key: cord-328046-5us4se5o
authors: Xu, H. Y.; Lim, K. P.; Shen, S.; Liu, D. X.
title: Further Identification and Characterization of Novel Intermediate and Mature Cleavage Products Released from the ORF 1b Region of the Avian Coronavirus Infectious Bronchitis Virus 1a/1b Polyprotein
date: 2001-09-30
journal: Virology
DOI: 10.1006/viro.2001.1098
sha: 
doc_id: 328046
cord_uid: 5us4se5o

Abstract The coronavirus 3C-like proteinase is one of the viral proteinases responsible for processing of the 1a and 1a/1b polyproteins to multiple mature products. In cells infected with avian coronavirus infectious bronchitis virus (IBV), three proteins of 100, 39, and 35 kDa, respectively, were previously identified as mature cleavage products released from the 1b region of the 1a/1b polyprotein by the 3C-like proteinase. In this report, we show the identification of two more cleavage products of 68 and 58 kDa released from the same region of the polyprotein. In addition, two stable intermediate cleavage products with molecular masses of 160 and 132 kDa, respectively, were identified in IBV-infected cells. The 160-kDa protein was shown to be an intermediate cleavage product covering the 100- and 68-kDa proteins, and the 132-kDa protein to be an intermediate cleavage product covering the 58-, 39-, and 35-kDa proteins. Immunofluorescent staining of IBV-infected cells and cells expressing individual cleavage products showed that the 100-, 68-, and 58-kDa proteins were associated with the membranes of the endoplasmic reticulum, and the 39- and 35-kDa proteins displayed diffuse distribution patterns.

Coronavirus gene expression involves the expression of six to seven mRNA species. In cells infected with the prototype species of the Coronaviridae, avian coronavirus infectious bronchitis virus (IBV), six mRNA species are detected. These include the genome-length mRNA (mRNA1) of 27.6 kilobases (kb) and five subgenomic mRNA species (mRNAs 2-6) with sizes ranging from 2 to 7 kb. The 5Ј-terminal unique region of mRNA 1 contains two large ORFs, 1a and 1b, encoding the 441-kDa 1a and 741-kDa 1a/1b polyproteins (Boursnell et al., 1987) (Fig.  1) . The two polyproteins are cleaved by two viral proteinases to produce functional products associated with viral replication (Ziebuhr et al., 2000) (Fig. 1) .

The first proteinase was identified to be included in a 195-kDa cleavage product, which contains a papain-like proteinase domain encoded by ORF 1a from nucleotides 4242 to 5553 (Lim et al., 2000) . This proteinase was shown to be involved in cleavage of the N-terminal region of the 1a and 1a/1b polyproteins at two G-G dipeptide bonds (G 673 -G 674 and G 2265 -G 2266 ) to release two mature products of 87 and 195 kDa Lim and Liu, 1998; Lim et al., 2000) . The second proteinase was identified as a 33-kDa protein in IBV-infected cells Lim et al., 2000; Ng and Liu, 2000) . This serine proteinase belongs to the picornavirus 3C proteinase group (3C-like proteinase) and was shown to mediate cleavage of the 1a and 1a/1b polyproteins at more than 10 Q-S(G, N) dipeptide bonds to release mature cleavage products (Liu et al., 1994 (Liu et al., , 1997 Liu, 1998, 2000) .

