Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus.

Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.

Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit.

The influenza virus RNA polymerase consists of a heterotrimeric complex of the PB1, PB2 and PA proteins, with the PB2 subunit responsible for recognizing 5' cap structures on the host cell RNAs used as primers for virus mRNA synthesis. To investigate further the role PB2 plays in mRNA synthesis, a set of polyclonal antisera raised against defined regions of the protein were tested for their ability to inhibit the virion transcriptase. All five sera were of sufficient titre to immunoprecipitate PB2 and four were capable of recognizing polymerase complexes containing PB1 and PA. However, only the serum raised against the carboxy terminus of PB2 (F5) substantially inhibited polymerase activity. This serum drastically reduced synthesis primed by globin mRNA, but only partially inhibited transcription primed by the dinucleotide ApG, or ApG and cap analogue. The preferential inhibition of globin-primed synthesis did not result from interference with cap recognition, as serum F5 did not reduce labelling of PB2 in a photoaffinity cap-binding assay. However, IgG and Fab fragments from F5 were found to inhibit virion endonuclease activity. This suggests that the C terminus of PB2 plays a crucial role in transcription initiation and implicates PB2 in endonuclease activity.

Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus.

A region of the infectious bronchitis virus (IBV) genome between nucleotide positions 8693 and 10927 which encodes the predicted 3C-like proteinase (3CLP) domain and several potential cleavage sites has been clones into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detectable processing activity of the nascent polypeptide unless the translation was carried out in the presence of microsomal membrane preparations. The processed products so obtained closely resembled in size those expected from cleavage at predicted glutamine-serine (Q/S) dipeptides and included a protein with a size of 35 kDa (p35) that corresponds to the predicted size of 3CLP. Efficient processing was dependent on the presence of membranes during translation; processing was found to occur when microsomes were added posttranslationally, but only after extended periods of incubation. C-terminal deletion analysis of the encoded polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hydrophobic region MP2. Dysfunctional mutagenesis of the putative active-site cysteine residue of 3CLP to either serine or alanine resulted in polypeptides that were impaired for processing, while mutagenesis at the predicted Q/S release sites implicated them in the release of the p35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3CLP is sensitive to inhibition by both serine and cysteine protease class-specific inhibitors. These data reveal the identity of the IBV 3C-like proteinase, which exhibits characteristics in common with the 3C proteinases of picornaviruses.

A region of the coronavirus infectious bronchitis virus 1a polyprotein encoding the 3C-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate.

In order to investigate the mechanisms involved in the processing of infectious bronchitis virus polyproteins, several candidate regions of the genome have been cloned and expressed in vitro. During these studies it was observed that the translation product encoded by one of these clones (pKT205) was poorly expressed. Biochemical and genetic analyses revealed that the basis for the poor expression was a post-translational event involving ubiquitination of the protein and degradation by an ATP-dependent system operating in the reticulocyte lysate used for the in vitro expression. Two independently acting regions which conferred instability were identified, one of which mapped to the predicted 3C protease domain, contained within the 5' end of the clone, while the other, more C-terminal region, was effective in conferring instability upon a heterologous protein to which it had been transferred. These regions may influence the stability of the authentic viral protein(s) in vivo and hence allow for the control of their expression and/or function at the level of proteolysis by cellular protease(s).

Identification, expression, and processing of an 87-kDa polypeptide encoded by ORF 1a of the coronavirus infectious bronchitis virus.

Nucleotide sequence analysis has shown previously that the genomic-length mRNA (mRNA1) of the coronavirus infectious bronchitis virus (IBV) contains two large open reading frames (ORFs), 1a and 1b, with the potential to encode polyproteins of approximately 441 and 300 kDa, respectively. We have characterized the specificity of a set of region-specific antisera raised against the 5'-portion of ORF 1a by immunoprecipitation of in vitro-synthesized, C-terminally truncated 1a polypeptides and used these antisera to detect virus-specific proteins in IBV-infected Vero cells. Two antisera, which had specificity for IBV sequences from nucleotides 710 to 2079 and 1355 to 2433, respectively, immunoprecipitated a polypeptide of approximately 87 kDa from IBV-infected Vero cells. In vitro translation of ORF 1a sequence terminating at nucleotide 5763 did not produce this protein unless the in vitro translation products were incubated with Vero cell S10 extracts prepared from either IBV-infected or mock-infected Vero cells. However, processing of the 87-kDa protein was also observed when the same region was expressed in Vero cells using the vaccinia virus/T7 expression system. This observation indicates that the 87-kDa polypeptide is encoded within the 5'-most 3000 nucleotides of mRNA 1 and that it might be cleaved from the 1a polyprotein by viral and cellular proteinases.

Generation of a defective RNA of avian coronavirus infectious bronchitis virus (IBV). Defective RNA of coronavirus IBV.

The Beaudette strain of IBV was passaged 16 times in chick kidney (CK) cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage six. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12423 to 18744 in the IBV genome and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products.

The genome-length mRNA (mRNA 1) of the coronavirus infectious bronchitis virus (IBV) contains two large open reading frames (ORFs), 1a and 1b, with the potential to encode polypeptides of 441 and 300 kDa, respectively. The downstream ORF, ORF 1b, is expressed by a ribosomal frameshifting mechanism. In an effort to detect viral polypeptides encoded by ORF 1b in virus-infected cells, immunoprecipitations were carried out with a panel of region-specific antisera. A polypeptide of approximately 100 kDa was precipitated from IBV-infected, but not mock-infected, Vero cells by one of these antisera (V58). Antiserum V58 was raised against a bacterially expressed fusion protein containing polypeptide sequences encoded by ORF 1b nucleotides 14492 to 15520; it recognizes specifically the corresponding in vitro-synthesized target protein. A polypeptide comigrating with the 100,000-molecular-weight protein (100K protein) identified in infected cells was also detected when the IBV sequence from nucleotides 8693 to 16980 was expressed in Vero cells by using a vaccinia virus-T7 expression system. Deletion analysis revealed that the sequence encoding the C terminus of the 100K polypeptide lies close to nucleotide 15120; it may therefore be generated by proteolysis at a potential QS cleavage site encoded by nucleotides 15129 to 15135. In contrast, expression of IBV sequences from nucleotides 10752 to 16980 generated two polypeptides of approximately 62 and 235 kDa, which represent the ORF 1a stop product and the 1a-1b fused product generated by a frameshifting mechanism, respectively, but no processed products were observed. Since the putative picornavirus 3C-like proteinase domain is located in ORF 1a between nucleotides 8937 and 9357, this observation suggests that deletion of the picornavirus 3C-like proteinase domain and surrounding regions abolishes processing of the 1b polyprotein. In addition, the in vitro translation and in vivo transfection studies also indicate that the ORF 1a region between nucleotides 8763 and 10720 contains elements that down-regulate the expression of ORF 1b.