Journal of General Virology (1990), 71, 2725-2729.
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Expression of the full-length products of the human papillomavirus type 6b (HPV-6b) and HPV-11 L2 open reading frames by recombinant baculovirus, and antigenic comparisons with HPV-11 whole virus particles Robert C. Rose,* William Bonnez, David G. Striker and Richard C. Reichman Infectious Diseases Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, New York 14642, U.S.A.
The L2 open reading frames (ORFs) of human papillomavirus (HPV) types 6b and 11 were expressed as full-length non-fusion proteins in Spodoptera frugiperda (Sf-9) cells using recombinant baculovirus. Both proteins were detected on Western blots as immunoreactive bands which migrated with apparent Mrs of 76K and 78K, respectively, and contained both crossreactive and type-specific epitopes, as determined by polyclonal antisera directed against defined subregions of the HPV-6b and HPV-11 L 2 0 R F s . In addition, the
minor capsid protein of HPV-11 particles co-migrates with the HPV-11 L 2 0 R F product and is immunoreactive with HPV-11 L2-specific antisera. These observations indicate that the anomalous electrophoretic mobilities of papillomavirus L 2 0 R F proteins can be explained without invoking post-transcriptional processing events and that the minor capsid protein of HPV-11 is antigenically and biophysically related to the HPV-11 L 2 0 R F product.
Human papillomavirus (HPV) types 6 and 11 generally produce benign anogenital warts (Syrjanen et al., 1987). Like other papillomaviruses (PV), these viruses have not been propagated in vitro. In addition, intact virions are generally not present in large numbers in naturally infected tissues, and biophysical and antigenic characterization of HPV particles has been difficult to perform. Therefore, HPV proteins have been produced by recombinant DNA technology in prokaryotic expression systems (Bonnez et al., 1989; Doorbar & Gallimore, 1987; Komly et al., 1986; Strike et al., 1989; Tomita et al., 1987) in order to study the molecular biology of HPV and evaluate host immune responses to infection. A number of previous studies have provided evidence that the major constituent of PV capsids is a protein with an average Mr of 54K to 57K encoded by the L1 open reading frame (ORF) (Roseto et al., 1984; Favre et al., 1975; Meinke & Meinke, 1981 ; Pilacinski et al., 1984). In these experiments, L 1 0 R F coding capacities have been closely correlated with the size of recombinant L1 products and the major capsid protein present in viral particles. In addition, polyclonal antisera raised against these recombinant L1 ORF products demonstrate the antigenic similarity of these products to the major capsid protein. Similarly obtained evidence has indicated that the L2
ORF encodes a minor structural component of the PV capsid. However, the coding capacity of this ORF does not correlate well with the mobility of the antigenically related minor capsid protein .present in viral particles. Observations of the protein's mobility place it in an Mr range of 64 to 78K, which exceeds the L 2 0 R F ' s coding capacity by approximately 25K (Doorbar & Gallimore, 1987; Jin et al., 1989; Rippe & Meinke, 1989). Possible explanations for this discrepancy include post-transcriptional and/or post-translational modifications, as well as inherent structural properties of the protein. Some previous observations indicate that modification of the L2 product may not be required to explain its electrophoretic mobility. For example, L2 ORF products expressed in bacterial systems exhibit anomalous migration patterns that are consistent with the observed mobilities of the minor capsid protein (Komly et al., 1987; Tomita et al., 1987). To define more closely the relationship between the L2 ORF and the minor capsid protein of viral particles, we expressed the products of the entire L 2 0 R F s of HPV-6b and HPV-11 as non-fused proteins in a baculovirus expression vector (BEV) system and compared their electrophoretic mobilities with an immunologically cross-reactive protein present in HPV-11 whole viral particles. Cloned HPV-6b DNA was provided by L. Gissmann (Heidelberg, F.R.G.). The HPV-6b L 2 0 R F was reconstructed by subcloning the 5' 300 bp fragment
1"Present address: 2244 Sargeant Avenue, St Paul, Minnesota 55105, U.S.A. 0000-9590 © t990 SGM
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Fig. 2. Mobility comparison of recombinant HPV-11L2ORFproduct with proteins present in HPV-I 1 whole viral particles. Lane 1 contains recombinant HPV-11 L2 and lane 2 contains SDS-solubilized HPV-11 whole viral particles. Blot was reacted with anti-llL2-C rabbit antiserum at a dilution of 1:500. Arrow indicates the locations of recombinant HPV-11 L2 and the minor capsid protein doublet bands. Lower Mr immunoreactive bands in BEV-L2 preparations (e.g. lane 1) have been observed repeatedly and may represent alternative forms of L2. Similar migration patterns of L2 species in BPV-1 and HPV-la particle preparations have been observed (Jin et al., 1989: Doorbar & Gallimore, 1987). Fig. 1. Western blot detection of recombinant L 2 0 R F products in infected Sf-9 cell lysates. Lanes 1 and 4 contain uninfected cell lysates; lanes 2 and 5, wild-type AcNPV-infected cell lysates; lane 3, recombinant HPV-6b L2 (H6L2)-infected cell lysate; lane 6, recombinant HPV-11 L2 (H 11L2)-infected cell lysate. Panel (a) was probed with rabbit antisera directed against the carboxy-terminal portion of the HPV-6b L2 ORF and (b) was probed with rabbit antisera directed against the carboxy-terminal portion of the HPV-11 L2 ORF (see Fig. 4). Arrow indicates the location of the L2 bands.