In addition to the viral proteinases, understanding of the functions of other cleavage products from the 1a and 1a/1b polyproteins is emerging. For example, the 71-kDa protein released from the human coronavirus 1a/1b polyprotein was shown to possess ATPase and RNA duplex-unwinding activities, confirming the previous predication that the protein may be the viral helicase (Heusipp et al., 1997a; Seybert et al., 2000a,b) . Immunofluorescence and biochemical studies demonstrated that several cleavage products are membrane-associated and colocalize with the viral RNA replication-transcription machinery (Bost et al., 2000; Denison et al., 1999; Ng and Liu, 2000; Schiller et al., 1998; Shi et al., 1999; Sims et al., 2000; Ziebuhr and Siddell, 1999; , suggesting the involvement of these products in viral RNA replication. More recent studies showed that the first cleavage product of mouse hepatitis virus-A59 (MHV-A59) 1a and 1a/1b polyproteins, p28, might play a direct role in viral RNA synthesis together with polymerase and helicase (Bost et al., 2000; Sims et al., 2000) . Other cleavage products, such as the MHV 22-kDa protein, may segregate into different but tightly associated membrane populations which may serve independent functions during viral replication (Bost et al., 2000; Sims et al., 2000) .

In previous reports, we demonstrated that four previously predicted Q-S(G) dipeptide bonds located in the 1b region of the 1a/1b polyprotein are genuine cleavage sites of the 3C-like proteinase (Liu et al., 1994 . Taken together with the N-terminal cleavage site identified for releasing the 100-kDa protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kDa, respectively (Fig. 1 ). Among them, the 100-, 39-, and 35-kDa proteins were specifically identified in IBV-infected cells. In this communication, we report the identification of the two previously unidentified products, the 68-and 58-kDa proteins, in IBVinfected cells with newly raised region-specific antisera. Meanwhile, two relatively stable intermediate cleavage products with molecular masses of approximately 160 and 132 kDa were identified. Analysis of the expression and processing kinetics showed that both the 160-and 132-kDa intermediate cleavage products coexist with their final cleavage products during the viral replication cycle. Furthermore, immunofluorescence analysis showed that the 100-, 68-, and 58-kDa proteins were associated with the membranes of the endoplasmic reticulum (ER), while the 39-and 35-kDa proteins displayed diffuse distribution patterns.

In our previous reports, four Q-S(G) dipeptide bonds, Q 891(1b) -S 892(1b) , Q 1492(1b) -G 1493(1b) , Q 2012(1b) -S 2013(1b) , and Q 2350(1b) -S 2351(1b) , located in the ORF 1b region and encoded by nucleotides 15129-15134, 16929-16934, 18492-18497, and 19506-19511, respectively , were demonstrated to be the cleavage sites of the 3C-like proteinase (Liu et al., 1994 (Fig. 1 ). Taken together with the Q 3928 -S 2929 dipeptide bond (encoded by nucleotides 12310-12315) identified as the N-terminal cleavage site of the 100-kDa protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kDa. Among them, the 100-, 39-, and 35-kDa proteins were specifically identified in IBV-infected cells (Liu et al., 1994 . To further identify and characterize the cleavage products, four new region-specific antisera, anti-100, anti-68, anti-58, and anti-35, were raised in rabbits against bacterially expressed viral proteins. The IBV proteins used to raise these antisera were encoded by nucleotides 12447-15131, 15536-16787, 16932-18494, and 19509-20414, respectively (Fig. 1) . Anti-100 was raised to replace V-58, which was raised against the IBV sequence encoded by nucleotides 14492-15520, and was used to identify the 100-kDa protein in IBV- infected cells (Liu et al., 1994) . The specificities of these antisera were tested by immunoprecipitation assay, showing that they could specifically precipitate their target proteins synthesized in both the in vitro system and intact cells (data not shown).

