- Email: [email protected]
P38
ELSEVIER
Virus Research
Virus Research 51 (1997) 197-201
Identification of bovine viral diarrheavirus nonstructural polypeptide NS4B/P38 Alberto L. van Olphen, Ruben O. Donis
*
Department of Veterinary and Biomedical Sciences, IANR, University of Nebraska-Lincoln, Lincoln NE 68583-0905, USA
Received 27 May 1997; received in revised form 7 August 1997; accepted 13 August 1997
Abstract
The bovine pestivirus polyprotein is processed into at least 11 mature polypeptides. Previous studies with polyprotein region-specific antiserum raised against fl-galactosidase fusion proteins or synthetic peptides allowed the assignment of viral non-structural proteins to specific segments of the BVDV genome. However, the gene product from the NS4B/P38 region of the viral genome remained to be demonstrated directly. BVDV cDNA fragments predicted to encode part of NS4B/P38 (from codon 2521 to 2838 of the BVDV open reading frame) and a portion of NS5A/P58 (from codon 3008 to 3340) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Polyclonal rabbit antibodies prepared against each of the purified fusion proteins, GST-2521-2838 and GST-3008-3340, were used to immunoprecipitate viral polypeptides present in BVDV-infected cell lysates. Rabbit antiserum to GST-2521-2838 bound a polypeptide of 38 kDa identified as the mature NS4B/P38 polypeptide; while anti GST-3008-3340 lacked this specificity and bound NS5A/P58. Moreover, both antisera recognized a 96 kDa polypeptide, previously identified as a NS4B-NS5A/PP96 precursor. The function of the newly identified and highly conserved NS4B/P38 protein in viral replication remains to be determined. © 1997 Elsevier Science B.V.
Complete nucleotide sequences of the bovine viral diarrhea virus (BVDV) genomic R N A allowed elucidation of its genomic organization, leading to its classification within genus Pestivirus of the Flaviviridae (Renard et al., 1985; Collett et al., 1988a,b; Francki et al., 1991). The replication strategy of BVDV adheres to the paradigm of the * Corresponding author. Tel.: + 1 402 4726063; fax: + 1 402 4729690; e-mail: [email protected]
positive strand R N A virus superfamily (Collett et al., 1989). Following endocytosis and membrane fusion, genomic R N A is delivered to the cytosol for translation by a cap-independent initiation mechanism (Flores and Donis, 1995; Poole et al., 1995; Flores et al., 1996). Translation yields a polyprotein which is cleaved by cellular and viral endopeptidases co-translationally and post-translationally (Collett et al., 1989; Akkina, 1991; Wiskerchen and Collett, 1991; R u m e n a p f et al.,
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A.L. van OIphen, R.O. Donis
1993; Stark et al., 1993; Tautz et al., 1993; Thiel et al., 1993; Elbers et al., 1996). Although the exact location of the BVDV NS3 cleavages to generate NS3/P80, NS4A/P10, NS4B/P38, NS5A/P58 and NS5B/P75 from the polyprotein are unknown, all these mature products have been identified unequivocally, with the exception of NS4B/P38 (Collett et al., 1989; Wiskerchen and Collett, 1991; Rumenapf et al., 1993; Stark et al., 1993; Tautz et al., 1993; Thiel et al., 1993; Elbers et al., 1996). The NS4B/P38 protein has been previously referred to as p37, p32, and p30 by various authors, and should be called NS4B in the future (Collett et al., 1989; Akkina, 1991; Wiskerchen and Collett, 1991; Tautz et al., 1993). Pulse-chase studies combined with immunoprecipitation using region-specific antiserum indicated that NS4B/P38 was probably derived from the N-terminus of a 96 kDa precursor polypeptide which gave rise to a 58 kDa polypeptide (NS5A/P58) from its C-terminus (Collett et al., 1988a; Akkina, 1991). The predicted ~ 33 38 kDa cleavage product (NS4B/ P38) could not be identified because a suitable monospecific antibody was not available (Collett et al., 1988a; Akkina, 1991). A polypeptide of this size was identified in BVDV-infected cells by antibodies present in convalescent BVDV calf serum but its precise identity could not be definitively established (Donis and Dubovi, 1987a). To identify the gene product(s) derived from this region of the BVDV genome, codons 25212838 (approximate boundaries of NS4B) and 3008-3040 (middle one-half of NS5A/P58) were cloned into a prokaryotic expression vector to express these genes as glutathione S-transferase fusion proteins (Fig. 1A and B). Expressed GST2521 2838 and GST-3008-3340 fusion proteins were purified by separation in preparative SDSPAGE and electroelution (Fig. 1C, lanes 1 4) (Macfarlane, 1989). Purified GST-BVDV proteins ( > 90% purity, 250/~g) were emulsified in oil with muramyl dipeptide adjuvant (RIBI, ImmunoChem Research) and injected into rabbits to produce antiserum. The specificity of the resulting rabbit antibodies was ascertained by radioimmunoprecipitation (RIP) of lysates prepared with denaturing detergents from BVDV-infected cells metabolically labeled with 35S-methionine. Rabbit
Virus Research 51 (1997) 197 201
antiserum to GST-2521 2838 bound NS4B/P38 (Fig. 2, lane 2) as well as NS5A/P58. A trace of BVDV precursor polypeptides NS4B-NS5A/P96 and NS5A-NS5B/P133 was also observed (Fig. 2, lane 2). In contrast, antiserum to GST-3008-3340 A
.24 ! 4
~2800
v
--3380
y
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~- NS4A NS4B
B
NS5A
2521 2838 3008 3340 ...... ~ --t ~ J 7945 8895 /9406 .10401 Nhel " X ~ n d l l ~ N h e l . , ,."" , /'" Hindlll .
