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Immunolocalization of the pseudorabies virus immediate-early protein IE180 by immunoperoxidase staining
Journal of Virological Methods
ELSEVIER
Journal of Virological Methods 66 (1997) 219-226
Immunolocalization of the pseudorabies virus immediate-early protein IE180 by immunoperoxidase staining Chienjin
Huang a,*, Ying-Shiow
Lin a, Jing-Wen
Cheng a, Tim-Jye
Chang
b
a Graduate Institute of Veterinary Microbiology, National Chung Hsing University, 2.50 Kuo Kuang Road, Taichung 40227, Taiwan, ROC b Department of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC Accepted 3 April 1997
Abstract The immediate-early (IE) gene of pseudorabies virus (PRV) expresses immediately upon infection, a phosphorylated protein (immediate-early protein, IE180) that can transactivate viral other genes and plays an essential role in regulating viral gene expression. In order to detect and localize IE180 in infected cells early on, this gene was cloned for overexpression, and the expressed products were applied to generate specific antibodies against IE180 protein. Two recombinant expression plasmids pN and pNB were constructed by cloning the IE gene onto PET 30a( + ) expression vector via NcoI and BumHI sites. Plasmid pN contains the 1.8-kb NcoI-NcoI fragment of IE gene coding for the N-terminus of 616 amino acid residues, while pNB contains the 2%kb NcoI-BarnHI fragment coding for the rest of the IE180 protein. Both pN and pNB were transformed, respectively, into E. coli cells and produced large amounts of IE protein products during induction with 1 mM IPTG. The expressed IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively. These expression products were purified and then used as antigens to immunize mice for preparing specific antibodies against PRV IE180 protein. The specificities of the mice immune sera were confirmed by their abilities to react with IE180 protein present in the PRV infected cells in the Western immunoblotting assay. Furthermore, immunoperoxidase staining of PRV infected cells undertaken with these antisera revealed the subcellular distribution of the IE proteins in the infected cells and also demonstrated their transportation from the cytoplasm to the nucleus during infection. 0 1997 Elsevier Science B.V. Keywords:
Pseudorabies
* Corresponding
virus; Immediate-early
protein; Immunoperoxidase
author. Tel: + 886 4 2840751 ext. 48; fax + 886 4 2859222.
0166-0934/97/$17.00 0 1!>97Elsevier Science B.V. All rights reserved. PIZ SO1 66-0934(97)00069-4
staining
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of Virological Methods 66 (1997) 219-226
1. Introduction
2. Materials and methods
Pseudorabies virus (PRV) is a herpesvirus causing severe disease in swine and leading to latent infection. PRV is classified into the genus I/avicellovirus of subfamily Alphaherpesvirinae (Roizman, 1991). Similar to other herpesviruses, the PRV genes express in a sequential cascade manner and divided into three classes defined as immediate-early (IE), early, and late genes on the basis of the time of expression (Feldman et al., 1979; 1982). IE gene is expressed immediately upon infection and does not require prior viral protein synthesis for its expression. Only one major IE gene has been identified in PRV (Ihara et al., 1983), which is located at the inverted repeat region of the viral genome (Cheung, 1989). The coding region of this gene is 4380 nucleotides in length and codes for 1460 amino acid residues (Cheung et al., 1990). The product of the IE gene (IE protein) is a phosphorylated protein with a molecular weight of 180 kDa (IE180) and accumulates in nuclei of infected cells (Ben-Porat et al., 1975). IE180 can transactivate the transcription of early and late genes as well as other viral and cellular genes, and downregulates its own transcription (Ihara et al., 1983; Green et al., 1983; Ono et al., 1995). Thus, the PRV IE protein plays an indispensable role in regulating viral gene expression. However, IE proteins are synthesized during the normal course of infection in relatively small amounts early after infection (l-3 h). Recovery of IE protein from infected cells in large quantities and quality is extremely difficult. Recently, by using recombinant DNA techniques, the PRV IE gene has been cloned and expressed in different expression systems; and the expressed proteins have been found to be functional (Chlan et al., 1987; Yamada and Shimiza, 1994). DNA cloning approaches were adopted to construct the PET 30a( + ) recombinant expression vectors containing PRV IE gene in order to produce a large amount of IE protein in E. coli. The expression products were then used as antigens to immunize mice for preparing specific antisera against IE180 protein, so that PRV infection can be detected early and the IE protein structure and function can be examined further.