To identify the cleavage products in IBV-infected cells, confluent monolayers of Vero cells were infected with IBV at a m.o.i. of approximately 3 PFU per cell. To reduce the background, 5 g/ml of actinomycin D was added to the culture medium at 2 h postinfection and cells were labeled with [ 35 S]methionine and cysteine at 6 h postinfection. Cell lysates were prepared from cells harvested at 8 h postinfection and subjected to immunoprecipitation with anti-100, anti-68, anti-58, V17, and anti-35. V17 was raised against the IBV polypeptides encoded by nucleotides 19154-20414 and used to detect the 39-and 35-kDa proteins in IBV-infected cells in a previous report . Immunoprecipitation with anti-100 resulted in the detection of the 100-kDa protein and a protein with an apparent molecular mass of 160 kDa (Fig.  2a, lane 10) . The 160-kDa protein was also detected by anti-68 (Fig. 2a, lane 9) . No product corresponding to the 68-kDa putative helicase domain-containing protein was detected by anti-68 (Fig. 2a, lane 9) . The detection of the 160-kDa protein with the two N-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product containing the 100-kDa and the putative 68-kDa proteins. Immunoprecipitation of the same lysates with anti-58 resulted in the detection of two products with apparent molecular masses of 58 and 132 kDa (Fig. 2a, lane 8) . The 132-kDa protein was also immunoprecipitated by V17 and anti-35 (Fig. 2a, lanes 6 and 7) . In addition, V17 also precipitated specifically the 39-and 35-kDa proteins (Fig. 2a, lane 7) . Once again, very weak immunoprecipitation of the 35-kDa protein was observed (Fig. 2a, lane  7) . The 35-kDa protein was also weakly immunoprecipitated by anti-35 (Fig. 2a, lane 6 ). The detection of the 132-kDa protein by the three C-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product derived from the C-terminal region of the polyprotein. Interestingly, the three C-terminally specific antisera also weakly immunoprecipitated the 160-kDa protein (Fig. 2a , lanes 6-8). The reason for this result is currently unclear, but it may reflect the interaction among the cleavage products.

As immunoprecipitation with anti-68 failed to detect the putative 68-kDa protein in IBV-infected Vero cells, we then tried to detect the protein by Western blot with the same antiserum. For this purpose, cell lysates were prepared from IBV-infected Vero cells harvested at 8, 24, and 32 h postinfection, respectively, and subjected to Western blotting analysis. As shown in Fig. 2b , a polypeptide with an apparent molecular mass of 68 kDa was specifically detected in lysates prepared from cells harvested at 24 h postinfection (lane 2), and the expression of the protein was dramatically increased at 32 h postinfection (lane 3). Expression and processing kinetics of the ORF 1b region of the 1a/1b polyprotein in IBV-infected Vero cells

As the 160-and 132-kDa proteins may represent stable intermediate cleavage products, time-course experiments were carried out to investigate the expression and processing kinetics of the two products in IBV-infected cells. For this purpose, confluent monolayers of Vero cells were infected with IBV at a m.o.i. of approximately 3 PFU per cell and were labeled for 2 h with [ 35 S]methionine at 6 h postinfection. The cells were then washed with complete medium and chased with a 10-fold excess of cold methionine until they were harvested at appropriate times. Immunoprecipitation of cell lysates with anti-100 showed the detection of both the 100-and 160-kDa proteins throughout the time course (Fig. 3a) . The 100-kDa protein appeared at the beginning of the time course, peaked after chase for 1.5 h, and remained stable at the end of the time course (Fig. 3a, lane 2) . The 160-kDa protein peaked at the beginning of the time course and remained detectable after chase for 7.5 h (Fig. 3a, lanes 1-6) . Slight and gradual reduction of the 160-kDa protein over time was observed (Fig. 3a) . These results demonstrate that the 160-kDa protein is a relatively stable intermediate cleavage product coexisting with the 100-kDa mature cleavage product during the IBV infection cycle.

Similarly, immunoprecipitation of cell lysates with anti- 58 showed the detection of both the 132-and 58-kDa proteins (Fig. 3b) . The 132-kDa protein appeared at the beginning of the time course, peaked after chased for 3 h, and remained detectable at the end of the time course (Fig. 3b, lanes 9-12) . The 58-kDa protein was first seen after chased for 3 h and gradually increased over time (Fig. 3b, lanes 9-12) . Interestingly, a product with an apparent molecular mass of 97 kDa, representing an intermediate cleavage product containing the 58-and 39-kDa proteins , was weakly detected and briefly observed during the time course (Fig. 3b , lanes 9 and 10), indicating that it is not a stable intermediate cleavage product. The coexistence of the 132-and 58-kDa proteins over time suggests that the 132-kDa protein is a stable intermediate cleavage product.