.
.
.
.
~faST>
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Xbal
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4
5
-145 -92 -84 -62 -34 -26
Fig. 1. Expression and purification of GST-BVDV fusion proteins. (A) Diagram of the NS4B/P38 NS5A/P58 region of the BVDV polyprotein, indicating approximate location of the cleavage sites to yield mature polypeptides, codon numbering of Collett et al. (Collett et al., 1988b, 1989). (B) Two cDNA fragments derived from BVDV, strain NADL, were cloned into prokaryotic GST fusion expression plasmid vector to obtain GST-BVDV hybrid proteins (Guan and Dixon, 1991). Nhel-HindlIl fragments encompassing codons 2521--2838 (which approximately corresponds to NS4B/P38) and codons 3008 3340 (which approximately correspond to the C-terminal region of NS5A/P58) were cloned into expression vector pGEX-KG yielding pGST-2521 2838 and pGST-3008 3340 (Guan and Dixon, 1991). (C) GST-BVDV fusion protein expression in E.coli strain BL21 after induction with isopropylfl-D-thiogalactopyranoside; whole bacterial lysates from pGST-2521-2838 (lane 2) and pGST-3008 3340 (lane 4) transformants show overexpressed fusion proteins. Proteins separated by SDS-PAGE were visualized by staining with Coomassie blue. Regions of the preparative SDS-PAGE containing bands from lysates shown in lanes 2 (pGST-2521 2838) and 4 (pGST-3008-3340) were excised and proteins eluted. Purified fusion proteins were separated in lanes I (GST-4B-5A) and 3 (GST-5A), respectively. Standard proteins for molecular mass (shown in kDa) were separated in lane 5.
A.L. van Olphen, R.O. Donis / Virus Research 51 (1997) 197-201
1
2
3
4 5
6
1459262-
-NS3 -NS5A
34-
-NS4B ....
Fig. 2. Viral specificity of rabbit antiserum to GST-BVDV proteins assessed by radioimmunoprecipitation. BVDV, strain NADL-infected and mock-infected bovine cells labeled metabolically with 35S-methionine from 14 to 16 h after infection were solubilized in SDS-PAGE sample buffer (65 mM Tris, 2% SDS, 10 mM DTT, 0.001% bromophenol blue; pH 8.0), heated at 90°C for 4 min and diluted 10-fold in TNE-RIP buffer (50 mM tris, 150 mM NaCI, 5 mM EDTA, pH 7.4 (TNE buffer) supplemented with 0.5% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, and 100 /iM phenylmethysulfonylfluoride (TNE-RIP buffer)). Lysates from mock-infected (lanes 1, 3 and 5) and infected (lanes 2, 4 and 6) cells were incubated with hyperimmune rabbits sera or a bovine polyclonal antiserum from a BVDV convalescent calf prepared as described previously (Donis and Dubovi, 1987a). Immune complexes were harvested using fixed protein A-bearing strain Cowan I Staphylococcus aureus (Pansorbin ®, CalbiochemBehring, San Diego, CA) as described (Donis and Dubovi, 1987a). Rabbit anti-GST-2521 2838 (lanes 1 and 2) and rabbit anti GST-3008-3340 (lane 3 and 4) show specific recognition of unique polypeptides (NS4B and NS5A) not precipitated from mock-infected cell lysates (lanes 1 and 3). Bovine polyclonal antiserum (lanes 5 and 6) reveals the precipitation of several non-structural BVDV proteins and some precursors in the infected cell lysate shown in lane 6 (NS3 and NS4B). Arrows in lane 2 denote the position of precursor polypeptides NS5A-5B (solid) or NS4B-5A (hatched).