2.1. Virus and cell culture The PRV TNL strain was an isolate recovered in 1976 at a commercial pig farm in southern Taiwan, the Republic of China, and passaged continuously in the laboratory. MDBK (MadinDarby bovine kidney) cells were kindly provided by Dr Y.C. Zee (University of California at Davis) and grown in Dulbeceo’s modified Eagle medium supplemented with 10% fetal bovine serum. PRV was purified from the supernatant of infected cells by sucrose-gradient centrifugation and viral DNA was purified essentially as described previously (Todd and Mcferran, 1985). 2.2. Plasmids PRV DNA was subjected to BamHI digestion and the 8th restriction fragment, containing the complete open reading frame (ORF) of IE gene, were gel-purified and cloned on plasmid pUC19 to generate plasmid pUC/IE. The IE DNA was then amplified and recovered by BamHI digestion of pUC/IE, and treated with NcoI to generate two large IE fragments: a 1.8-kb NcoI-NcoI fragment and a 2.8-kb NcoI-BamHI fragment. Both IE fragments were gel-purified and inserted into the NcoI or NcoI/BamHI sites of the expression vector pET30a( + ) (Novagen) to construct expression plasmids pN and pNB respectively (Fig. 1). The IE180 ORF both in pN and pNB had been further confirmed to be cloned in frame by DNA sequencing. 2.3. Expression
of IE protein
Plasmids pN and pNB were trasnsformed into E. coli BL21(DE3, pLysS) competent cells according to the manifacturer’s manual. A single colony of each transformant was grown in Luria-Bertani (LB) medium containing 30 pg/ml kanamycin at 37°C until the OD,,, reached 1.0. Isopropyl P-Dthiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM. The culture was incubated for an additional 1 to 5 h(s) at 37°C. The cells were harvested by centrifugation and
C. Huang et al. /Journal
221
of Virological Methods 66 (1997) 219-226
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Fig. 1. Construction of PRV IE expression vectors. (A) Diagram of PRV genome and BamHI restriction enzyme map of TNL strain. The genome is organized into unique long (UL), internal repeat (IR), unique short (US), and terminal repeat (TR) sequences. Below the genome is an expanded diagram of the BarnHI- fragment. (B) The BamHI-8 fragment was cloned into pUC19 to construct plasmid pUC/IE. (C). The 1.8 kb NcoI-NcoI fragment and the 2.8 kb NcoI-BarnHI fragment of BamHI-8 fragment were cloned into PET 30a( + 11to construct expression plasmid pN and pNB respectively.
resuspended in 100 mM Tris-HCl (pH 8.0) containing 1 mM EDT.A. Cells were broken by sonication and insoluble material was collected by centrifugating at lfi 000 x g for 10 min at 4’C, and solubilized proteins were analyzed by SDSpolyacrylamide gel Ielectrophoresis (SDS/PAGE).
2.4. Preparation IE protein
of specljic antibody
against PRV
The protein bands represented the pN-expressed 60 kDa protein and pNB-expressed 100 kDa protein were excised and eluted from SDS-
222
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of Virological Methods 66 (1997) 219-226
polyacrylamide gel, and then concentrated by centricon-50 (Amicon). Five BALB/c mice were injected interperitoneally at biweekly interval three times with 0.2 mg of protein mixed with Freund’s adjuvant. The animals were bled and the sera were absorbed with E.coli lysate mobilized on nitrocellulose paper.
stained slightly with Meyer’s hematoxylin, which stains the nuclei blue to visualize the background tissue cells. After rinsing, the stained cells were allowed to dry completely and one drop of Permount was added to the coverslips and quickly replaced invertedly onto glass slides and examined by light microscopy.