into a pATH TrpE fusion vector as previously described (Strike et al., 1989) and then adding the remainder of the L 2 0 R F , contained in a 1291 bp BamHI/XhoII fragment [nucleotide (nt) coordinates 4722 to 6013]. The full-length HPV-6b L2 construct was expressed as a TrpE fusion protein and also transferred to a baculovirus transfer vector (pAcC5, provided by E. Kawasaki, Cetus, Emeryville, California, U.S.A.) via common NcoI/blunt cloning sites. HPV-11 viral particles were produced in the athymic mouse xenograft system described by Kreider (Kreider et al., 1986). Particles were isolated and purified (Favreet al., 1975) from experimentally induced tumours and HPV-11 genomic DNA was extracted and cloned into the BamHI site of pBluescript (Stratagene). The HPV-11 L 2 0 R F was converted to a BamHI/XbaI cassette by digesting the cloned isolate with AvrII (which cuts this isolate once only at nt position 4423), filling the ends with the Klenow fragment of DNA polymerase I and inserting a BamHI adapter which contained the first seven nucleotides (including the first ATG codon) of the putative L2 coding sequence. The entire ORF was transferred to a pATH vector for expression as a TrpE
fusion protein, and also to a baculovirus transfer vector (pVL-1393, provided by M. Summers, Texas A & M University, College Station, Texas, U.S.A.) via the common BamHI and XbaI sites. Expression in the BEV system was accomplished by standard methods (Summers & Smith, 1987) and resulted in the production of full-length non-fused proteins that were immunoreactive with rabbit polyclonal antisera directed against the HPV-6b and HPV-11 L 2 0 R F s (Fig. 1). These proteins, which were not present in uninfected or wild-type Autographa californica nuclear polyhedrosis virus (AcNPV)-infected cell lysates, migrate with apparent MrS of 76K (6L2) and 78K (llL2). DNA sequence analyses confirmed the presence of the HPV-6b and HPV-11 L2 start and stop codons (data not shown). Direct electrophoretic comparison of the BEV HPV-11 L 2 0 R F recombinant product with an immunologically cross-reactive 78K Mr protein present in HPV-11 viral particles indicates that the minor capsid protein is closely associated with this ORF (Fig. 2). These observations imply that RNA splicing is not required to produce a protein with an apparent Mr within this range. The appearance of an immunoreactive protein doublet in the usual location of the minor capsid protein has not been previously reported and may represent an artefact of the athymic mouse system. Although evidence derived from prokaryotic studies strongly suggests that glycosylation is not involved in anomalous L 2 0 R F product mobilities, the fact that Sf-9 cells are eukaroyotic presents the possibility that recombinant L2 proteins expressed in this system could
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Fig. 3. Western blot analysis of deglycosylation reactions. (a) Baculovirus-produced HIV gpl60 (MicroGeneSys) which was probed with an HIV antiserum at a dilution of I : 100. (b) Baculovirus-produced HPV-6b L 2 0 R F product probed with rabbit anti-6L2C antiserum at a dilution of 1:1000. Reactions in lanes 1 were not treated with Nglycanase. Reactions in lanes 2 were treated with N-glycanase as discussed in the text. Arrows indicate the positions of the reaction products.