did not bind NS4B/P38 and recognized mature NS5A/P58 (Fig. 2, lane 4). Faint bands from precursor polypeptides and NS4B-NS5A/P96 and NS5A-NS5B/P133 were also detected (Fig. 2, lane 4). Pre-immune sera from these rabbits did not selectively precipitate any polypeptide from BVDV-infected cell lysates (data not shown). RIP of cell lysates prepared with non-ionic detergent, which preserve protein folding (native lysates), revealed no qualitative differences in the viral polypeptides identified, as compared to denatured lysates (data not shown). However, the efficiency of NS4B/P38 and NS5A/P58 precipitation (and their precursors) from denatured cell lysates is
199
superior, in agreement with the unfolded state of the purified proteins used for immunization. The finding that antiserum to GST-2521-2838 recognized NS5A/P58 as effectively as NS4B/P38 was unexpected, since only a few amino acids of the latter were represented in the immunizing polypeptide. One possible explanation would be co-precipitation of NS4B/P38-NS5A/P58 complexes by the antiserum to NS4B/P38. However, this seems unlikely because antibodies to NS5A/ P58 did not precipitate NS4B/P38 (Fig. 2, lane 4). In addition, the GST-2521-2838 antiserum detected the same two proteins, NS5A/P58 and NS4B/P38, in Western blots (data not shown). Thus, we concluded that the cDNA fragment comprising codons 2521-2838 encodes not only the predicted NS4B/P38 protein but also a stretch of amino acids from the N-terminus of NS5A/ P58. Contrary to our assumptions, the N-terminus of NS5A/P58 is located N-terminal of amino acid residue 2838, and probably upstream of residue 2830 since, theoretically, a minimum of about eight N-terminal amino acids are necessary for antibodies to precipitate NS5A/P58 (Hodges et al., 1988). Regardless of the exact location of the processing site, the N-terminal region of NS5A/P58 appears to be highly immunogenic and surface-exposed. Indeed, hydropathy analysis of NS4B/P38 and the flanking regions of the BVDV polyprotein indicates that residues 2806-2838 (the C-terminal 33 amino acids of GST-2521-2838) are predominantly hydrophilic (data not shown) (Kyte and Doolitle, 1982). Although the bovine anti-BVDV serum used in these experiments (Fig. 2) was from a calf experimentally inoculated with BVDV, cattle recovered from natural BVDV infections also have low levels of circulating antibodies to NS4B/P38 and NS5A/P58, in addition to high concentrations of immunoglobulins specific for the major targets of the humoral response: the envelope glycoproteins and NS23/P125 (Donis and Dubovi, 1987b). To further explore the specificity of the rabbit polyclonal antibodies and the precursor-product relationships among the NS4B/P38, NS5A/P58 and NS5B we performed RIP analyses of a set of lysates from infected cells pulse-labeled with 35Smethionine and either harvested immediately or
200
A.L. van Olphen, R.O. Donis ,' Virus Research 51 (1997) 197 201
A
1
2
3
4
5
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14592-
-NS5A-5B -NS4B-5A
62-
-NS5A ~
34-
o ~
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in agreement with the finding that NS4B/P38 is one of the two most conserved nonstructural proteins a m o n g pestiviruses (Deng and Brock, 1992; Donis, 1995). The function of this newly identified NS4B/P38 in BVDV replication remains to be established. Antisera to NS4B/P38 and NS5A/P58 may help elucidate their role in the BVDV life cycle.