2.5. Western immunoblotting 3. Results MDBK cells were infected with PRV at 10 m.o.i. in the presence of cycloheximide (100 pug/ml) and incubated for 5 h. The cultures were then washed to remove the drug and incubated for 1.5, 3 and 4 h. The cells were harvested and resuspended in an equal volume of 2 x SDS/PAGE sample buffer. Proteins were separated by 10% SDS/PAGE according to the method of Laemmli (1970) and transferred by electroblotting onto nitrocellulose sheets as described by Towbin et al. (1979). The sheet was then treated sequentially with blocking solution (PBS containing 1% BSA) with lOO-fold dilution of mouse anti-IE protein serum, and with anti-mouse IgG goat antibody conjugated to peroxidase (Zymed). Finally, the sheet was soaked in a substrate solution (ECL western blotting detection reagent, Amersham), and exposed to X-film according to the manufacturer’s instruction. 2.6. Immunoperoxidase staining MDBK cells grown on coverslips in a 24-well culture plate were infected with PRV at 1 m.o.i. in the presence of cycloheximide. The cells were fixed with 95% ethanol and 5% glacial acetic acid for 3 min. After washing with dH,O, the fixed cells were treated with PBS buffer containing 0.25% Triton X-100 and 5% DMSO to increase the cellular membrane permeability. The coverslips were then reacted with the mouse anti-IE protein antiserum, rinsed with PBS, and the enzyme was detected by the adding two drops of a mixture of one drop of a 3% hydrogen peroxide solution and 1 drop of a 2.2% solution of 3-amino-9-ethylcarbazole (AEC) in 2.5 ml of 0.1 M acetate buffer, pH 5.2. The AEC chromogen was converted by the enzyme within 5-8 min to red, water insoluble precipitate. The coverslips, after rinsing with dH,O, were counter
3.1. Expression of PR V IE protein in E. coli The expression plasmids pN and pNB described above were used to transform E. coli BL21(DE3, pLysS) cells respectively. The cultures were grown at 37 in liquid LB medium containing kanamycin, and the recombinant proteins were induced to overexpression by 1 mM IPTG. SDS/PAGE analysis of cellular proteins form IPTG-induced cells revealed that the expressed recombinant IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively. In addition, the intensities of the expressed proteins were found to be increasing with induction times (Fig. 2). The 60 kDa and 100 kDa proteins were then gel-purified and used as antigens to immunize mice for preparing specific antibodies against PRV IE protein. 3.2. Western immunoblotting analysis of PR V-expressed IE protein The mouse antiserum against expressed IE protein was used to detect the viral IE protein in PRV infected-MDBK cells. Mouse antiserum prepared against pN-expressed 60 kDa protein detected clearly the 180 kDa protein (IE180) present in PRV infected cells which was not present in uninfected cells (Fig. 3). While mouse antiserum against pNB-expressed 100 kDa protein recognized PRV IE180 protein but with a weak signal (Data not shown). 3.3. Localization of PRV IE protein by immunoperoxidase staining Subcellular distribution of PRV IE protein was visulized by immunoperoxidase staining using mouse antiserum against pN-expressed 60 kDa
C. Huang et al. /Journal
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of Virological Methods 66 (1997) 219-226
-
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Fig. 2. Coomassie blue-stained SDS/PAGE (10%) dem,onstrating expression of the PRV IE protein in (A) pN-transformed E. coli BL21(DE3, pLysS) cells and (B) pNB-transformed cells. Experiments were performed as described in Section 2. Lanes 1-6: crude extract of the transformed cells after 0 to 5 h-IPTG induction respectively. Lanes 7- 11: crude extract of the transformed cells after 1 to 5 h without IPTG induction respectively. Molecular weight markers are shown in lane M. The 90 kDa, 60 kDa, and 100 kDa bands are indicated by the arrow a, b, and c respectively.
protein and the immunoperoxidase staining patterns are showed in Fig. 4. IE180 molecules were synthesized initially in the cytoplasm of MDBK
cells and then translocated gradually into nuclei at 1.5 h post removal of cycloheximide and accumulated predominantly in the nuclei thereafter.
c.
224
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Huang et al. /Journal
M
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of Virological Methods 66 (1997) 219-226
2
3
4
5
6
22D180-
Fig. 3. Western immunoblotting analysis of PRV IE180 protein by antiserum raised against E. co&expressed IE protein. Antiserum derived from mice that had been immunized and boosted with the plasmid pN-expressed 60 kDa truncated IE protein was used to detect PRV IE180 in the viral infected cell at 1.5, 3, and 4 h after removal of cycloheximide (Lanes l-3). Lanes 4-6 were uninfected cell with the same cycloheximide treatment and served as mock cell control for lanes 1 to 3 respectively.
