and p40, to treatment with glycosidases was assessed .... Cell 46, 63-74. BALL, J. M., RAO, V. S. V., ROBEY, W. G., ISSEL, C. J. & MONTELARO,. R. C. (1988).
Journal of General Virology (1990), 71, 701-706.
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Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus Robin Steinman, 1 Jim Dombrowski, 1 Thomas O'Connor, 1 Ronald C. Montelaro, 2 Quentin Tonelli, 1 Karen Lawrence, 1 Cynthia Seymour, 1 Joel Goodness, 1 Neils C. Pedersen 3 and Philip R. Andersen 1. x I D E X X Corporation, 100 Fore Street, Portland, Maine 04101, 2 Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803 and 3 School of Veterinary Medicine, University of California, Davis, California 95616, U.S.A.
Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase H P L C and the aminoterminal sequences of p l 0 and p24 were determined.
Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 35[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of50K (Pr50°~). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosidase H and glycopeptidase F revealed that p130 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.
Introduction
infectious anaemia virus (EIAV) (Parekh et aL, 1980). Recently this group of viruses has been the focus of an intensive study to provide a better understanding of their pathogenicity and persistence (Haase, 1986). To understand better the nature of FIV and its relationship to other lentiviruses, as well as to participate in the development of a manageable laboratory animal model for HIV infection, we have undertaken a study of FIV structural proteins. The results presented in this paper identify the major internal proteins of the virus. The envelope proteins of the virus are identified as glycoproteins, which play a major role in the development of an immune response to viral infection. Our results also provide additional evidence to support the classification of FIV as a lentivirus.
Feline immunodeficiency virus (FIV) is a retrovirus that was originally isolated from cats which exhibited a syndrome similar to AIDS (Pedersen et al., 1987). More recently additional isolates have been identified in both the United Kingdom and Japan (Harbour et al., 1988; Ishida et al., 1988). The virus can be propagated in peripheral blood lymphocytes, or in the permanent feline T cell line LSA-1 (Yamamoto et al., 1986). Infection of these cells with FIV leads to the production of virus, formation of giant syncytia and cell death. The particular cellular tropism of this virus, the preferential utilization of magnesium as a divalent cation by the virus-associated D N A polymerase and the unique morphology of the virus all suggest that FIV should be subclassified as a lentivirus. Members of the lentivirus subgroup include the human immunodeficiency viruses (HIV) (Montagnier et al., 1984; Popovic et al., 1984), simian isolates (Benveniste et al., 1986; Kanki et al., 1986) and isolates from various ungulates, including visna virus (Narayan etal., 1977), caprine arthritis-encephalitis virus (CAEV) (Narayan et al., 1980; Dahlberg et al., 1981), bovine immunodeficiency virus (Gonda et al., 1987) and equine 0000-9137 © 1990 SGM
Methods Virus and cell culture. FIV was propagated in chronically infected Crandall feline kidney (CRFK) cells (Crandall & Despeaux, 1959). The virus was concentrated from tissue culture fluids by precipitation with polyethylene glycol (Bishop et al., 197I) and purified by density gradient centrifugation on glycerol gradients, as previously described (Montelaro et al., 1982).