Note added in proof
B
1
145-
2
3
4
5
6
92-
-NS5A-5B -NS4B-5A
62-
-NS5A
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Fig. 3. Processing of NS4B/P38 precursors. Bovine cells were infected with BVDV (lanes 3 6) or mock infected (lanes 1 and 2), were pulsed with 35S-methionine for 15 rain in methioninedeficient medium at 14 h after infection. Following the labeling pulse, cell monolayers either lysed immediately(lanes 1 and 3) in SDS-PAGE sample buffer or chased with medium containing 10-fold excess methionine for intervals of 15 (lane 4), 60 (lane 5) and 120 rain (lanes 2 and 6) prior to harvesting. Lysates were immune precipitated with rabbit antiserum to GST-2521-2838 (Panel A) or rabbit anti GST-3008 3340 (Panel B). Arrows in lane 3 denote the position of precursor polypeptides as indicated in Fig. 2. The migration positions of molecular standards (mass in kDa,) are marked.
after a cold-methionine chase for different time periods. In agreement with a previous report by Akkina, NS5A can be generated by cleavage from two alternative precursor polypeptides; NS5ANS5B/PPI33 and NS4B-NS5A/PP96 (Fig. 3, panels A and B) (Akkina, 1991). Quantitative image analysis of pulse-chase R I P autoradiograms indicated that the precursor polypeptides NS5ANS5B/PP133 and NS4B-NS5A/PP96 have a half-life of 1 h and their decrease corresponds with increasing levels of the mature NS4B/P38 and NS5A/P58 products (Fig. 3, panels A and B). Rabbit antisera to GST-2521 2838 precipitated NS4B/P38 and NS5A/P58 from antigenically divergent strains of BVDV (data not shown). This is
These results are entirely consistent with two reports on the BVDV nonstructural protein processing sites published while this manuscript was under review: [1] Tautz, N., Elbers, K., Stoll, D., Meyers, G., Thiel, H.J., 1997. Serine protease of pestiviruses: determination of cleavage sites. J. Virol. 71(7), 5415-22. [2] Xu, J., Mendez, E., Caron, P.R., Lin, C., Murcko, M.A., Collett, M.S., Rice, C.M., 1997. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J. Virol. 71(7), 5312-22.
Acknowledgements We thank Donald MacFarlane for suggestions with preparative electrophoresis and J. Dixon for plasmids. Our thanks to Fernando Osorio and Clinton Jones for critical reading of the manuscript. This work was supported in part by a N R I grant 92-37204-7959 from the U S D A and funds from the Center for Biotechnology of the University of Nebraska-Lincoln. This manuscript has been assigned Journal Series # 11974 by the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln.
References Akkina, R.K., 1991. Pestivirus bovine viral diarrhea virus polypeptides: identification of new precursor proteins and alternative cleavage pathways. Virus Res. 19 (1), 67 81.
A.L. van Olphen, R.O. Donis / Virus Research 51 (1997) 197-201
Collett, M.S., Larson, R., Belzer, S.K., Retzel, E., 1988a. Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology 165 (1), 200208. Collett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K., Purchio, A.F., 1988b. Molecular cloning and nucleotide sequence of the pestivirus Bovine Viral Diarrhea virus. Virology 165, 191 199. Collett, M.S., Moennig, V., Horzinek, M.C., 1989. Recent advances in pestivirus research. J. Gen. Virol. 70 (Pt 2), 253-266. Deng, R., Brock, K.V., 1992. Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1. Virology 191 (2), 867- 869. Donis, R., 1995. Molecular biology of Bovine Viral Diarrhea virus and its interactions with the host. In: Baker, J., Houe, H. (Eds.), The Veterinary Clinics of North America, vol. 11. Saunders, Philadelphia, PA, pp. 393-424. Donis, R.O., Dubovi, E.J., 1987a. Characterization of bovine viral diarrhoea-mucosal disease virus-specific proteins in bovine cells. J. Gen. Virol. 68, 1597-1605. Donis, R.O., Dubovi, E.J., 1987b. Molecular specificity of the antibody responses of cattle naturally and experimentally infected with cytopathic and noncytopathic bovine viral diarrhea virus biotypes. Am. J. Vet. Res. 48 (11), 15491554. Elbers, K., Tautz, N., Becher, P., Stoll, D., Rumenapf, T., Thiel, H.J., 1996. Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7. J. Virol. 70 (6), 4131-4135. Flores, E., Donis, R., 1995. Isolation of a mutant MDBK cell line resistant to bovine viral diarrhea virus infection due to a block in viral entry. Virology 208, 565-575. Flores, E., Kreutz, L., Donis, R., 1996. Swine and ruminant pestiviruses require the same cellular factor to enter bovine cells. J. Gen. Virol. 77, 1295-1303. Francki, R., Fauquet, C., Knudson, D., Brown, F., 1991. Classification and nomenclature of viruses. Fifth report of the ICTV. Archives of Virology (Suppl 2). Springer, New York, 450 pp. Guan, K.L., Dixon, J.E., 1991. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and