4. Discussion
This study demonstrated that the PRV IE protein can be expressed successfully in E.coli PET expression system. Also, the mouse antisera generated against these expressed IE proteins can be used to detect specifically the PRV IE protein in viral infected cells. Since IE180 protein is a high moleculer weight protein containing more then 50% of alanine, argine, proline, and glycine residues (Cheung, 1989), expression of IE 180 typically results in an aggregated product in a preliminary trial. To circumvent this problem, we modified the cloning approach to express IE180 protein into two smaller molecules (60 kDa and 100 kDa) and prepared antisera against them. The antigenicity of both truncated expressed proteins was apprently unaltered since the antibodies generated could recognize specifically PRV IE180 protein in infected cells by Western immunoblotting and immunoperoxidase staining. However, the biological functions due to modifications in protein structure associated with 60 kDa and 100 kDa IE proteins requires further study. Addition-
ally, another protein with a molecular weight of 90 kDa was shown to be induced specifically by IPTG in E. coli cells transfected with pN (Fig. 2A, arrow a). Since its molecular weight was much higher than the expected 60 kDa and was constitutely synthesized in relatively a small amount before induction, this 90 kDa protein should be an IPTG-induced bacterial protein rather than a pN-expressed product. In fact, the mouse immune serum raised against this particular protein did not recognize the PRV IEl80 protein by immunoperoxidase staining of viral infected cells (data not shown). Furthermore, since the PRV IE mRNA is synthesized immediately upon the virus entering the cell and produced continuously only for 20 min (Feldman et al., 1979), a low level of IE180 produced in the PRV infected cells interfered with the immunoassays. Cyclohemide is an inhibitor for protein synthesis which has been used to accumulate the IE mRNA in cells infected with herpes simplex virus (HSV) (Fenwick and Clark, 1983). Experiments were therefore also undertaked using cycloheximide to treat cells during PRV infection
C. Huang et al. /Journal
of Virological Methods 66 (1997) 219-226
225
Fig. 4. Immunoperoxidat#e staining of PRV-infected cells fixed on coverslips. Experiments were performed as described in Materials and Methods. Mouse immune serum against plasmid pN-expressed 60 kDa IE protein was used to detect the expression and distribution of PRV IEl80 in the infected cell that resulted in a red reaction product. These coverslips have also been counterstained with hematoxylin which stains the nuclei blue. (a) Uninfected cell at 4 h after removal of cycloheximide, (b-d) PRV-infected cells at 1.5, 3, 4 h after removal of cycloheximide respectively. Magnification 200 x
period for increasing IE mRNA accumulation. As soon as the drug was removed, the IE transcripts were translated to produce a large amount of IE180 porteins which could be detected easily by the mouse antisera. As shown in Fig. 3, IE180 proteins were synthesized after 1.5 h following removal of cycloheximide, and the amount of IE180 protein was increased dramatically during the next few hours. The specificities of mouse antisera against the expressed PRV IE proteins were demonstrated further by immunostaining the virus infected cells. Immunoperoxidase staining has been employed commonly for light microscopic immunolocalization of cellular proteins particularly for sparse antigens with several advantages over the immunofluoresence antibody technique such as improving sensitivity, counterstaining to show the cellular morphology, and the use of nonfluorescent materials and equipment (Wright and Scholey, 1993). By applying this technique, the PRV IE portein was localized in infected cells using the mouse antisera. According to the position and density of staining, PRV IE180 protein was first synthesized and distributed widely in the cytoplasma and then gr,adually translocated into nucli and accumulated in this compartment for transactivating the viral other genes. In conclusion, irnmunoperoxidase staining offers a novel and rapid method for detecting and localizing the PRV IE protein early after viral
infection. Furthermore, mouse immune sera prepared against bacterially expressed PRV IE proteins were found to have specificities to IE180 protein which may function as useful diagnostic reagents for detecting infection or for investigating further the function of IE180 protein as well as PRV pathogenesis.
Acknowledgements
This work was supported by grant NSC 8% 2321-B-005-061 from the National Science Council, Taiwan, Republic of China.
References Ben-Porat, T., Kervina, M., Kaplan, AS., 1975. Early functions of the genome of herpesrirus. Virology 65, 355-362. Cheung, A.K., 1989. DNA nucleotide sequence analysis of the immediate-early gene of pseudorabies virus. J. Virol. 63, 29082913. Cheung, A.K., Vlcek, C., Paces, V., Schwyser, M., 1990. Update and comparison of the immediate-early gene DNA sequences of two pseudorabies virus isolates. Virus Genes 4, 261-265. Chlan, C.A., Coulter, C., Feldman, L.T., 1987. Binding of the pseudorabies virus immediate-early protein to singlestranded DNA. J. Virol. 61, 185551860. Feldman, L.T., Rixon, F.J., Jean, J-H., Ben-Porat, T., Kaplan, AS., 1979. Transcription of the genome of pseudorabies virus (a herpesvirus) is strictly controlled. Virology 97, 316-327.