be glycosylated. However, endoglycosidase treatment of BEV-expressed 6L2 (N-glycanase; Genzyme) had no effect on the mobility of the HPV-6b L2 product (Fig. 3) which suggests that N-glycosylation sites present in the primary sequence of this protein are not glycosylated in this system. The positive control used for this experiment, human immunodeficiency virus (HIV) glycoprotein gpl60, was also produced in the BEV system (MicroGeneSys) and underwent a large mobility change, which indicates that carbohydrate moeities added by Sf9 cell mechanisms can be efficiently removed with this enzyme. Expression of these L 2 0 R F s as pATH TrpE fusion proteins in Escherichia coli resulted in production of recombinant L2 fusion proteins, each with an apparent Mr of 115K, which is approximately 28K larger than the 87K Mr predicted from the fusion of these coding sequences to a 37K TrpE moiety (data not shown). In previous experiments (Strike et al., 1989) HPV-6b L2 sub-ORF fragments, also expressed as TrpE fusion proteins, exhibited similar anomalous migration patterns in SDS-PAGE systems, with the exception of the fragment containing the amino-terminal 100 amino acids of the ORF (6L2-N ; Fig. 4) which migrated at its predicted M r. These results were obtained under denaturing conditions from peptides produced in a prokaryo-
Fig. 4. Sub-ORF fragments of the HPV-6b and HPV-I 1 L 2 0 R F s used to produce rabbit polyclonal antisera. The indicated restriction sites were used to clone and express the L2 sub-ORF fragments as ]~galactosidase fusion proteins in E. coil (Strike et al., 1989) and, in the present study, llL2-C. Nucleotide coordinates of restriction endonuclease sites used are as indicated in parentheses and correspond to sequence data obtained from GenBank. 6L2-N refers to the aminoterminal 100 amino acid residues, 6L2-MID refers to the middle 230 amino acid residues and 6L2-C refers to the carboxy-terminal 129 amino acid residues of the HPV-6b L2 ORF. I IL2-C refers to the carboxy-terminal 126 amino acid residues of the HPV-I 1 L2 ORF.
tic expression system, which provides additional evidence that higher-order protein structure and/or posttranscriptional or post-translational processing steps are not required to explain the migration patterns of L2 products. In addition, these observations suggest that these anomalies are not derived from the primary structure of the'hig, hly conserved amino-terminal region of the L 2 0 R F ipr.oduct. The othergoal of this study was to define the locations of immunologically cross-reactive and type-specific epitopes in the HPV-6b and HPV-11 L 2 0 R F s . Rabbit polyclonal antisera were raised against three recombinant fusion proteins derived from fragments 6L2-N, 6L2MID and 6L2-C (Fig. 4) of the HPV-6b L 2 0 R F , as previously described (Strike et al., 1989). Preliminary experiments with full-length L2-TrpE fusion proteins indicated that the antiserum directed against the 6L2-C region did not cross-react with the HPV-11 L 2 0 R F product (data not shown). To investigate this observation further, the carboxy-terminal portion of the HPV-11 L 2 0 R F (llL2-C) was cloned in a similar manner to that used for the 6L2-C fragment. Briefly, a 450 bp PstI/XbaI fragment (HPV-11 nt coordinates 5435 to 5885) was cloned in a p E X vector (Stanley & Luzio, 1984) and expressed as a//-galactosidase fusion protein. The fusion protein was purified by SDS-PAGE, excised and emulsified with Freund's complete adjuvant and used to produce polyclonal antisera as previously described (Strike et al., 1989). Pre-immune sera from all animals used in these experiments were HPV L2
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Fig. 5. Localization of cross-reactive and type-specific epitopes of the HPV-6b and HPV-11 L20RFs. Lanes 1 contain recombinant HPV-6b L2-infected cell lysates; lanes 2 contain recombinant HPV-11 L2infected cell lysates. Panels (a) to (d) were probed with rabbit polyclonal antisera directed against (a) the amino-terminal portion of the HPV-6b L20RF, (b) the middle portion of the HPV-6b L20RF, (c) the carboxy-terminal portion of the HPV-6b L 2 0 R F and (d) the carboxy-terminal portion of the HPV-11 L20RF.
seronegative, as j u d g e d by W e s t e r n blot l m m u n o a s s a y s ( d a t a not shown). Fig. 5 is a W e s t e r n blot w h i c h d e m o n s t r a t e s t h a t the r e c o m b i n a n t L 2 0 R F proteins c a r r y b o t h cross-reactive a n d type-specific epitopes. Polyclonal a n t i s e r a d i r e c t e d a g a i n s t the 6L2-N a n d 6L2M I D H P V - 6 b L 2 0 R F f r a g m e n t s cross-react w i t h the B E V HPV-11 L 2 0 R F p r o d u c t (Fig. 5 a a n d b). T h e a n t i s e r a raised a g a i n s t the c a r b o x y - t e r m i n a l p o r t i o n s o f the H P V - 6 b a n d HPV-11 L 2 0 R F s (6L2-C a n d 11L2-C) do not cross-react, however. This o b s e r v a t i o n suggests the presence o f one or m o r e type-specific e p i t o p e s in this region o f the H P V - 6 b a n d HPV-11 L 2 0 R F s a