Further characterization and definition of the coding sequences of products processed from the Cterminal 200-kDa region of the 1a/1b polyprotein Two dicistronic constructs, p3C-CITE-IBV20 and p3C-CITE-IBV8, were made and expressed to characterize the expression and processing patterns of the C-terminal 200-kDa region of the 1a/1b polyprotein. In these two constructs, the region coding for the 3C-like proteinase was placed between the T7 promoter and the internal ribosome entry site (IRES) of encephomyolitis virus (EMCV), and the IBV sequences from nucleotides 15132-20506 and 15132-18495, respectively, were cloned downstream of IRES (Fig. 1) .

Expression of both constructs in Cos-7 cells resulted in the detection of the 33-kDa 3C-like proteinase, which comigrates on SDS-PAGE with the 33-kDa protein detected in IBV-infected cells (Fig. 4, lanes 2-4) . Immunoprecipitation of cell lysates prepared from p3C-CITE-IBV20-transfected cells with anti-58 led to the detection of three protein species with apparent molecular masses of 200, 132, and 58 kDa, respectively (Fig. 4, lane 7) . The 200-kDa protein represents the full-length product encoded by the IBV 1b sequence present in this plasmid, and the 132-and 58-kDa proteins comigrated with the 132-and 58-kDa proteins detected in IBV-infected cells (Fig. 4, lanes 6 and 7) . Immunoprecipitation of cell lysates prepared from p3C-CITE-IBV8-transfected cells with anti-58 led to the detection of the 58-kDa protein and a product with an apparent molecular mass of 125 kDa (Fig. 4, lane 8) . The 125-kDa protein may represent the full-length product encoded by the 1b sequence present in this construct. Immunoprecipitation of cell lysates prepared from p3C-CITE-IBV20-transfected cells with antiserum V17 led to the detection of the 200-, 132-, 39-, and 35-kDa proteins (Fig. 4, lane 11) . The 132-, 39-, and 35-kDa proteins comigrated with the three equivalent FIG. 4 . Comparative analysis of the 132-and 58-kDa proteins expressed and processed in cells transfected with p3C-CITE-IBV20, p3C-CITE-IBV8, and pIBV1b4 and in IBV-infected cells. Plasmid DNAs were expressed in Cos-7 cells using the vaccinia virus-T7 expression system (Fuerst et al., 1986) . Semiconfluent monolayers of Cos-7 cells were infected with a recombinant vaccinia virus (vTF7-3), transfected with plasmid DNA using DOTAP according to the instructions of the manufacturer (Roche), and labeled with [ 35 S]methionine and cysteine, and lysates were prepared. Cell lysates prepared from mock-infected (lanes 1, 5, and 9) and IBV-infected (lanes 2, 6, and 10) Vero cells and transfected cells (lanes 3, 4, 7, 8, 11-13) were immunoprecipitated with anti-3C (lanes 1-4) , anti-58 (lanes 5-8, 12, and 13), and V17 (lanes 9-11). The radiolabeled polypeptides were separated on an SDS-12.5% polyacrylamide gel and detected by fluorography. Numbers indicate molecular mass in kilodaltons.

products detected in IBV-infected cells (Fig. 4, lanes  9-11) .

As mentioned earlier, the apparent molecular mass and processing pattern of the 132-kDa protein suggested that it may be derived from the C-terminal region of the 1a/1b polyprotein covering the 58-, 39-, and 35-kDa proteins. To further confirm this possibility, plasmid pIBV1b4, which covers nucleotides 16932-20490 and therefore encodes the 132-kDa product , was expressed in Cos-7 cells. As expected, immunoprecipitation of cell lysates prepared from cells transfected with pIBV1b4 with anti-58 resulted in the detection of the full-length 132-kDa protein, which comigrated on SDS-PAGE with the 132-kDa intermediate cleavage detected from cells transfected with p3C-CITE-IBV20 (Fig. 4, lanes  12 and 13) .