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Feldman, L.T., Demarchi, J.M., Ben-Porat, T., Kaplan, AS., 1982. Control of abundance of immediate-early mRNA in herpesvirus (pseudorabies)-infected cell. Virology 116,250263. Fenwick, M.L., Clark, J., 1983. The effect of cycloheximide on the accumulation and stability of functional cc-mRNA in cells infected with herpes simplex virus. J. Gen. Virol. 64, 1955-1963. Green, M.R., Treisman, R., Maniatis, T., 1983. Transcriptional activation of cloned human /3-globin genes by viral immediate-early gene products. Cell 35, 137-148. Ihara, S., Feldman, L., Watanabe, S., Ben-Porat, T., 1983. Characterization of the immediate-early functions of pseudorabies virus. Virology 131, 437-454. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Ono, E., Sakoda, Y., Taharaguchi, S., Watanabe, S., Tonomura, N., Kida, H., Shimisu, Y., 1995. Inhibition of pseudorabies virus replication by a chimeric trans-gene
product repressing transcription of the immediate-early gene. Virology 210, 128-140. Roizman, B., 1991. Herpesviridae. In: Francki, R.I.B., Fanguet, C.M., Knudson, D.L., Brown, F. (Eds.), Classification and Nomenclature of Viruses: Fifth Report of the International Committee on Toxonomy of Viruses. Springer, Wien New York, pp. 103-123. Todd, D., Mcferran, J.B., 1985. Restriction endonuclease analysis of Aujeszky’s diseases (pseudorabies) virus DNA: comparison of Northern Ireland isolates and isolates from other countries. Arch. Virol. 88, 167-176. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Wright, B.D., Scholey, J.M., 1993. Nonfluorescent immunolocalization of antigens in mitotic sea urchin blastomeres. Methods Cell Biol. 37, 223-239. Yamada, S., Shimiza, M., 1994. Isolation and characterization of mutants of pseudorabies virus with deletion in the immediate-early regulatory gene. Virology 199, 3666375.
ELSEVIER
Journal of Virological Methods 66 (1997) 219-226
Immunolocalization of the pseudorabies virus immediate-early protein IE180 by immunoperoxidase staining Chienjin
Huang a,*, Ying-Shiow
Lin a, Jing-Wen
Cheng a, Tim-Jye
Chang
b
a Graduate Institute of Veterinary Microbiology, National Chung Hsing University, 2.50 Kuo Kuang Road, Taichung 40227, Taiwan, ROC b Department of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC Accepted 3 April 1997
Abstract The immediate-early (IE) gene of pseudorabies virus (PRV) expresses immediately upon infection, a phosphorylated protein (immediate-early protein, IE180) that can transactivate viral other genes and plays an essential role in regulating viral gene expression. In order to detect and localize IE180 in infected cells early on, this gene was cloned for overexpression, and the expressed products were applied to generate specific antibodies against IE180 protein. Two recombinant expression plasmids pN and pNB were constructed by cloning the IE gene onto PET 30a( + ) expression vector via NcoI and BumHI sites. Plasmid pN contains the 1.8-kb NcoI-NcoI fragment of IE gene coding for the N-terminus of 616 amino acid residues, while pNB contains the 2%kb NcoI-BarnHI fragment coding for the rest of the IE180 protein. Both pN and pNB were transformed, respectively, into E. coli cells and produced large amounts of IE protein products during induction with 1 mM IPTG. The expressed IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively. These expression products were purified and then used as antigens to immunize mice for preparing specific antibodies against PRV IE180 protein. The specificities of the mice immune sera were confirmed by their abilities to react with IE180 protein present in the PRV infected cells in the Western immunoblotting assay. Furthermore, immunoperoxidase staining of PRV infected cells undertaken with these antisera revealed the subcellular distribution of the IE proteins in the infected cells and also demonstrated their transportation from the cytoplasm to the nucleus during infection. 0 1997 Elsevier Science B.V. Keywords:
Pseudorabies
* Corresponding
virus; Immediate-early
protein; Immunoperoxidase
author. Tel: + 886 4 2840751 ext. 48; fax + 886 4 2859222.
