Journal of Virological Methods, 29 (1990) 13-22
13
Etsevier VDRMET 01031
Specific enzyme-linked immunosorbent assay for the detection of antibodies to the human spumavirus Christoph Mahnke, Martin L&helt, Helmut Bannert and Rolf M. F%igel institut f@r~rusforschu~g,
Deutsches Krebsforschungszentruna,
(Accepted
Heidelberg,
F.R.G.
20 March 1990)
Summary Recombinant plasmid clones were constructed harbouring the central domains of the outer membrane protein and the transmembrane protein of the env gene of human spumaretrovirus (HSRV), The corresponding fusion proteins were expressed in E. cdi, purified and used sub~quendy to produce antibodies against the HSRV env proteins in rabbits. The a~~enticity of the bacte~~ly produced domain of the HSRV env proteins was shown by radioimmunoprecipitation of the viral env glycoprotein from HSRV-infected human cells with rabbit antibodies raised against the recombinant antigens. The recombinant viral antigens were used to establish a sensitive and spumavirus-specific enzyme-linked immunosorbent assay (ELISA). This anti HSRV antibody ELISA makes it possible to screen human sera for the presence of spumavirus infections. Foamy virus; ELISA screening test, env; Recombinant antigen
Retroviruses have been implicated in the etiology of a number of diseases of man. Such viruses include the human immunodeficiency viruses HIV-l and HIV-2 that have been shown to be the etiological agents of the acquired immunodeficiency syn~ome, AIDS (Be-S~oussi et al., 1983; Popovic et al., 1984; Clavel et al., 1986). In addition, the human T-cell lymphotropic viruses HTLV-I and HTLV-II are considered to be responsible for adult T-cell leukemia and, furthermore, for a Correspondence
to: Rolf M. Fliigel, projektgruppe Humane Retroviren, Institut fiir Virusforschung, DKFZ, Im Neuenheimer Feld 280, 6900 Heidelberg, F.R.G. 01~0934/90/?§03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
14
rare form of myopa~y, tropic spastic paresis (U~hiy~a et al., 1977; Poiesz et al., 1980; Gessain et al., 1985). The corresponding virus-specific ELISAs have proved essential for establishing the prevalence of viral infections in man and in correlating the presence of a given virus with clinically defined groups of patients. A third subfamily or group of retroviruses consists of spumaviruses also called foamy viruses (Weiss et al., 1982; Maurer and Fhigel, 1988; Lewe and Fliigel, 1989). Previously, we have cloned molecularly and characterized extensively the genome of one member of this group of viruses, the human spumaretrovirus, HSRV (Fliigel et al., 1987; Maurer et al., 1988; Rethwilm et al., 1987) that had originally been isolated from a patient with a nasopharyngeal carcinoma (Achong et al., 1972). Since this is a virus in search of a disease (Weiss, 1988), so to speak, it will be necessary to screen a relatively large number of human sera. HSRV-specific ELBA was, therefore, developed.
Materials and Methods Construction of recombinant plasmids expressing HSRV env gene regions Plasmid clones that harbor foamy viral inserts including the env gene have been described previously (Fliigel et al., 1987; Maurer et al., 1988; Rethwilm et al., 1987). To construct a recombinant clone that contained the central region of the env protein, pHSRV-HH-C55 (F&gel et al., 1987) was digested with PstI and XbaI and the resultant DNA fragment was inserted into the co~esponding sites of the expression vector pEx34 with T4 DNA ligase (Strebel et al., 1986). (The only difference between vector pEx34 and pEx31 is that the PstI site in the ampicillin resistance gene was deleted). The resulting recombinant plasmid, pEx34PX was characterized by restriction enzyme analysis and nucleotide sequencing of the vector/insert border. The results showed uneqivocally that the viral insert DNA had the expected size of 825 bp and was oriented in the proper direction for expression. Moreover, the nucleotide sequence analysis proved that the HSRV env domain was in reading frame ‘a’, the first of the three possible frames, so that it was fused to the MS2 polymerase protein (Strebel et al., 1986). Another recombinant plasmid was constructed that contained the central part of the coding sequences of the tr~smembr~e protein of the HSRV env region. Plasmid clone pHSRV-HH-C55 was digested with NIzeI and the ends filled up with four dNTPs by the Klenow DNA polymerase thereby creating blunt ends. Subsequently, the plasmid was digested with XbaI. The plasmid vector pEx34 was separately digested with Hindi and the Hind111 site filled up as described above to generate blunt ends, followed by a digestion with XbaI. Subsequently purified vector and insert DNAs were ligated with T4 DNA ligase. The resulting recombinant, pEx34XN, was characterized as described above and shown to ha&our a viral insert of 801 bp as expected.