To gain clues of the functions of the five cleavage products in the viral replication cycle, indirect immunofluorescence analysis of cells expressing individual cleavage products was carried out and representative confocal microscopy images are present in The proteins were labeled with the FITC-conjugated secondary antibodies. Panels B, E, H, K, and N refer to cells stained with R6, a dye for the ER. The green images represent FITC-derived green fluorescence, and red images represent rhodamine and Texas red-derived red fluorescence. Colocalization of viral proteins with the organelle markers is represented by the yellow region within each cell in the merged images (C, F, I, L, and O). Panels a-o show Cos-7 cells transfected with the empty vector pKT0 and stained with antisera or R6 as indicated. The fluorescence was viewed using a confocal scanning Zeiss microscope. nofluorescent pattern of anti-PDI (data not shown). These results suggest that the three proteins may be associated with the ER membranes. In cells expressing the 39and 35-kDa proteins, a diffuse staining pattern was observed for each protein (Figs. 5J and 5M), which does not coalign with the staining pattern of R6 (Figs. 5J-5O). This diffuse distribution pattern of the 39-and 35-kDa proteins was unexpected, as the majority of cleavage products were shown to be membrane-associated. The same antisera were used to stain cells transfected with the empty vector pKT0 (Liu et al., 1994) , showing weak background staining (Figs. 5a-5o) .

The subcellular localization patterns of the five proteins were then analyzed in IBV-infected cells (Fig. 6) . Immunofluorescent staining of IBV-infected Vero cells with anti-100, anti-68, and anti-58, respectively, showed similar ER localization profiles (Figs. 6A-6I), and a diffuse staining pattern was observed in cells stained with antiserum V17, which reacts with both the 39-and 35-kDa proteins (Figs. 6J-6L) . Similarly, a diffuse distribution pattern was observed in cells stained with anti-35 (Figs. 6M-6O). The same antisera were used to stain mockinfected cells, showing weak background staining (Figs. 6a-6o).

In our previous reports, we showed the identification of three mature cleavage products of 100, 39, and 35 kDa, processed from the 1b region of the 1a/1b polyprotein (Liu et al., 1994 . However, we were unable to detect the two other cleavage products of 68 and 58 kDa. In this study, we report the identification of the 68-and 58-kDa proteins in IBV-infected cells. In addition, two stable intermediate cleavage products of 160 and 132 kDa, respectively, were identified, which coexist with the mature cleavage products in virus-infected cells. Immunofluorescence analysis showed that the polymerase domain-containing 100-kDa protein and the helicase domain-containing 68-kDa protein as well as the 58-kDa protein may be associated with the ER and IC membranes, the viral replication and assembly sites. However, the 39-and 35-kDa proteins display diffuse distribution patterns in both IBV-infected cells and cells expressing each of the proteins.

It is intriguing that immunoprecipitation and Western blot of IBV-infected cells harvested at 8 h postinfection failed to detect the 68-kDa protein. The protein was detected by Western blot in IBV-infected cells harvested at 24 and 32 h postinfection. As the equivalent human coronavirus 71-kDa protein was shown to be an RNA helicase (Heusipp et al., 1997a; Seybert et al., 2000a,b) , this protein is likely the IBV helicase. It is expected that such a functional product directly involved in viral RNA replication would be expressed at early stages of the viral replication cycle. In fact, the equivalent 71-kDa protein of human coronavirus was first seen in virus-infected cells at 5 h postinfection (Heusipp et al., 1997a) . The reason for the failure to detect the 68-kDa protein in IBV-infected cells at earlier time point is uncertain, but it might reflect the folding property, as discussed later, of the protein. The dramatic increase in the detection of the 68-kDa protein at 24 and 32 h postinfection may partially be due to the secondary infection of cells that remain uninfected during the primary infection. As significantly more cells (over 95% of cells) were infected at 24-32 h postinfection, it is understandable that more protein would be detected. A slight, but gradual increase of the accumulation of the human coronavirus 71-kDa protein over a time course of 15 h was also observed (Heusipp et al., 1997a) . Alternatively, the increase in the detection of the 68-kDa protein at 24-32 h postinfection may reflect the genuine accumulation pattern of the protein in virusinfected cells at late stages of the viral replication cycle. If this were the case, it may indicate that the protein might also be involved in processes other than viral RNA replication.