n d the a b s e n c e o f cross-reacting e p i t o p e s (Fig. 5c a n d d) even t h o u g h the regions used to p r o d u c e these a n t i s e r a are m o r e t h a n 75% i d e n t i c a l at the a m i n o a c i d level. Previous r e p o r t s b a s e d on studies o f P V - i n f e c t e d tissue s p e c i m e n s ( K o m l y et al., 1986; J i n et al., 1989) p r o v i d e d i m m u n o h i s t o c h e m i c a l e v i d e n c e t h a t type-specific epitopes are c o n t a i n e d w i t h i n the L2 p r o d u c t s o f H P V - l a a n d b o v i n e p a p i l l o m a v i r u s type 1 (BPV-1), respectively. O u r results p r o v i d e d i r e c t e v i d e n c e for the existence o f s i m i l a r type-specific e p i t o p e s w i t h i n the p r o t e i n c o d i n g sequences o f the L 2 0 R F s o f H P V - 6 b a n d H P V - 1 1 , two closely related genital H P V s . T h i s i n f o r m a t i o n m a y p r o v e to be useful in the d e v e l o p m e n t o f tests for the d i a g n o s i s o f genital H P V infections. W e c o n c l u d e t h a t the L 2 0 R F of HPV-6b, with a c o d i n g p o t e n t i a l o f 51 K , e n c o d e s a p r o t e i n w i t h an Mr o f a p p r o x i m a t e l y 76K a n d t h a t the L 2 0 R F o f H P V - I 1 , with a coding capacity of 49K, produces a protein of 78K Mr. O u r o b s e r v a t i o n s i n d i c a t e t h a t p o s t - t r a n s c r i p t i o n a l
a n d p o s t - t r a n s l a t i o n a l processing systems d o not a p p e a r to be n e c e s s a r y to p r o d u c e the unusual e l e c t r o p h o r e t i c m o b i l i t i e s o f L 2 0 R F p r o t e i n s in S D S - p o l y a c r y l a m i d e gel systems. I n a d d i t i o n , c o m p a r i s o n o f the b i o p h y s i c a l a n d a n t i g e n i c c h a r a c t e r i s t i c s o f r e c o m b i n a n t HPV-11 L2 p r o t e i n w i t h p r o t e i n s p r e s e n t in H P V - I 1 p a r t i c l e s i n d i c a t e s t h a t the m i n o r c a p s i d p r o t e i n o f HPV-11 is closely related to the HPV-11 L 2 0 R F product. W e h a v e also d e m o n s t r a t e d t h a t the H P V - 6 b a n d HPV-11 L2 O R F p r o d u c t s c o n t a i n b o t h cross-reactive a n d typespecific e p i t o p e s ; studies designed to further c h a r a c t e r ize a n d d e l i n e a t e these e p i t o p e s are in progress. Purified H P V - 6 b a n d HPV-11 L 2 0 R F p r o t e i n s p r o d u c e d in the B E V system will be used in o t h e r i m m u n o l o g i c a l studies. sf-9 cells, wild-type AcNPV strain E2 and several baculovirus transfer vectors were graciously provided by Max Summers, Texas A & M University, College Station, Texas, U.S.A. Our appreciation also goes to John J. Treanor for helpful discussions and critical review of the manuscript and to Carrie Harvey for expert technical assistance. This work was supported by AI-23418 and AI-27658 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.
References
BONNEZ,W., ROSE,R., REICHMAN,R. & STRIKE,D. (1989). The PstIXholl restriction fragment of the HPV-6b L10RF lacks immunological specificity as determined by sera from HPV-6 condyloma acuminatum patients and controls. In UCLA Symposia on Molecular and Cellular Biology, New Series, vol. 124, pp. 77--80. Edited by P. Howley & T. Broker. New York: Alan R. Liss. DOORBAR, J. & GALLIMORE, P. (1987). Identification of proteins encoded by the L1 and L2 open reading frames of human papillomavirus la. Journal of Virology 61, 2793 2799. FAVRE, M., BREITBURD, F., CROISSANT,O. & ORTH, G. (1975). Structural polypeptides of rabbit, bovine, and human papillomaviruses. Journal of Virology 15, 1239-1247. JIN, X. W., COWSERT,L. M., PILACINSKI,W. P. & JENSON,A. B. (1989). Identification of L2 open reading frame gene products of bovine papillomavirus type 1 using monoclonal antibodies. Journal of General Virology 70, l 133-1140. KOMLY,C., BREITBURO,F., CROISSANT,O. & STREECK,R. (1986). The L2 open reading frame of human papillomavirus type la encodes a minor structural protein carying type-specific antigens. Journal of Virology 60, 813-816. KREIDER,J. W., HOWETT,M. K., LILL,N. L., BARTLETT,G. L., ZAINO, R. J., SEDLACEK,T. V. & MORTEL,R. (1986). In vivo transformation of human skin with human papillomavirus type 11 from condylomata acuminata. Journal of Virology 59, 369 376. MEINKE,W. & MEINKE,G. C. (1981). Isolation and characterization of the major capsid protein of bovine papilloma virus type 1. Journal of General Virology 52, 15 24. PILACINSKI, W., GLASSMAN,D., KRZYZEK, R., SADOWSKI, P. & ROBBINS,A. (1984). Cloning and expression in Escherichia coli of the bovine papillomavirus LI and L2 open reading frames. Bio/Technology 1, 356-360. RIPPE, R. & MEINKE,W. (1989). Identification and characterization of the BPV-2 L2 protein. Virology' 171, 298-301. ROSETO, A., PO'fHIER,P., GUILLEMIN,M.-C., PERIES,J., BREITBURD, F., BONNEAUD,N. & OATH,G. (1984). Monoclonal antibodies to the major capsid protein of human papillomavirus type 1. Journal of General Virology 65, 1319-1324.