15
Expression of recombinant antigen in E. coli and purification offusion protein envpx Expression and purification of fusion proteins was carried out according to Strebel et al. (1986) with the following modification. To visualize the protein bands after preparative SDS-polyacrylamide gel electrophoresis, precipitation of protein with 4 M sodium acetate was used instead of staining with Coomassie blue. After eluting the fusion proteins from the gel, the extracted proteins were rerun on 18% polyacrylamide gels and eluted from the gels through a Centricon microconcentrator (Amicon Co.). The purity of the recombinant antigens was finally reevaluated on analytical gels. Production of antisera against envpx and envxn Rabbits were initially immunized with about 100 pg of the purified fusion proteins suspended in Freund’s complete adjuvant and further stimulated by immunizing five times at intervals of 2 to 4 weeks with the same amount of recombinant antigen in Freund’s incomplete adjuvant. Sera were titrated by ELISA and immunoblotting with the purified recombinant antigen that was used for immunization. ELISA test for spumaviral env antibodies The assays were performed according to Voller et al. (1976) but omitting the fixation with glutaraldehyde. Recombinant envpx antigen (0.05-0.1 &well) were pipetted onto 96-well microtiter plates (Flow Labs. and Greiner Co.). After incubation at room temperature for 1 h the wells were washed three times with 0.15 ml of 0.1% Tween 20 in PBS. After drying, 100 ~1 of 3% bovine serum albumin in 0.1% Tween/PBS solution were added to each well followed by an incubation at 4°C overnight. Subsequently, the plates were dried and washed as before and test serum samples or rabbit antibodies against envpx were added at increasing dilutions in 0.1% tieen 2O/PBS. The microtiterplates were tightly sealed and incubated at 37°C for 1 h. After decanting and washing with 0.1% Tween 2O/PBS, the plates were dried and subsequently 50 ,~l protein A-peroxidase solution (1 mg/ml) diluted 1:250 were added in PBS. Thereafter the microtiterplates were sealed in plastic bags and incubated at 37°C for 60 min. After washing and drying 100 ~1 of color solution were added to each well. To prepare the color solution 28 mg of o-phenylenediamine dihydrochloride (Sigma Co., Deisenhofen) was dissolved in 10 ml of 100 mM citric acid, pH 5.0; 5 ~1 30% hydrogen peroxide were added shortly before use. After an incubation for 15 min at 25°C the color reaction was stopped by adding 100 ~1 2 M sulphuric acid Optical densities were determined by an automated ELISA reader (Biotec Model EL310). As negative controls for the ELISA, human sera from symptom-free individuals were used. Additionally, human sera that reacted with the envpx antigen under the assay conditions used gave negative results when the bacterially expressed fusion protein was omitted. Diluted rabbit hyperimmune sera against the HSRV env fusion proteins served as positive controls. Routinely, test sera from individuals were diluted 1: 150.
16
Labeling and immunoprecipitation of viral proteins in HSRV-infected human embryonic lung (HEL) cells
Monolayers of HEL cells grown in 25cm* tissue culture flasks were infected as described previously (Fltigel et al., 1987) and washed with medium free of methionine or cysteine. L-[35S]methionine (spec. activity 1129 Ci/mmol, NEN, Dreieich, F.R.G.) or cysteine (or both) were added 1 h after infection to a final concentration of 10 &i/ml each. Labeling was stopped by lysing cells eight days post-infection in 0.5 ml of RIPA-buffer (150 mM NaCl, 10 mM sodium phosphate, pH 7.5, 1% Nonidet P-40, 0.5% sodium desoxycholate, and 0.1% SDS). To avoid protein degradation, 10 ,@ml aprotinin were added. Extracts were incubated for 10 min at 65°C to denature virions. To immunoprecipitate viral proteins, the method of Strebel et al. was used (1986). Briefly, a suspension of Staphylococcus protein A (Sigma Co.) was incubated with antiserum for 1 h at room temperature in RIPA-buffer (200 ~1). After washing to remove unbound antibodies, 100 ,ul of the radioactive viral antigen was incubated in 500 ~1 for 45 min at 25°C. Unbound material was removed by washing with RIPA-buffer. Immunoprecipitated proteins were treated by heating to 95°C for 5 min in an equal volume of sample buffer and detected by fluorography after SDS-polyacrylamide gel electrophoresis.