0166-0934/97/$17.00 0 1!>97Elsevier Science B.V. All rights reserved. PIZ SO1 66-0934(97)00069-4
staining
220
C. Huang et al. /Journal
of Virological Methods 66 (1997) 219-226
1. Introduction
2. Materials and methods
Pseudorabies virus (PRV) is a herpesvirus causing severe disease in swine and leading to latent infection. PRV is classified into the genus I/avicellovirus of subfamily Alphaherpesvirinae (Roizman, 1991). Similar to other herpesviruses, the PRV genes express in a sequential cascade manner and divided into three classes defined as immediate-early (IE), early, and late genes on the basis of the time of expression (Feldman et al., 1979; 1982). IE gene is expressed immediately upon infection and does not require prior viral protein synthesis for its expression. Only one major IE gene has been identified in PRV (Ihara et al., 1983), which is located at the inverted repeat region of the viral genome (Cheung, 1989). The coding region of this gene is 4380 nucleotides in length and codes for 1460 amino acid residues (Cheung et al., 1990). The product of the IE gene (IE protein) is a phosphorylated protein with a molecular weight of 180 kDa (IE180) and accumulates in nuclei of infected cells (Ben-Porat et al., 1975). IE180 can transactivate the transcription of early and late genes as well as other viral and cellular genes, and downregulates its own transcription (Ihara et al., 1983; Green et al., 1983; Ono et al., 1995). Thus, the PRV IE protein plays an indispensable role in regulating viral gene expression. However, IE proteins are synthesized during the normal course of infection in relatively small amounts early after infection (l-3 h). Recovery of IE protein from infected cells in large quantities and quality is extremely difficult. Recently, by using recombinant DNA techniques, the PRV IE gene has been cloned and expressed in different expression systems; and the expressed proteins have been found to be functional (Chlan et al., 1987; Yamada and Shimiza, 1994). DNA cloning approaches were adopted to construct the PET 30a( + ) recombinant expression vectors containing PRV IE gene in order to produce a large amount of IE protein in E. coli. The expression products were then used as antigens to immunize mice for preparing specific antisera against IE180 protein, so that PRV infection can be detected early and the IE protein structure and function can be examined further.
2.1. Virus and cell culture The PRV TNL strain was an isolate recovered in 1976 at a commercial pig farm in southern Taiwan, the Republic of China, and passaged continuously in the laboratory. MDBK (MadinDarby bovine kidney) cells were kindly provided by Dr Y.C. Zee (University of California at Davis) and grown in Dulbeceo’s modified Eagle medium supplemented with 10% fetal bovine serum. PRV was purified from the supernatant of infected cells by sucrose-gradient centrifugation and viral DNA was purified essentially as described previously (Todd and Mcferran, 1985). 2.2. Plasmids PRV DNA was subjected to BamHI digestion and the 8th restriction fragment, containing the complete open reading frame (ORF) of IE gene, were gel-purified and cloned on plasmid pUC19 to generate plasmid pUC/IE. The IE DNA was then amplified and recovered by BamHI digestion of pUC/IE, and treated with NcoI to generate two large IE fragments: a 1.8-kb NcoI-NcoI fragment and a 2.8-kb NcoI-BamHI fragment. Both IE fragments were gel-purified and inserted into the NcoI or NcoI/BamHI sites of the expression vector pET30a( + ) (Novagen) to construct expression plasmids pN and pNB respectively (Fig. 1). The IE180 ORF both in pN and pNB had been further confirmed to be cloned in frame by DNA sequencing. 2.3. Expression
of IE protein
Plasmids pN and pNB were trasnsformed into E. coli BL21(DE3, pLysS) competent cells according to the manifacturer’s manual. A single colony of each transformant was grown in Luria-Bertani (LB) medium containing 30 pg/ml kanamycin at 37°C until the OD,,, reached 1.0. Isopropyl P-Dthiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM. The culture was incubated for an additional 1 to 5 h(s) at 37°C. The cells were harvested by centrifugation and
C. Huang et al. /Journal
221
of Virological Methods 66 (1997) 219-226
(Al us
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UL
TR
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I 0
0. I
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Fig. 1. Construction of PRV IE expression vectors. (A) Diagram of PRV genome and BamHI restriction enzyme map of TNL strain. The genome is organized into unique long (UL), internal repeat (IR), unique short (US), and terminal repeat (TR) sequences. Below the genome is an expanded diagram of the BarnHI- fragment. (B) The BamHI-8 fragment was cloned into pUC19 to construct plasmid pUC/IE. (C). The 1.8 kb NcoI-NcoI fragment and the 2.8 kb NcoI-BarnHI fragment of BamHI-8 fragment were cloned into PET 30a( + 11to construct expression plasmid pN and pNB respectively.
resuspended in 100 mM Tris-HCl (pH 8.0) containing 1 mM EDT.A. Cells were broken by sonication and insoluble material was collected by centrifugating at lfi 000 x g for 10 min at 4’C, and solubilized proteins were analyzed by SDSpolyacrylamide gel Ielectrophoresis (SDS/PAGE).