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STANLEY, K. K. & LUZIO, J. P. (1984). Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO Journal 3, 1429-1434. STRIKE, D. G., BONNEZ, W., ROSE, R. C. & REICHMAN, R. C. (1989). Expression in Escherichia coil of seven DNA fragments comprising the complete L1 and L2 open reading frames of human papillomavirus type 6b and localization of the 'common antigen' region. Journal of General Virology 70, 543-555. SUMMERS, M. D. & SMITH, G. E. (1987). A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin, no. 1555.
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SYRJANEN, K., GISSMANN, L. & KOSS, L. G. (editors) (1987). Papillomaviruses and Human Disease. Berlin: Springer-Verlag. TOMITA, Y., SHIRASAWA, H., SEKINE, H. & SIMIZU, B. (1987). Expression of the human papillomavirus type 6b L2 open reading frame in Escherichia coil: L2-fl-galactosidase fusion proteins and their antigenic properties. Virology 158, 8 14.
(Received 26 March 1990; Accepted 12 July 1990)
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Expression of the full-length products of the human papillomavirus type 6b (HPV-6b) and HPV-11 L2 open reading frames by recombinant baculovirus, and antigenic comparisons with HPV-11 whole virus particles Robert C. Rose,* William Bonnez, David G. Striker and Richard C. Reichman Infectious Diseases Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, New York 14642, U.S.A.
The L2 open reading frames (ORFs) of human papillomavirus (HPV) types 6b and 11 were expressed as full-length non-fusion proteins in Spodoptera frugiperda (Sf-9) cells using recombinant baculovirus. Both proteins were detected on Western blots as immunoreactive bands which migrated with apparent Mrs of 76K and 78K, respectively, and contained both crossreactive and type-specific epitopes, as determined by polyclonal antisera directed against defined subregions of the HPV-6b and HPV-11 L 2 0 R F s . In addition, the
minor capsid protein of HPV-11 particles co-migrates with the HPV-11 L 2 0 R F product and is immunoreactive with HPV-11 L2-specific antisera. These observations indicate that the anomalous electrophoretic mobilities of papillomavirus L 2 0 R F proteins can be explained without invoking post-transcriptional processing events and that the minor capsid protein of HPV-11 is antigenically and biophysically related to the HPV-11 L 2 0 R F product.
Human papillomavirus (HPV) types 6 and 11 generally produce benign anogenital warts (Syrjanen et al., 1987). Like other papillomaviruses (PV), these viruses have not been propagated in vitro. In addition, intact virions are generally not present in large numbers in naturally infected tissues, and biophysical and antigenic characterization of HPV particles has been difficult to perform. Therefore, HPV proteins have been produced by recombinant DNA technology in prokaryotic expression systems (Bonnez et al., 1989; Doorbar & Gallimore, 1987; Komly et al., 1986; Strike et al., 1989; Tomita et al., 1987) in order to study the molecular biology of HPV and evaluate host immune responses to infection. A number of previous studies have provided evidence that the major constituent of PV capsids is a protein with an average Mr of 54K to 57K encoded by the L1 open reading frame (ORF) (Roseto et al., 1984; Favre et al., 1975; Meinke & Meinke, 1981 ; Pilacinski et al., 1984). In these experiments, L 1 0 R F coding capacities have been closely correlated with the size of recombinant L1 products and the major capsid protein present in viral particles. In addition, polyclonal antisera raised against these recombinant L1 ORF products demonstrate the antigenic similarity of these products to the major capsid protein. Similarly obtained evidence has indicated that the L2
ORF encodes a minor structural component of the PV capsid. However, the coding capacity of this ORF does not correlate well with the mobility of the antigenically related minor capsid protein .present in viral particles. Observations of the protein's mobility place it in an Mr range of 64 to 78K, which exceeds the L 2 0 R F ' s coding capacity by approximately 25K (Doorbar & Gallimore, 1987; Jin et al., 1989; Rippe & Meinke, 1989). Possible explanations for this discrepancy include post-transcriptional and/or post-translational modifications, as well as inherent structural properties of the protein. Some previous observations indicate that modification of the L2 product may not be required to explain its electrophoretic mobility. For example, L2 ORF products expressed in bacterial systems exhibit anomalous migration patterns that are consistent with the observed mobilities of the minor capsid protein (Komly et al., 1987; Tomita et al., 1987). To define more closely the relationship between the L2 ORF and the minor capsid protein of viral particles, we expressed the products of the entire L 2 0 R F s of HPV-6b and HPV-11 as non-fused proteins in a baculovirus expression vector (BEV) system and compared their electrophoretic mobilities with an immunologically cross-reactive protein present in HPV-11 whole viral particles. Cloned HPV-6b DNA was provided by L. Gissmann (Heidelberg, F.R.G.). The HPV-6b L 2 0 R F was reconstructed by subcloning the 5' 300 bp fragment