The identification of two stable intermediate cleavage products of 160 and 132 kDa coexisting with the mature cleavage products raised two interesting questions. First, the two products may have certain functions during the viral replication cycle. The 160-kDa product is particularly interesting in this aspect, considering the fact that it contains both the polymerase and helicase domains and the 68-kDa helicase protein is undetectable at early stages of infection. It is plausible that the 160-kDa protein might have both polymerase and helicase functions in the replication of viral RNA at early stages of the infection. In fact, some smaller positive-stranded RNA viruses encode single proteins containing both the polymerase and helicase domains (Buck, 1996) . The second interesting question is why only these two intermediate cleavage products were detectable in IBV-infected cells. As shown in Fig. 4 as well as in our previous report , other intermediate cleavage products were also observed when this region was expressed in intact cells. One obvious distinct feature of the cleavage site between these two products is that it is a Q-G dipeptide bond, while all the other cleavage sites in this region of the polyprotein are Q-S dipeptide bonds (Fig. 1) . However, no experimental data indicate that cleavage at the Q-G site is more efficient than at the Q-S sites.

The 68-kDa protein migrated on SDS-PAGE as a multiprotein species, heterogeneous smear, probably due to the formation of protein aggregates, and deletion analysis showed that a stretch of 30 amino acid residues in the C-terminal region was responsible for the aberrant migration property of the protein (data not shown). It suggests that the 68-kDa protein may misfold when expressed in intact cells in the absence of other viral components. In recent years, it was found that the proper folding of certain proteins requires the assistance of molecular chaperones (Ellis and van der Vies, 1991; Gething and Sambrook, 1992) . As the 68-kDa protein may be a component of the viral RNA replication complex, the protein is expected to interact with viral RNA and other viral proteins. Those viral RNA/proteins may act as a chaperone for the correct folding of the 68-kDa protein in IBV-infected cells. It would be of interest to define if the region of the 30 amino acid residues that were shown to be responsible for the aberrant migration of the 68-kDa protein on SDS-PAGE contain either RNA binding or protein interacting domains. However, no such domains were found in this or the neighboring regions by computer analysis using relevant programs.

The 58-kDa protein was recently shown to be able to induce programmed cell death when expressed alone in intact cells . This is the first IBV product that was demonstrated to be a proapoptotic protein. In our previous report, we were unable to identify the 58-kDa protein in IBV-infected cells due to the cross-reactivity of the antiserum used with a cellular protein . A newly raised antiserum was used in this study, leading to the successful identification of the protein in virus-infected cells. Understanding of the expression, processing, and subcellular distribution pattern of the 58-kDa protein would help us to further characterize the proapoptotic property of the protein and to study the functions of the protein in the pathogenesis of IBV-induced infection in chicken, the natural host of IBV.

Currently, no functions have been assigned to the 39and 35-kDa proteins. A counterpart of the IBV 39-kDa protein, the 41-kDa protein of human coronavirus, was shown to exhibit a punctate, perinuclear distribution pat-tern in virus-infected cells (Heusipp et al., 1997b) . Interestingly, the 39-and 35-kDa proteins display a diffuse distribution pattern in both IBV-infected cells and in cells overexpressing the proteins. As the majority of the cleavage products from the 1a and 1a/1b polyproteins were shown to be associated with cellular membranes at or near the viral replication and assembly sites, the diffuse distribution pattern may exclude the direct involvement of the two proteins in the formation of viral replication complexes.