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purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192 (2), 262-267. Hodges, R.S., Heaton, R.J., Parker, J.M., Molday, L., Molday, R.S., 1988. Antigenantibody interaction. Synthetic peptides define linear antigenic determinants recongnized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin. J. Biol. Chem. 263 (24), 11768-11775. Kyte, J., Doolitle, R.F., 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157 (I), 105-132. Macfarlane, D.E., 1989. Two dimensional benzyldimethyl-nhexadecylammonium chloride--sodium dodecyl sulfate preparative polyacrylamide gel electrphoresis: a high capacity high resolution technique for the purification of proteins from complex mixtures [published erratum appears in Anal. Biochem. 1989 July, 180 (1), 194]. Anal. Biochem. 176 (2), 457-463. Poole, T., Wang, C., Popp, R., Potgeiter, L., Siddiqui, A., Collett, M., 1995. Pestivirus translation initiation occurs by internal ribosome entry. Virology 206, 750-754. Renard, A., Guiot, C., Schmetz, D., Dagenais, L., Pastoret, P.P., Dina, D., Martial, J.A., 1985. Molecular cloning of bovine viral diarrhea viral sequences. DNA 4, 429-438. Rumenapf, T., Unger, G., Strauss, J.H., Thiel, H.J., 1993. Processing of the envelope glycoproteins of pestiviruses. J. Virol. 67 (6), 3288-3294. Stark, R., Meyers, G., Rumenapf, T., Thiel, H.J., 1993. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus. J. Virol. 67 (12), 7088-7095. Tautz, N., Meyers, G., Thiel, H.J., 1993. Processing of polyubiquitin in the polyprotein of an RNA virus. Virology 197 (1), 74-85. Thiel, H.J., Meyers, G., Stark, R., Tautz, N., Rumenapf, T., Unger, G., Conzelmann, K.K., 1993. Molecular characterization of positive-strand RNA viruses: pestiviruses and the porcine reproductive and respiratory syndrome virus (PRRSV). Arch. Virol. Suppl. 7, 41-52. Wiskerchen, M., Collett, M.S., 1991. Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology 1984 (1), 341-350.
Virus Research
Virus Research 51 (1997) 197-201
Identification of bovine viral diarrheavirus nonstructural polypeptide NS4B/P38 Alberto L. van Olphen, Ruben O. Donis
*
Department of Veterinary and Biomedical Sciences, IANR, University of Nebraska-Lincoln, Lincoln NE 68583-0905, USA
Received 27 May 1997; received in revised form 7 August 1997; accepted 13 August 1997
Abstract
The bovine pestivirus polyprotein is processed into at least 11 mature polypeptides. Previous studies with polyprotein region-specific antiserum raised against fl-galactosidase fusion proteins or synthetic peptides allowed the assignment of viral non-structural proteins to specific segments of the BVDV genome. However, the gene product from the NS4B/P38 region of the viral genome remained to be demonstrated directly. BVDV cDNA fragments predicted to encode part of NS4B/P38 (from codon 2521 to 2838 of the BVDV open reading frame) and a portion of NS5A/P58 (from codon 3008 to 3340) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Polyclonal rabbit antibodies prepared against each of the purified fusion proteins, GST-2521-2838 and GST-3008-3340, were used to immunoprecipitate viral polypeptides present in BVDV-infected cell lysates. Rabbit antiserum to GST-2521-2838 bound a polypeptide of 38 kDa identified as the mature NS4B/P38 polypeptide; while anti GST-3008-3340 lacked this specificity and bound NS5A/P58. Moreover, both antisera recognized a 96 kDa polypeptide, previously identified as a NS4B-NS5A/PP96 precursor. The function of the newly identified and highly conserved NS4B/P38 protein in viral replication remains to be determined. © 1997 Elsevier Science B.V.