1"Present address: 2244 Sargeant Avenue, St Paul, Minnesota 55105, U.S.A. 0000-9590 © t990 SGM
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Fig. 2. Mobility comparison of recombinant HPV-11L2ORFproduct with proteins present in HPV-I 1 whole viral particles. Lane 1 contains recombinant HPV-11 L2 and lane 2 contains SDS-solubilized HPV-11 whole viral particles. Blot was reacted with anti-llL2-C rabbit antiserum at a dilution of 1:500. Arrow indicates the locations of recombinant HPV-11 L2 and the minor capsid protein doublet bands. Lower Mr immunoreactive bands in BEV-L2 preparations (e.g. lane 1) have been observed repeatedly and may represent alternative forms of L2. Similar migration patterns of L2 species in BPV-1 and HPV-la particle preparations have been observed (Jin et al., 1989: Doorbar & Gallimore, 1987). Fig. 1. Western blot detection of recombinant L 2 0 R F products in infected Sf-9 cell lysates. Lanes 1 and 4 contain uninfected cell lysates; lanes 2 and 5, wild-type AcNPV-infected cell lysates; lane 3, recombinant HPV-6b L2 (H6L2)-infected cell lysate; lane 6, recombinant HPV-11 L2 (H 11L2)-infected cell lysate. Panel (a) was probed with rabbit antisera directed against the carboxy-terminal portion of the HPV-6b L2 ORF and (b) was probed with rabbit antisera directed against the carboxy-terminal portion of the HPV-11 L2 ORF (see Fig. 4). Arrow indicates the location of the L2 bands.
into a pATH TrpE fusion vector as previously described (Strike et al., 1989) and then adding the remainder of the L 2 0 R F , contained in a 1291 bp BamHI/XhoII fragment [nucleotide (nt) coordinates 4722 to 6013]. The full-length HPV-6b L2 construct was expressed as a TrpE fusion protein and also transferred to a baculovirus transfer vector (pAcC5, provided by E. Kawasaki, Cetus, Emeryville, California, U.S.A.) via common NcoI/blunt cloning sites. HPV-11 viral particles were produced in the athymic mouse xenograft system described by Kreider (Kreider et al., 1986). Particles were isolated and purified (Favreet al., 1975) from experimentally induced tumours and HPV-11 genomic DNA was extracted and cloned into the BamHI site of pBluescript (Stratagene). The HPV-11 L 2 0 R F was converted to a BamHI/XbaI cassette by digesting the cloned isolate with AvrII (which cuts this isolate once only at nt position 4423), filling the ends with the Klenow fragment of DNA polymerase I and inserting a BamHI adapter which contained the first seven nucleotides (including the first ATG codon) of the putative L2 coding sequence. The entire ORF was transferred to a pATH vector for expression as a TrpE
fusion protein, and also to a baculovirus transfer vector (pVL-1393, provided by M. Summers, Texas A & M University, College Station, Texas, U.S.A.) via the common BamHI and XbaI sites. Expression in the BEV system was accomplished by standard methods (Summers & Smith, 1987) and resulted in the production of full-length non-fused proteins that were immunoreactive with rabbit polyclonal antisera directed against the HPV-6b and HPV-11 L 2 0 R F s (Fig. 1). These proteins, which were not present in uninfected or wild-type Autographa californica nuclear polyhedrosis virus (AcNPV)-infected cell lysates, migrate with apparent MrS of 76K (6L2) and 78K (llL2). DNA sequence analyses confirmed the presence of the HPV-6b and HPV-11 L2 start and stop codons (data not shown). Direct electrophoretic comparison of the BEV HPV-11 L 2 0 R F recombinant product with an immunologically cross-reactive 78K Mr protein present in HPV-11 viral particles indicates that the minor capsid protein is closely associated with this ORF (Fig. 2). These observations imply that RNA splicing is not required to produce a protein with an apparent Mr within this range. The appearance of an immunoreactive protein doublet in the usual location of the minor capsid protein has not been previously reported and may represent an artefact of the athymic mouse system. Although evidence derived from prokaryotic studies strongly suggests that glycosylation is not involved in anomalous L 2 0 R F product mobilities, the fact that Sf-9 cells are eukaroyotic presents the possibility that recombinant L2 proteins expressed in this system could
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XbaI (5885)
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Fig. 3. Western blot analysis of deglycosylation reactions. (a) Baculovirus-produced HIV gpl60 (MicroGeneSys) which was probed with an HIV antiserum at a dilution of I : 100. (b) Baculovirus-produced HPV-6b L 2 0 R F product probed with rabbit anti-6L2C antiserum at a dilution of 1:1000. Reactions in lanes 1 were not treated with Nglycanase. Reactions in lanes 2 were treated with N-glycanase as discussed in the text. Arrows indicate the positions of the reaction products.