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Sera. Feline antisera to FIV and feline syncytium-forming virus (FeSFV) were obtained from experimentally infected specific pathogen-free (SPF) cats. Antiserum to FIV was prepared in rabbits by immunizing the animals with detergent-disrupted, inactivated preparations of FIV. Goat anti-feline leukaemia virus (FeLV) serum was obtained from the BCB Repository (Microbiological Associates). Horse antisera to EIAV were kindly provided by Dr J. Pearson (Diagnostic Virology, Ames, Ia, U.S.A). Pooled human sera containing a high titre of HIV antibody, as well as rabbit antiserum prepared against HIV, were provided by Dr J. Carlson, University of California, Davis, Ca., U.S.A. Serum from goats that were naturally infected with CAEV was a gift from Dr N. East, University of California, Davis; Dr P. Marx, California Primate Research Center, Davis, provided serum from a rhesus monkey persistently infected with the Davis isolate of simian immunodeficiency virus (SIV-sooty mangabey) as well as rabbit antiserum prepared against this virus. Gel electrophoresis. SDS-PAGE was performed as described by Laemmli (1970). Proteins were visualized by staining with Coomassie blue R-250. ELISA. FIV microassay plates were prepared by coating microtitre wells with an inactivated, disrupted preparation of FIV antigen. Assay kits for determination of anti-FIV antibody were used (O'Connor et al., 1989). Radiolabelling of cells. Confluent cultures of CRFK cells productively infected with FIV, or uninfected control cells, in 75 cmz flasks were incubated for 30 min at 37 °C in methionine- and cysteine-free Dulbecco's modified Eagle's medium. Cell cultures were then incubated for 4 h in 8 ml of the same medium containing 100 ~tCi/ml [35S]methionine/cysteine (sp. act. 1200 Ci/mmol, New England Nuclear). The radioactive tissue culture fluids were removed and the cells were lysed with 5 ml of 10 mM-sodium phosphate pH 7.5, 100 mMNaCI, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mM-PMSF and 100 kallikrein-inactivating units of aprotinin per ml (Sigma). Before use the cell lysates were clarified by centrifugation at 100000 g for 30 min at 4 °C and the pellets discarded. Immunopreeipitation of labelled cell extracts. Samples of labelled cell lysates (0.1 ml) and 5 ~tl of the tested serum were mixed in microcentrifuge tubes and incubated overnight at 4 °C. Next 0.2 ml of a 5% suspension of Protein A-Sepharose CL-4B beads (Pharmacia) in 10 mM-phosphate buffer pH 7.5, containing 100 mM-NaCI, 1% Triton X-100 and 0.1% SDS, was added to each tube and mixed continuously for 30 min at 4 °C. The antigen antibody complexes bound to the Protein A-Sepharose beads were collected by centrifugation (2 min at 20000 g) and washed three times with lysis buffer. The final pellet was resuspended in 25 ~tl SDS-PAGE loading buffer and heated for 3 min at 100 °C. Sepharose beads were then removed by centrifugation and the supernatant was used in SDS PAGE. Gels were processed for fluorography using Enlightening (DuPont) and exposed at - 7 0 °C to Kodak XAR-5 film. Western immunoblot. The Western blot procedure (Towbin et al., 1979) was utilized with the following modifications. Gradient-purified FIV was disrupted and proteins (approximately 2 ~tg per strip) were resolved on a 10% polyacrylamide gel and then transferred to a sheet of nitrocellulose. The nitrocellulose sheet was then blocked with 30% calf serum, 1% bovine serum albumin, 0-05% Tween-20 in Dulbecco's phosphate-buffered saline (PBS). The nitrocellulose sheets were cut into 0-5 cm strips and incubated with a 1 : 100 dilution of the serum sample in blocking buffer for 2 h at room temperature. Strips were repeatedly rinsed with wash buffer (0.05% Tween-20 in PBS) and then incubated for 1 h at room temperature with anti-feline, horseradish peroxidase-labelled conjugate. The strips were repeatedly rinsed with wash buffer again and then incubated with the precipitating substrate,
4-chloro-l-naphthol, for 10 min. Strips were partially dried and the results were interpreted immediately.
HPLC and gas phase sequencing. The procedures used to isolate and determine the N-terminal amino acid sequence of isolated FIV structural proteins were those described by Ball et al. (1988). Monoelonal antibody production. BALB/c mice (Jackson Laboratories) were immunized with 50 gg per mouse of Triton X-100-disrupted, inactivated FIV mixed with an equal volume of complete Bactoadjuvant H37Ra (Difco). Mice were boosted 2 weeks later with the same antigen preparation at a dose of 100 I-tgper mouse in incomplete adjuvant. Fusions were performed 3 days after the last injection with mouse myeloma line P3X63-Ag8.653 (K6hler & Milstein, 1975; Kearney et al., 1979). Hybridomas were selected in HAT medium and screened in FIV-coated microwell plates.