Complete nucleotide sequences of the bovine viral diarrhea virus (BVDV) genomic R N A allowed elucidation of its genomic organization, leading to its classification within genus Pestivirus of the Flaviviridae (Renard et al., 1985; Collett et al., 1988a,b; Francki et al., 1991). The replication strategy of BVDV adheres to the paradigm of the * Corresponding author. Tel.: + 1 402 4726063; fax: + 1 402 4729690; e-mail: [email protected]
positive strand R N A virus superfamily (Collett et al., 1989). Following endocytosis and membrane fusion, genomic R N A is delivered to the cytosol for translation by a cap-independent initiation mechanism (Flores and Donis, 1995; Poole et al., 1995; Flores et al., 1996). Translation yields a polyprotein which is cleaved by cellular and viral endopeptidases co-translationally and post-translationally (Collett et al., 1989; Akkina, 1991; Wiskerchen and Collett, 1991; R u m e n a p f et al.,
0168-1702/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0168-1702(97)00093 -2
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A.L. van OIphen, R.O. Donis
1993; Stark et al., 1993; Tautz et al., 1993; Thiel et al., 1993; Elbers et al., 1996). Although the exact location of the BVDV NS3 cleavages to generate NS3/P80, NS4A/P10, NS4B/P38, NS5A/P58 and NS5B/P75 from the polyprotein are unknown, all these mature products have been identified unequivocally, with the exception of NS4B/P38 (Collett et al., 1989; Wiskerchen and Collett, 1991; Rumenapf et al., 1993; Stark et al., 1993; Tautz et al., 1993; Thiel et al., 1993; Elbers et al., 1996). The NS4B/P38 protein has been previously referred to as p37, p32, and p30 by various authors, and should be called NS4B in the future (Collett et al., 1989; Akkina, 1991; Wiskerchen and Collett, 1991; Tautz et al., 1993). Pulse-chase studies combined with immunoprecipitation using region-specific antiserum indicated that NS4B/P38 was probably derived from the N-terminus of a 96 kDa precursor polypeptide which gave rise to a 58 kDa polypeptide (NS5A/P58) from its C-terminus (Collett et al., 1988a; Akkina, 1991). The predicted ~ 33 38 kDa cleavage product (NS4B/ P38) could not be identified because a suitable monospecific antibody was not available (Collett et al., 1988a; Akkina, 1991). A polypeptide of this size was identified in BVDV-infected cells by antibodies present in convalescent BVDV calf serum but its precise identity could not be definitively established (Donis and Dubovi, 1987a). To identify the gene product(s) derived from this region of the BVDV genome, codons 25212838 (approximate boundaries of NS4B) and 3008-3040 (middle one-half of NS5A/P58) were cloned into a prokaryotic expression vector to express these genes as glutathione S-transferase fusion proteins (Fig. 1A and B). Expressed GST2521 2838 and GST-3008-3340 fusion proteins were purified by separation in preparative SDSPAGE and electroelution (Fig. 1C, lanes 1 4) (Macfarlane, 1989). Purified GST-BVDV proteins ( > 90% purity, 250/~g) were emulsified in oil with muramyl dipeptide adjuvant (RIBI, ImmunoChem Research) and injected into rabbits to produce antiserum. The specificity of the resulting rabbit antibodies was ascertained by radioimmunoprecipitation (RIP) of lysates prepared with denaturing detergents from BVDV-infected cells metabolically labeled with 35S-methionine. Rabbit
Virus Research 51 (1997) 197 201
antiserum to GST-2521 2838 bound NS4B/P38 (Fig. 2, lane 2) as well as NS5A/P58. A trace of BVDV precursor polypeptides NS4B-NS5A/P96 and NS5A-NS5B/P133 was also observed (Fig. 2, lane 2). In contrast, antiserum to GST-3008-3340 A
.24 ! 4
~2800
v
--3380
y
¥
~- NS4A NS4B
B
NS5A
2521 2838 3008 3340 ...... ~ --t ~ J 7945 8895 /9406 .10401 Nhel " X ~ n d l l ~ N h e l . , ,."" , /'" Hindlll .
.
.
.
.
~faST>
f:i
t"
Xbal
C
GS
NS5B-~
1
2
H{ndlll
3
4
5
-145 -92 -84 -62 -34 -26
Fig. 1. Expression and purification of GST-BVDV fusion proteins. (A) Diagram of the NS4B/P38 NS5A/P58 region of the BVDV polyprotein, indicating approximate location of the cleavage sites to yield mature polypeptides, codon numbering of Collett et al. (Collett et al., 1988b, 1989). (B) Two cDNA fragments derived from BVDV, strain NADL, were cloned into prokaryotic GST fusion expression plasmid vector to obtain GST-BVDV hybrid proteins (Guan and Dixon, 1991). Nhel-HindlIl fragments encompassing codons 2521--2838 (which approximately corresponds to NS4B/P38) and codons 3008 3340 (which approximately correspond to the C-terminal region of NS5A/P58) were cloned into expression vector pGEX-KG yielding pGST-2521 2838 and pGST-3008 3340 (Guan and Dixon, 1991). (C) GST-BVDV fusion protein expression in E.coli strain BL21 after induction with isopropylfl-D-thiogalactopyranoside; whole bacterial lysates from pGST-2521-2838 (lane 2) and pGST-3008 3340 (lane 4) transformants show overexpressed fusion proteins. Proteins separated by SDS-PAGE were visualized by staining with Coomassie blue. Regions of the preparative SDS-PAGE containing bands from lysates shown in lanes 2 (pGST-2521 2838) and 4 (pGST-3008-3340) were excised and proteins eluted. Purified fusion proteins were separated in lanes I (GST-4B-5A) and 3 (GST-5A), respectively. Standard proteins for molecular mass (shown in kDa) were separated in lane 5.