be glycosylated. However, endoglycosidase treatment of BEV-expressed 6L2 (N-glycanase; Genzyme) had no effect on the mobility of the HPV-6b L2 product (Fig. 3) which suggests that N-glycosylation sites present in the primary sequence of this protein are not glycosylated in this system. The positive control used for this experiment, human immunodeficiency virus (HIV) glycoprotein gpl60, was also produced in the BEV system (MicroGeneSys) and underwent a large mobility change, which indicates that carbohydrate moeities added by Sf9 cell mechanisms can be efficiently removed with this enzyme. Expression of these L 2 0 R F s as pATH TrpE fusion proteins in Escherichia coli resulted in production of recombinant L2 fusion proteins, each with an apparent Mr of 115K, which is approximately 28K larger than the 87K Mr predicted from the fusion of these coding sequences to a 37K TrpE moiety (data not shown). In previous experiments (Strike et al., 1989) HPV-6b L2 sub-ORF fragments, also expressed as TrpE fusion proteins, exhibited similar anomalous migration patterns in SDS-PAGE systems, with the exception of the fragment containing the amino-terminal 100 amino acids of the ORF (6L2-N ; Fig. 4) which migrated at its predicted M r. These results were obtained under denaturing conditions from peptides produced in a prokaryo-
Fig. 4. Sub-ORF fragments of the HPV-6b and HPV-I 1 L 2 0 R F s used to produce rabbit polyclonal antisera. The indicated restriction sites were used to clone and express the L2 sub-ORF fragments as ]~galactosidase fusion proteins in E. coil (Strike et al., 1989) and, in the present study, llL2-C. Nucleotide coordinates of restriction endonuclease sites used are as indicated in parentheses and correspond to sequence data obtained from GenBank. 6L2-N refers to the aminoterminal 100 amino acid residues, 6L2-MID refers to the middle 230 amino acid residues and 6L2-C refers to the carboxy-terminal 129 amino acid residues of the HPV-6b L2 ORF. I IL2-C refers to the carboxy-terminal 126 amino acid residues of the HPV-I 1 L2 ORF.
tic expression system, which provides additional evidence that higher-order protein structure and/or posttranscriptional or post-translational processing steps are not required to explain the migration patterns of L2 products. In addition, these observations suggest that these anomalies are not derived from the primary structure of the'hig, hly conserved amino-terminal region of the L 2 0 R F ipr.oduct. The othergoal of this study was to define the locations of immunologically cross-reactive and type-specific epitopes in the HPV-6b and HPV-11 L 2 0 R F s . Rabbit polyclonal antisera were raised against three recombinant fusion proteins derived from fragments 6L2-N, 6L2MID and 6L2-C (Fig. 4) of the HPV-6b L 2 0 R F , as previously described (Strike et al., 1989). Preliminary experiments with full-length L2-TrpE fusion proteins indicated that the antiserum directed against the 6L2-C region did not cross-react with the HPV-11 L 2 0 R F product (data not shown). To investigate this observation further, the carboxy-terminal portion of the HPV-11 L 2 0 R F (llL2-C) was cloned in a similar manner to that used for the 6L2-C fragment. Briefly, a 450 bp PstI/XbaI fragment (HPV-11 nt coordinates 5435 to 5885) was cloned in a p E X vector (Stanley & Luzio, 1984) and expressed as a//-galactosidase fusion protein. The fusion protein was purified by SDS-PAGE, excised and emulsified with Freund's complete adjuvant and used to produce polyclonal antisera as previously described (Strike et al., 1989). Pre-immune sera from all animals used in these experiments were HPV L2
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(a) 1
(b) 2
1
(c) 2
1
(d) 2
1
2
(~78K) I~76K)
Fig. 5. Localization of cross-reactive and type-specific epitopes of the HPV-6b and HPV-11 L20RFs. Lanes 1 contain recombinant HPV-6b L2-infected cell lysates; lanes 2 contain recombinant HPV-11 L2infected cell lysates. Panels (a) to (d) were probed with rabbit polyclonal antisera directed against (a) the amino-terminal portion of the HPV-6b L20RF, (b) the middle portion of the HPV-6b L20RF, (c) the carboxy-terminal portion of the HPV-6b L 2 0 R F and (d) the carboxy-terminal portion of the HPV-11 L20RF.