The egg-adapted Beaudette strain of IBV (ATCC VR-22) was obtained from the American Type Culture Collection (ATCC) and was adapted to Vero cells as described by Alonso-Caplen et al. (1984) . Briefly, the virus was passaged three times in 11-day-old chicken embryos and then adapted to Vero cells (ATCC CCL-81) by a series of passages at 24-48 h intervals. The cytopathic effects, including syncytium formation and rounding up of cells, were initially observed after three passages in Vero cells. Virus stocks were prepared after the 36th passage by infecting monolayers of Vero cells at a m.o.i. of approximately 0.1 PFU/cell. The virus was harvested at 24 h postinfection and the titer of the virus preparation was determined by plaque assay on Vero cells.

Vero cells were grown at 37°C in 5% CO 2 and maintained in Glasgow's modified minimal essential medium (GMEM) supplemented with 10% newborn calf serum.

Confluent monolayers of Vero cells were infected with IBV at a m.o.i. of approximately 3 PFU/cell. Prior to being labeled, the cells were incubated in methionine-free medium for 30 min. After 4 h of labeling with 25 Ci of [ 35 S]methionine, the cells were scraped off the dishes in phosphate-buffered saline (PBS), recovered by centrifugation, and stored at Ϫ80°C.

Open reading frames placed under control of the T7 promoter were expressed transiently in eukaryotic cells as described previously (Liu et al., 1994) . Briefly, semiconfluent monolayers of Vero cells were infected with 10 PFU/cell of a recombinant vaccinia virus (vTF7-3) which expresses the bacteriophage T7 RNA polymerase and then transfected with appropriate plasmid DNA using the DOTAP transfection reagent according to the instructions of the manufacturer (Roche). After incubation of the cells at 37°C for 4 h, 25 Ci/ml of [ 35 S]methionine was added directly to the medium. The radiolabeled cells were harvested at 18 h posttransfection.

Appropriate primers and template DNAs were used in amplification reactions with Pfu DNA polymerase (Stratagene) under standard buffer conditions with 2 mM MgCl 2 . The PCR conditions were 30 cycles of 95°C for 45 s, X°C for 45 s, and 72°C for X min. The annealing temperature (X°C) and the extension time (X min) were adjusted according to the melting temperature of the primers used and the length of the PCR fragments synthesized.

Plasmid DNA-transfected Vero cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% NP-40) and precleared by centrifugation at 12,000 rpm for 5 min at 4°C in a microfuge. Immunoprecipitation was carried out as described previously (Liu et al., 1994) .

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of virus polypeptides was carried out using 12.5% polyacrylamide gels (Laemmli, 1970) . Labeled polypeptides were detected by autoradiography or fluorography of dried gels.

Cells were grown on four-well chamber slides (Iwaki) and infected with IBV or transfected with appropriate plasmid DNAs. After washing with PBS, the cells were fixed with 4% paraformaldehyde (in PBS) for 15 min at room temperature and permeabilized with 0.2% Triton X-100 (in PBS), followed by incubation with specific antiserum at room temperature for 2 h. Antibodies were diluted in fluorescence dilution buffer (PBS with 5% normal goat serum). The cells were then washed with PBS and incubated with anti-rabbit IgG conjugated to fluorescein isothiocynate (FITC) (Sigma) in the fluorescence dilution buffer at 4°C for 1 h before mounting.

Confocal microscopy was performed on a Zeiss Axioplan microscope connected to a Bio-Rad MRC 1024 laser scanner equipped with an argon laser with appropriate filters. Fluorescent images were superimposed to allow fine comparison and colocalization of green (FITC) and red (TRITC) signals in a single pixel produces yellow, while separated signals are green or red.