A.L. van Olphen, R.O. Donis / Virus Research 51 (1997) 197-201
1
2
3
4 5
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1459262-
-NS3 -NS5A
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-NS4B ....
Fig. 2. Viral specificity of rabbit antiserum to GST-BVDV proteins assessed by radioimmunoprecipitation. BVDV, strain NADL-infected and mock-infected bovine cells labeled metabolically with 35S-methionine from 14 to 16 h after infection were solubilized in SDS-PAGE sample buffer (65 mM Tris, 2% SDS, 10 mM DTT, 0.001% bromophenol blue; pH 8.0), heated at 90°C for 4 min and diluted 10-fold in TNE-RIP buffer (50 mM tris, 150 mM NaCI, 5 mM EDTA, pH 7.4 (TNE buffer) supplemented with 0.5% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, and 100 /iM phenylmethysulfonylfluoride (TNE-RIP buffer)). Lysates from mock-infected (lanes 1, 3 and 5) and infected (lanes 2, 4 and 6) cells were incubated with hyperimmune rabbits sera or a bovine polyclonal antiserum from a BVDV convalescent calf prepared as described previously (Donis and Dubovi, 1987a). Immune complexes were harvested using fixed protein A-bearing strain Cowan I Staphylococcus aureus (Pansorbin ®, CalbiochemBehring, San Diego, CA) as described (Donis and Dubovi, 1987a). Rabbit anti-GST-2521 2838 (lanes 1 and 2) and rabbit anti GST-3008-3340 (lane 3 and 4) show specific recognition of unique polypeptides (NS4B and NS5A) not precipitated from mock-infected cell lysates (lanes 1 and 3). Bovine polyclonal antiserum (lanes 5 and 6) reveals the precipitation of several non-structural BVDV proteins and some precursors in the infected cell lysate shown in lane 6 (NS3 and NS4B). Arrows in lane 2 denote the position of precursor polypeptides NS5A-5B (solid) or NS4B-5A (hatched).
did not bind NS4B/P38 and recognized mature NS5A/P58 (Fig. 2, lane 4). Faint bands from precursor polypeptides and NS4B-NS5A/P96 and NS5A-NS5B/P133 were also detected (Fig. 2, lane 4). Pre-immune sera from these rabbits did not selectively precipitate any polypeptide from BVDV-infected cell lysates (data not shown). RIP of cell lysates prepared with non-ionic detergent, which preserve protein folding (native lysates), revealed no qualitative differences in the viral polypeptides identified, as compared to denatured lysates (data not shown). However, the efficiency of NS4B/P38 and NS5A/P58 precipitation (and their precursors) from denatured cell lysates is
199
superior, in agreement with the unfolded state of the purified proteins used for immunization. The finding that antiserum to GST-2521-2838 recognized NS5A/P58 as effectively as NS4B/P38 was unexpected, since only a few amino acids of the latter were represented in the immunizing polypeptide. One possible explanation would be co-precipitation of NS4B/P38-NS5A/P58 complexes by the antiserum to NS4B/P38. However, this seems unlikely because antibodies to NS5A/ P58 did not precipitate NS4B/P38 (Fig. 2, lane 4). In addition, the GST-2521-2838 antiserum detected the same two proteins, NS5A/P58 and NS4B/P38, in Western blots (data not shown). Thus, we concluded that the cDNA fragment comprising codons 2521-2838 encodes not only the predicted NS4B/P38 protein but also a stretch of amino acids from the N-terminus of NS5A/ P58. Contrary to our assumptions, the N-terminus of NS5A/P58 is located N-terminal of amino acid residue 2838, and probably upstream of residue 2830 since, theoretically, a minimum of about eight N-terminal amino acids are necessary for antibodies to precipitate NS5A/P58 (Hodges et al., 1988). Regardless of the exact location of the processing site, the N-terminal region of NS5A/P58 appears to be highly immunogenic and surface-exposed. Indeed, hydropathy analysis of NS4B/P38 and the flanking regions of the BVDV polyprotein indicates that residues 2806-2838 (the C-terminal 33 amino acids of GST-2521-2838) are predominantly hydrophilic (data not shown) (Kyte and Doolitle, 