seronegative, as j u d g e d by W e s t e r n blot l m m u n o a s s a y s ( d a t a not shown). Fig. 5 is a W e s t e r n blot w h i c h d e m o n s t r a t e s t h a t the r e c o m b i n a n t L 2 0 R F proteins c a r r y b o t h cross-reactive a n d type-specific epitopes. Polyclonal a n t i s e r a d i r e c t e d a g a i n s t the 6L2-N a n d 6L2M I D H P V - 6 b L 2 0 R F f r a g m e n t s cross-react w i t h the B E V HPV-11 L 2 0 R F p r o d u c t (Fig. 5 a a n d b). T h e a n t i s e r a raised a g a i n s t the c a r b o x y - t e r m i n a l p o r t i o n s o f the H P V - 6 b a n d HPV-11 L 2 0 R F s (6L2-C a n d 11L2-C) do not cross-react, however. This o b s e r v a t i o n suggests the presence o f one or m o r e type-specific e p i t o p e s in this region o f the H P V - 6 b a n d HPV-11 L 2 0 R F s a n d the a b s e n c e o f cross-reacting e p i t o p e s (Fig. 5c a n d d) even t h o u g h the regions used to p r o d u c e these a n t i s e r a are m o r e t h a n 75% i d e n t i c a l at the a m i n o a c i d level. Previous r e p o r t s b a s e d on studies o f P V - i n f e c t e d tissue s p e c i m e n s ( K o m l y et al., 1986; J i n et al., 1989) p r o v i d e d i m m u n o h i s t o c h e m i c a l e v i d e n c e t h a t type-specific epitopes are c o n t a i n e d w i t h i n the L2 p r o d u c t s o f H P V - l a a n d b o v i n e p a p i l l o m a v i r u s type 1 (BPV-1), respectively. O u r results p r o v i d e d i r e c t e v i d e n c e for the existence o f s i m i l a r type-specific e p i t o p e s w i t h i n the p r o t e i n c o d i n g sequences o f the L 2 0 R F s o f H P V - 6 b a n d H P V - 1 1 , two closely related genital H P V s . T h i s i n f o r m a t i o n m a y p r o v e to be useful in the d e v e l o p m e n t o f tests for the d i a g n o s i s o f genital H P V infections. W e c o n c l u d e t h a t the L 2 0 R F of HPV-6b, with a c o d i n g p o t e n t i a l o f 51 K , e n c o d e s a p r o t e i n w i t h an Mr o f a p p r o x i m a t e l y 76K a n d t h a t the L 2 0 R F o f H P V - I 1 , with a coding capacity of 49K, produces a protein of 78K Mr. O u r o b s e r v a t i o n s i n d i c a t e t h a t p o s t - t r a n s c r i p t i o n a l
a n d p o s t - t r a n s l a t i o n a l processing systems d o not a p p e a r to be n e c e s s a r y to p r o d u c e the unusual e l e c t r o p h o r e t i c m o b i l i t i e s o f L 2 0 R F p r o t e i n s in S D S - p o l y a c r y l a m i d e gel systems. I n a d d i t i o n , c o m p a r i s o n o f the b i o p h y s i c a l a n d a n t i g e n i c c h a r a c t e r i s t i c s o f r e c o m b i n a n t HPV-11 L2 p r o t e i n w i t h p r o t e i n s p r e s e n t in H P V - I 1 p a r t i c l e s i n d i c a t e s t h a t the m i n o r c a p s i d p r o t e i n o f HPV-11 is closely related to the HPV-11 L 2 0 R F product. W e h a v e also d e m o n s t r a t e d t h a t the H P V - 6 b a n d HPV-11 L2 O R F p r o d u c t s c o n t a i n b o t h cross-reactive a n d typespecific e p i t o p e s ; studies designed to further c h a r a c t e r ize a n d d e l i n e a t e these e p i t o p e s are in progress. Purified H P V - 6 b a n d HPV-11 L 2 0 R F p r o t e i n s p r o d u c e d in the B E V system will be used in o t h e r i m m u n o l o g i c a l studies. sf-9 cells, wild-type AcNPV strain E2 and several baculovirus transfer vectors were graciously provided by Max Summers, Texas A & M University, College Station, Texas, U.S.A. Our appreciation also goes to John J. Treanor for helpful discussions and critical review of the manuscript and to Carrie Harvey for expert technical assistance. This work was supported by AI-23418 and AI-27658 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.
References
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(Received 26 March 1990; Accepted 12 July 1990)
