a 33-kDa protein in IBV-infected cells, and its catalytic center was shown to consist of H(2820) and C(2922) residues. It is released from the 1a and 1a/1b polyproteins by autoprocessing at two Q-S dipeptide bonds (Q(2779)-S(2780) and Q(3086)-S(3087)). In this report, further characterization of the two cleavage sites demonstrates that the N-terminal Q(2779)-S(2780) site is tolerant to mutations at the P1 position. Deletion of the C-terminal region of the proteinase shows that a significant amount of the enzymatic activity is maintained upon deletion of up to 67 amino acids, suggesting that the extreme C-terminal region may be dispensable for the proteolytic activity of the proteinase. Analysis of the autoprocessing kinetics in vitro reveals that proteolysis at the Q(2779)-S(2780) site is the first cleavage event mediated by this proteinase. This is followed by cleavage at the Q(3086)-S(3087) site. The occurrence of both cleavage events in intact cells is potentially rapid and efficient, as no intermediate cleavage products covering the proteinase were detected in either IBV-infected or transfected cells. Immunofluorescence microscopy and subcellular fractionation studies further show differential subcellular localization of the proteinase in IBV-infected cells and in cells expressing the 3C-like proteinase alone, indicating that additional roles in viral replication might be played by this protein. Finally, a Q-A (Q(3379)-A(3380)) dipeptide bond encoded by nucleotides 10,663 to 10,668 was demonstrated to be a cleavage site of the proteinase. (+info)