Results Virus-associated proteins and their reactivity on Western immunoblots P r o t e i n s a s s o c i a t e d w i t h p u r i f i e d F I V w e r e a n a l y s e d by S D S - P A G E a n d c o m p a r e d w i t h p r o t e i n s i s o l a t e d in an i d e n t i c a l m a n n e r f r o m the s p e n t c u l t u r e m e d i u m o f u n i n f e c t e d C R F K cells. A n a l y s i s o f s t a i n e d gels r e v e a l e d t h r e e m a j o r p r o t e i n s (p24, p15 a n d p l 0 ) r e a d i l y identifia b l e in p u r i f i e d F I V p r e p a r a t i o n s (Fig. l a). W e s t e r n i m m u n o b l o t t i n g o f v i r u s - a s s o c i a t e d p r o t e i n s , u s i n g sera f r o m cats e x p e r i m e n t a l l y i n f e c t e d w i t h F I V , r e v e a l e d s t r o n g r e a c t i v i t y w i t h p24 a n d p15 a n d a lesser d e g r e e o f reactivity with pl0 and proteins of 32K, 40K, 47K and 65K. T h i s p a t t e r n c o u l d be r e p e a t e d l y d e m o n s t r a t e d w i t h serial s e r u m s a m p l e s o b t a i n e d f r o m e x p e r i m e n t a l l y i n f e c t e d cats. S e r u m f r o m cats e x p o s e d to F e L V or F e S F V f a i l e d to r e c o g n i z e t h e p r o t e i n s i d e n t i f i e d by sera f r o m F I V - i n f e c t e d cats ( O ' C o n n o r et al., 1989).
Analysis o f F I V core structural proteins I n d i v i d u a l p r o t e i n s f r o m i s o l a t e d v i r u s w e r e r e s o l v e d by r e v e r s e p h a s e H P L C . T h r e e m a j o r p r o t e i n p e a k s (A, B a n d C) w e r e o b s e r v e d (Fig. l b ) . O n S D S - P A G E gels p e a k s A , B a n d C e x h i b i t e d M r v a l u e s o f 10K, 2 4 K a n d 15K, r e s p e c t i v e l y . M o n o c l o n a l a n t i b o d i e s to F I V p24 a n d p 15 also r e a c t e d w i t h t h e i s o l a t e d p r o t e i n s o f p e a k s B a n d C by W e s t e r n b l o t t i n g ( d a t a n o t shown). T h e N t e r m i n a l a m i n o a c i d s e q u e n c e s for p24 a n d p l 0 w e r e d e t e r m i n e d (Fig. 1 c). T h e p r o t e i n p 15 w a s n o t s e q u e n c e d because phenylthiohydantoin derivatives were not detected. A s i g n i f i c a n t d e g r e e o f h o m o l o g y w a s o b s e r v e d bet w e e n t h e N - t e r m i n a l a m i n o a c i d s o f F I V p24 a n d t h o s e o f t h e m a j o r c o r e p r o t e i n s o f o t h e r l e n t i v i r u s isolates.