2.4. Preparation IE protein
of specljic antibody
against PRV
The protein bands represented the pN-expressed 60 kDa protein and pNB-expressed 100 kDa protein were excised and eluted from SDS-
222
C. Huang et al. /Journal
of Virological Methods 66 (1997) 219-226
polyacrylamide gel, and then concentrated by centricon-50 (Amicon). Five BALB/c mice were injected interperitoneally at biweekly interval three times with 0.2 mg of protein mixed with Freund’s adjuvant. The animals were bled and the sera were absorbed with E.coli lysate mobilized on nitrocellulose paper.
stained slightly with Meyer’s hematoxylin, which stains the nuclei blue to visualize the background tissue cells. After rinsing, the stained cells were allowed to dry completely and one drop of Permount was added to the coverslips and quickly replaced invertedly onto glass slides and examined by light microscopy.
2.5. Western immunoblotting 3. Results MDBK cells were infected with PRV at 10 m.o.i. in the presence of cycloheximide (100 pug/ml) and incubated for 5 h. The cultures were then washed to remove the drug and incubated for 1.5, 3 and 4 h. The cells were harvested and resuspended in an equal volume of 2 x SDS/PAGE sample buffer. Proteins were separated by 10% SDS/PAGE according to the method of Laemmli (1970) and transferred by electroblotting onto nitrocellulose sheets as described by Towbin et al. (1979). The sheet was then treated sequentially with blocking solution (PBS containing 1% BSA) with lOO-fold dilution of mouse anti-IE protein serum, and with anti-mouse IgG goat antibody conjugated to peroxidase (Zymed). Finally, the sheet was soaked in a substrate solution (ECL western blotting detection reagent, Amersham), and exposed to X-film according to the manufacturer’s instruction. 2.6. Immunoperoxidase staining MDBK cells grown on coverslips in a 24-well culture plate were infected with PRV at 1 m.o.i. in the presence of cycloheximide. The cells were fixed with 95% ethanol and 5% glacial acetic acid for 3 min. After washing with dH,O, the fixed cells were treated with PBS buffer containing 0.25% Triton X-100 and 5% DMSO to increase the cellular membrane permeability. The coverslips were then reacted with the mouse anti-IE protein antiserum, rinsed with PBS, and the enzyme was detected by the adding two drops of a mixture of one drop of a 3% hydrogen peroxide solution and 1 drop of a 2.2% solution of 3-amino-9-ethylcarbazole (AEC) in 2.5 ml of 0.1 M acetate buffer, pH 5.2. The AEC chromogen was converted by the enzyme within 5-8 min to red, water insoluble precipitate. The coverslips, after rinsing with dH,O, were counter
3.1. Expression of PR V IE protein in E. coli The expression plasmids pN and pNB described above were used to transform E. coli BL21(DE3, pLysS) cells respectively. The cultures were grown at 37 in liquid LB medium containing kanamycin, and the recombinant proteins were induced to overexpression by 1 mM IPTG. SDS/PAGE analysis of cellular proteins form IPTG-induced cells revealed that the expressed recombinant IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively. In addition, the intensities of the expressed proteins were found to be increasing with induction times (Fig. 2). The 60 kDa and 100 kDa proteins were then gel-purified and used as antigens to immunize mice for preparing specific antibodies against PRV IE protein. 3.2. Western immunoblotting analysis of PR V-expressed IE protein The mouse antiserum against expressed IE protein was used to detect the viral IE protein in PRV infected-MDBK cells. Mouse antiserum prepared against pN-expressed 60 kDa protein detected clearly the 180 kDa protein (IE180) present in PRV infected cells which was not present in uninfected cells (Fig. 3). While mouse antiserum against pNB-expressed 100 kDa protein recognized PRV IE180 protein but with a weak signal (Data not shown). 3.3. Localization of PRV IE protein by immunoperoxidase staining Subcellular distribution of PRV IE protein was visulized by immunoperoxidase staining using mouse antiserum against pN-expressed 60 kDa
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kDa 220
a -3
B
Ml2
3456
7
8
9
10
11
M
kDa
C-+ -66
-21.5
Fig. 2. Coomassie blue-stained SDS/PAGE (10%) dem,onstrating expression of the PRV IE protein in (A) pN-transformed E. coli BL21(DE3, pLysS) cells and (B) pNB-transformed cells. Experiments were performed as described in Section 2. Lanes 1-6: crude extract of the transformed cells after 0 to 5 h-IPTG induction respectively. Lanes 7- 11: crude extract of the transformed cells after 1 to 5 h without IPTG induction respectively. Molecular weight markers are shown in lane M. The 90 kDa, 60 kDa, and 100 kDa bands are indicated by the arrow a, b, and c respectively.
protein and the immunoperoxidase staining patterns are showed in Fig. 4. IE180 molecules were synthesized initially in the cytoplasm of MDBK
cells and then translocated gradually into nuclei at 1.5 h post removal of cycloheximide and accumulated predominantly in the nuclei thereafter.
c.