Plasmid pIBV1b4, which contains nucleotides 16932-20490, was previously described .

Plasmid pIBV1b3 contains nucleotides 15132-16931 and codes for the 68-kDa protein, pIBV1b6 contains nucleotides 16930-18495 and codes for the 58-kDa protein, pIBV1b9 contains nucleotides 18496-19508 and codes for the 39-kDa protein, and pIBV1b10 contains nucleotides 19509-20506 and codes for the 35-kDa protein. These constructs were made by cloning an NcoI-and BamHI-digested PCR fragment into NcoI-and BamHIdigested pKT0 (Liu et al., 1994) . The sequences of the two primers used to construct pIBV1b3 were 5Ј-CGACT-TCCATGGCTTGTGGCGTTЈ-3 and 5Ј-CCAAAGGATCCTA-TTGCAGACTTG-3Ј. The sequences of the two primers used to construct pIBV1b6 were 5Ј-ACAAGTCCCATGGG-TACAGGTT-3Ј and 5Ј-TATTGGATCCTACGGAGAGCTG-3Ј. The sequences of the two primers used to construct pIBV1b9 were 5Ј-GTTTTTCAGCTCCCATGGCTATCGAC-AAT-3Ј and 5Ј-AACCACACGTCGGATCCTATTGAAGCTG-TG-3Ј. The sequence of the two primers used to construct pIBV1b10 was 5Ј-CCACAGCTTCCCATGGCATG-GACGTG-3Ј.

Plasmid pIBVpol, which contains nucleotides 12451-15131 and codes for the 100-kDa protein with a 37-aminoacid truncation at the N-terminus, was constructed by cloning a BamHI-and XhoI-digested PCR fragment into BamHI-and XhoI-digested pET22b(ϩ) (Novagen). The sequences of the primers used were 5Ј-GTAATAAG-GATCCAGCTGGTATG-3Ј and 5Ј-AAGGCCTCGAGTTGTA-AAGTCGTAGGAGC-3Ј.

The two dicistronic constructs, p3C-CITE-IBV20 and p3C-CITE-IBV8, were constructed as follows. The IRES sequence was obtained by digestion of pCITE-1 (Novagen) with EcoRI, end-repair with Klenow, and redigestion with NcoI. This 592-bp fragment was then cloned into PvuII-and NcoI-digested pKT0, giving rise to pKT-CITE. The IBV sequence that codes for the 3C-like proteinase was obtained by digestion of pIBV3C (Liu et al., 1997) with BglII and BamHI and was cloned into BglII-digested pKT-CITE, giving rise to p3C-CITE. Digestion of p3C-CITE with NcoI produced a 1510-bp fragment containing both the IBV 3C-like proteinase and the IRES sequences. This fragment was then cloned into NcoI-digested pIBV1b8 and pIBV20, respectively, creating the two dicistronic constructs. Plasmids pIBV1b8 and pIBV20, which cover the IBV sequences from nucleotides 15132 to 18495 and 15132 to 20506, respectively, were constructed by cloning NcoI-and BamHI-digested PCR fragments covering the relevant regions into NcoI-and BamHI-digested pKT0.

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Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: Identification of a 10 kDa polypeptide and determination of its cleavage sites

Proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3C-like proteinase and identification of two novel cleavage products

Identification of a 24 kDa polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3C-like proteinase and determination of its cleavage sites

Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site

Processing of the coronavirus MHV-JHM polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of ORF1a

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplexunwinding activities with 5Ј-3Ј polarity

Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases

Colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral RNA in infected cells

Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes

Characterization of endoplasmic reticulum by colocalization of B-p and dicarbocyanine dyes

Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication

Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis

Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: Identification of proteolytic products and cleavage sites common to pp1a and pp1ab

Virus encoded proteinases and proteolytic processing in the Nidovirales

ACKNOWLEDGMENT This work was supported by the National Science and Technology Board of Singapore.