Identification o f F I V proteins in productively infected cells T o d e t e c t t h e v i r a l e n v e l o p e p r o t e i n s m o r e r e a d i l y , as well as t h e gag p r e c u r s o r p r o t e i n s o f F I V , cell e x t r a c t s
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labelled with [35 S]methionine/cysteine were examined by radioimmunoprecipitation assay (RIPA)-PAGE. Sera from experimentally infected cats recognized
703
proteins of Mr 130K, 50K, 40K, 36K, 22K and 15K in FIV-infected CRFK ceils or LSA-1 cells. Examination of serial bleeds from SPF-infected cats showed that most cats mount an immune response to p130, p50, p40 and p22 (Fig. 2a), although quantitative and qualitative differences in the immune response occur between the cats. This variability could be demonstrated with FIVinfected feline sera by the R I P A - P A G E assay (Fig. 2b). Immunoprecipitation of p 130 has been observed in every FIV antibody-positive serum, although in some samples this ability is greatly diminished. Similarly, the majority of sera immunoprecipitated p50. Whereas p50 and p130 are readily identifiable in infected cells using immune sera from FIV-infected cats, p50 is also recognized by monoclonal antibodies reactive with p15 and p24, which suggests that p50 is a precursor protein for these two viral proteins. The p130 is detectable in sera from all infected cats, it migrates as a broad band and is not observed in uninfected cell lysates. The p40 protein is identifiable both on Western blots and RIPA-PAGE, in both instances appearing as a very diffuse band. Antibodies to p40 were occasionally the earliest seen following viral infection and sometimes accompanied antibodies against p130, prior to the appearance of antibodies to core structural peptides (O'Connor et al., 1989). The similarity of these observations to the behaviour of gpl20 and gp41 of HIV (Sarngadharan et al., 1984; Veronese et al., 1985) led us to ascertain whether p130 and p40 were glycoproteins. The apparent Mr of p 130 was altered when labelling of FIV-infected cells with [35S]methionine/cysteine was carried out in the presence of tunicamycin (Fig. 2c), an inhibitor of N-glycosylation (Takatsuki et al., 1975). This caused a shift in the migration of p130 to a protein of 75K. The migration of p47 was unaffected by labelling under these conditions. The sensitivity of the presumptive glycoproteins, p 130 and p40, to treatment with glycosidases was assessed using endoglycosidase H (endo-/~-N-acetylglucosaminidase H) and glycopeptidase F (glycopeptide-N-glycosidase). Endoglycosidase H will cleave N-linked and some hybrid oligosaccharides from glycoproteins, but not complex oligosaccharides. Glycopeptidase F is capable of degrading biantennary hybrid and complex oligosaccharides (Tarentino et al., 1985). Major FIV proteins were visualized following immunoprecipitation of [35S]methionine/cysteine-labelled cell lysates and included p130, p50 and p40 (Fig. 2d). Treatment with endoglycosidase H for either 1 h, or overnight resulted in the disappearance of the p130 and the appearance of a protein with an Mr of 75K. The p50 band was unaffected by this treatment, which indicates that proteolytic activity was not responsible for the observed Mr change seen with p130. The migration of the p40 band through
704
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18 26 Fig. 2. (a) Immunoprecipitationof metabolically labelled FIV proteins by antibodies from experimentally infected cats. A serum sample was obtained prior to infection with FIV (lane 1) and at weekly intervals followinginfection with the virus (lanes 2 to 5). (b) Immunoprecipitation of metabolically labelled FIV proteins by feline sera previously identified as positive by an ELISA for FIV antibodies. (c) The effecton the migration of p130 by metabolicallylabelling cells in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of tunicamycin (1 p.g/ml). Lanes 1 and 3 show immunoprecipitations performed using normal cat sera; lanes 2 and 4 represent immunoprecipitations performedusing FIV-positiveantiserum. (d) Effectof endoglycosidaseH and glycopeptidaseF on p 130and p40. Lane 1 represents immunoprecipitated proteins from FIV-infectedcells using antibody-positivefeline serum. Lanes 2 and 3 are the same material followingtreatment with endoglycosidaseH for 1 h and 16 h, respectively, and lane 4 followinga 16 h treatment with glycopeptidase F. M~ are shown in the left-hand columns ( x 10-3).