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3
4
5
6
22D180-
Fig. 3. Western immunoblotting analysis of PRV IE180 protein by antiserum raised against E. co&expressed IE protein. Antiserum derived from mice that had been immunized and boosted with the plasmid pN-expressed 60 kDa truncated IE protein was used to detect PRV IE180 in the viral infected cell at 1.5, 3, and 4 h after removal of cycloheximide (Lanes l-3). Lanes 4-6 were uninfected cell with the same cycloheximide treatment and served as mock cell control for lanes 1 to 3 respectively.
4. Discussion
This study demonstrated that the PRV IE protein can be expressed successfully in E.coli PET expression system. Also, the mouse antisera generated against these expressed IE proteins can be used to detect specifically the PRV IE protein in viral infected cells. Since IE180 protein is a high moleculer weight protein containing more then 50% of alanine, argine, proline, and glycine residues (Cheung, 1989), expression of IE 180 typically results in an aggregated product in a preliminary trial. To circumvent this problem, we modified the cloning approach to express IE180 protein into two smaller molecules (60 kDa and 100 kDa) and prepared antisera against them. The antigenicity of both truncated expressed proteins was apprently unaltered since the antibodies generated could recognize specifically PRV IE180 protein in infected cells by Western immunoblotting and immunoperoxidase staining. However, the biological functions due to modifications in protein structure associated with 60 kDa and 100 kDa IE proteins requires further study. Addition-
ally, another protein with a molecular weight of 90 kDa was shown to be induced specifically by IPTG in E. coli cells transfected with pN (Fig. 2A, arrow a). Since its molecular weight was much higher than the expected 60 kDa and was constitutely synthesized in relatively a small amount before induction, this 90 kDa protein should be an IPTG-induced bacterial protein rather than a pN-expressed product. In fact, the mouse immune serum raised against this particular protein did not recognize the PRV IEl80 protein by immunoperoxidase staining of viral infected cells (data not shown). Furthermore, since the PRV IE mRNA is synthesized immediately upon the virus entering the cell and produced continuously only for 20 min (Feldman et al., 1979), a low level of IE180 produced in the PRV infected cells interfered with the immunoassays. Cyclohemide is an inhibitor for protein synthesis which has been used to accumulate the IE mRNA in cells infected with herpes simplex virus (HSV) (Fenwick and Clark, 1983). Experiments were therefore also undertaked using cycloheximide to treat cells during PRV infection
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Fig. 4. Immunoperoxidat#e staining of PRV-infected cells fixed on coverslips. Experiments were performed as described in Materials and Methods. Mouse immune serum against plasmid pN-expressed 60 kDa IE protein was used to detect the expression and distribution of PRV IEl80 in the infected cell that resulted in a red reaction product. These coverslips have also been counterstained with hematoxylin which stains the nuclei blue. (a) Uninfected cell at 4 h after removal of cycloheximide, (b-d) PRV-infected cells at 1.5, 3, 4 h after removal of cycloheximide respectively. Magnification 200 x
period for increasing IE mRNA accumulation. As soon as the drug was removed, the IE transcripts were translated to produce a large amount of IE180 porteins which could be detected easily by the mouse antisera. As shown in Fig. 3, IE180 proteins were synthesized after 1.5 h following removal of cycloheximide, and the amount of IE180 protein was increased dramatically during the next few hours. The specificities of mouse antisera against the expressed PRV IE proteins were demonstrated further by immunostaining the virus infected cells. Immunoperoxidase staining has been employed commonly for light microscopic immunolocalization of cellular proteins particularly for sparse antigens with several advantages over the immunofluoresence antibody technique such as improving sensitivity, counterstaining to show the cellular morphology, and the use of nonfluorescent materials and equipment (Wright and Scholey, 1993). By applying this technique, the PRV IE portein was localized in infected cells using the mouse antisera. According to the position and density of staining, PRV IE180 protein was first synthesized and distributed widely in the cytoplasma and then gr,adually translocated into nucli and accumulated in this compartment for transactivating the viral other genes. In conclusion, irnmunoperoxidase staining offers a novel and rapid method for detecting and localizing the PRV IE protein early after viral
infection. Furthermore, mouse immune sera prepared against bacterially expressed PRV IE proteins were found to have specificities to IE180 protein which may function as useful diagnostic reagents for detecting infection or for investigating further the function of IE180 protein as well as PRV pathogenesis.
Acknowledgements
This work was supported by grant NSC 8% 2321-B-005-061 from the National Science Council, Taiwan, Republic of China.
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