1982). Although the bovine anti-BVDV serum used in these experiments (Fig. 2) was from a calf experimentally inoculated with BVDV, cattle recovered from natural BVDV infections also have low levels of circulating antibodies to NS4B/P38 and NS5A/P58, in addition to high concentrations of immunoglobulins specific for the major targets of the humoral response: the envelope glycoproteins and NS23/P125 (Donis and Dubovi, 1987b). To further explore the specificity of the rabbit polyclonal antibodies and the precursor-product relationships among the NS4B/P38, NS5A/P58 and NS5B we performed RIP analyses of a set of lysates from infected cells pulse-labeled with 35Smethionine and either harvested immediately or
200
A.L. van Olphen, R.O. Donis ,' Virus Research 51 (1997) 197 201
A
1
2
3
4
5
6
14592-
-NS5A-5B -NS4B-5A
62-
-NS5A ~
34-
o ~
-NS4B
in agreement with the finding that NS4B/P38 is one of the two most conserved nonstructural proteins a m o n g pestiviruses (Deng and Brock, 1992; Donis, 1995). The function of this newly identified NS4B/P38 in BVDV replication remains to be established. Antisera to NS4B/P38 and NS5A/P58 may help elucidate their role in the BVDV life cycle.
Note added in proof
B
1
145-
2
3
4
5
6
92-
-NS5A-5B -NS4B-5A
62-
-NS5A
34-
Fig. 3. Processing of NS4B/P38 precursors. Bovine cells were infected with BVDV (lanes 3 6) or mock infected (lanes 1 and 2), were pulsed with 35S-methionine for 15 rain in methioninedeficient medium at 14 h after infection. Following the labeling pulse, cell monolayers either lysed immediately(lanes 1 and 3) in SDS-PAGE sample buffer or chased with medium containing 10-fold excess methionine for intervals of 15 (lane 4), 60 (lane 5) and 120 rain (lanes 2 and 6) prior to harvesting. Lysates were immune precipitated with rabbit antiserum to GST-2521-2838 (Panel A) or rabbit anti GST-3008 3340 (Panel B). Arrows in lane 3 denote the position of precursor polypeptides as indicated in Fig. 2. The migration positions of molecular standards (mass in kDa,) are marked.
after a cold-methionine chase for different time periods. In agreement with a previous report by Akkina, NS5A can be generated by cleavage from two alternative precursor polypeptides; NS5ANS5B/PPI33 and NS4B-NS5A/PP96 (Fig. 3, panels A and B) (Akkina, 1991). Quantitative image analysis of pulse-chase R I P autoradiograms indicated that the precursor polypeptides NS5ANS5B/PP133 and NS4B-NS5A/PP96 have a half-life of 1 h and their decrease corresponds with increasing levels of the mature NS4B/P38 and NS5A/P58 products (Fig. 3, panels A and B). Rabbit antisera to GST-2521 2838 precipitated NS4B/P38 and NS5A/P58 from antigenically divergent strains of BVDV (data not shown). This is
These results are entirely consistent with two reports on the BVDV nonstructural protein processing sites published while this manuscript was under review: [1] Tautz, N., Elbers, K., Stoll, D., Meyers, G., Thiel, H.J., 1997. Serine protease of pestiviruses: determination of cleavage sites. J. Virol. 71(7), 5415-22. [2] Xu, J., Mendez, E., Caron, P.R., Lin, C., Murcko, M.A., Collett, M.S., Rice, C.M., 1997. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J. Virol. 71(7), 5312-22.
Acknowledgements We thank Donald MacFarlane for suggestions with preparative electrophoresis and J. Dixon for plasmids. Our thanks to Fernando Osorio and Clinton Jones for critical reading of the manuscript. This work was supported in part by a N R I grant 92-37204-7959 from the U S D A and funds from the Center for Biotechnology of the University of Nebraska-Lincoln. This manuscript has been assigned Journal Series # 11974 by the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln.
References Akkina, R.K., 1991. Pestivirus bovine viral diarrhea virus polypeptides: identification of new precursor proteins and alternative cleavage pathways. Virus Res. 19 (1), 67 81.
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