the gel was slightly increased by endoglycosidase H treatment. Overnight treatment with glycopeptidase F produced the same alteration in p 130 and no alteration in p50, but this treatment did result in the disappearance of the p40 band and the appearance of a 22K protein. Immunological relatedness o f F I V to other lentiviruses Serological analyses have revealed a conservation of shared epitopes among lentivirus isolates (Stowring et al., 1979; Yaniv et al., 1986). We initially examined the possible relationship between FIV and other lentivir, uses, using R I P A - P A G E with [35S]methionine/cysteinelabelled, FIV-infected C R F K cells (Fig. 3a). Rabbit antiserum against E I A V p26 reacted strongly, thus recognizing the same series of related peptides (p50, p36 and p22) recognized by monoclonal antibodies directed against FIV p26. Similarly, rabbit antisera t o FIV: recognized the major gag precursor of E I A V p54 °( d a t a not shown). Other sera which were known to be positive for antibodies to HIV and SIV failed to react with FIV proteins in this assay. Western blot analysis revealed cross-reactivity of FIV with rabbit anti-EIAV p26,
rabbit anti-SIV and rabbit anti-HIV antibodies (Fig. 3b). Reactivity of FIV p130 and p50 was observed with serum obtained from horses infected with EIAV (Fig. 3 c). The reactivity of FIV p 130 was surprising, since the envelope sequences of lentiviruses are highly divergent (Montelaro et al., 1984; Payne et al., 1984; Alizon et al., 1986; Hahn et al., 1986). To determine whether the observed cross-reactivity of horse anti-EIAV to FIV p130 was due to oligosaccharide determinants [3SS]methionine/cysteine-labelled, FIV-infected C R F K cell lysates were pretreated overnight with endoglycosidase H. Immunoprecipitations performed with the deglycosylated cell extracts revealed the cross-reactivity observed for p 130 was a result of the oligosaccharides associated with this glycoprotein.
Discussion In the present study we have identified the major structural proteins of FIV. Both the biochemical nature and the immunological cross-reactivity of these proteins
705
Characterization o f F I V structural proteins
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strongly support the classification o f F I V as a m e m b e r o f the lentivirus subfamily o f retroviruses. The major core proteins of F I V (p24, p15 and pl0) closely correspond to the major core proteins of other lentiviruses (Parekh et al., 1980). The immunological cross-reactivity demonstrated for p24 and the conserved a m i n o acid sequences demonstrated between F I V p24 and the corresponding proteins of other lentiviruses suggest that the c o m m o n antigen is a major core protein. The inability to sequence p 15 suggested fatty acid acetylation of F I V p 15 similar to that seen for the pl5-1ike proteins isolated from other lentiviruses (Ball et al., 1988). The data imply that p47 is the putative precursor protein of these major core proteins. The glycoproteins of FIV, g p l 3 0 and gp40 were identified using standard biochemical techniques. The relative resistance o f gp40 to endoglycosidase H and its sensitivity to glycopeptidase F suggest there are distinct differences in the post-translational modification of gp40 and gpl30. Further work is needed to clarify the p r e c u r s o r - p r o d u c t relationship for the e n v e l o p e glycoproteins. The major glycoprotein gp 130 is a d o m i n a n t antigen in the immune response to F I V infection of cats. It was surprising to observe immunoreactivity to this protein in sera from horses infected with E I A V . This reactivity was shown to be dependent on the glycosylation o f p130, which implied i m m u n e cross-reactivity was due to sugar residues, or that glycosylation conferred a structure on
g p l 3 0 that would cross-react with antibodies from E I A V - i n f e c t e d horses (Alexander & Elder, 1984; Pinter & H o n n e n , 1988). Evaluation of further relatedness will be possible as gene sequence information on F I V becomes available for c o m p a r a t i v e analysis with E I A V and other lentiviruses. The structural proteins o f F I V clearly support its classification as a lentivirus. Additional studies elucidating the genomic organization and control o f gene expression of this virus will aid the development of F I V as a small animal model for H I V infection and in understanding F I V pathogenesis in cats.
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(Received 21 June 1989; Accepted 17 October 1989)