Results and Discussion Construction of recombinant plasmids expressing and expression of fusion proteins in E. coli
HSRV env domains
Since in natural retroviral infections antibodies are produced predominantly against the env and gag proteins, defined regions of the HSRV env gene were chosen to construct recombinant clones. The recombinant protein envpx was derived from the central domain of the HSRV env gene products as shown in Fig. 1. The region of the HSRV env gene that encompassed 815 bp was inserted into the PstI and XbaI sites of the expression vector pEX34 (Strebel et al., 1986). One of the resulting recombinant clones (pExPX) was established and characterized by restriction enzyme analysis. PstI-XbaI double-digestion resulted in DNA fragments of 3.3 kbp (vector) and 815 bp (viral insert) as expected. To ascertain that the reading frame of the HSRV env protein was in the same direction and correctly linked to the vector encoded MS2 polymerase protein, the vector/insert boundary of the clone pExPX was sequenced. The nucleotide sequence revealed that the viral insert was indeed in the proper reading frame fused to the MS2 polymerase peptide (data not shown). The recombinant plasmid pExPX was expressed as a fusion protein consisting of 98 amino acid residues that were derived from the MS2 phage polymerase (Strebel et al., 1986) plus 273 amino acids of the central domain of the HSRV env protein. The two combined domains would lead to a molecular mass of about 41 kDa. To determine the size of the fusion peptide, the envpx protein was separated from soluble bacterial proteins by two cycles of differential centrifugation and precipitation followed by polyacrylamide gel electrophoresis under denaturing
17
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Hind III
t
Fig. 1. Outline of strategy for molecular cloning of recombinant plasmid pEx34envPX. The arrow marks the position of the putative proteolytic cleavage site of the HSRV env precursor protein. For further details see &rebel et al. (1986).
conditions. Fig. 2A presents the migration pattern of the envpx protein (lane 7, marked by arrow) together with analyses of E. coli proteins without the vector pEx34 or transfected in alternative reading frames as controls. It is evident from Fig. 2A that only E. coli cells transfected with pEx34a (reading frame ‘a’ is the first of the three possible frames) expressed, an envpx fusion protein of molecular mass of approximately 42 kDa in agreement with the expected value of 41 kDa calculated above. A second recombinant plasmid that contained 267 amino acid residues of the HSRV transmembrane protein (‘IMP) as insert was separately constructed and established. Fig. 3 outlines the strategy for cloning the central part of the TMP of HSRV encompassing 267 residues out of the total 417. The recombinant plasmid, pEx34XN was characterized by restriction enzyme analysis and nucleotide sequencing of the vector/insert boundary, It was found that the HSRV env sequence was in the proper direction fused to the phage MS2 polymerase reading frame (data not shown). The resulting fusion protein, envxn, was expressed in E. culi and subsequently purified and used to raise antibodies in rabbits as described above for the recombinant antigen envpx. Characterization
of the fusion proteins
The recombinant fusion proteins were mainly found in the particulate fraction of the bacterial cell extracts and only partially soluble in 7 M urea. The E. coli
18
MLMl
2
34
56
76HM
kd
-66 -43 -25 -18 -15
Fig. 2. (A) SDS-polyactylamide gel electrophoresis of viral fusion proteins expressed in E. coli W537 after subsequent staining with Coomassie blue. Lanes were loaded with cell extracts as follows: E. co/i W537 only (lane I), E. coli W537 after transformation with the vector pEX34a but without any spumavira1 insert (lane Z), E. cooli W537 cells transformed with pEX34 in different reading frames that harbored a 2 kbp viral DNA fragment (lanes 3 to 6), E. coli W537 cells after ~~sfo~ation with
19
HSRV
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Fig. 3. Schematic outline of the strategy for the molecular cloning of recombinant plasmid pEx34envXN. The arrow marks the site of the putative proteolytic cleavage of the env precursor glycoprotein into the outer membrane and transmembrane proteins.
proteins that copurified with the HSRV env protein were eliminated by repeated preparative SDS-polyacrylamide gel electrophoresis (Fig. 2B). The HSRV-specific envxn fusion protein was eluted from the gel and used to raise antibodies in rabbits. To demonstrate that rabbit antibodies against the recombinant antigen envpx reacted specifically with native HSRV env proteins, total extracts from HSRV-infected HEL cells that had been labeled with radioactive methionine were immunoprecipitated with S. aureu~ protein A-bound anti-envpx antibodies. The result of the subsequent electrophoretic analysis on polyacrylamide gels under denaturing conditions is shown in Fig. 5. The appearance of an env glycoprotein band of about 135 kDa clearly documents that the rabbit antiserum directed against the recombinant viral protein envpx was immunoreactive with the authentic HSRV env glycoprotein of the expected size. the vector pEX34PX that contained the spumaviral env insert in the correct reading frame ‘a’ (lane 7). the alternative reading frame ‘c’ with the same viral envpx insert was not expressed (lane 8). M, LM, and HM are marker proteins as indicated on the right hand side in kDa. (B) SDS-polyacrylamide gel electrophoresis of recombinant proteins and purhlcation of meombinant antigen envpx by repeated runs of SDS-polyacrylamide gel electmphomsis. Conditions were as described under Fig. 2A. The lanes were loaded as follows: E. coli W537 only (lane 1). E. coli W ‘537 after transformation with pEx34a, pEx34b, and pEx34c without any viral insert (lanes 2.4, and 6)and with the HSRV envpx insert (lanes 3,5 and 7). The reextracted recombinant envpx after repeated rerun (lanes 8 and 9). As control for the precise mobility of the envpx fusion protein lane 10 was loaded with E. coli 537 extracts transformed with pExPX as in lane 3. M. low and high marker proteins.
20
10-3
10-L
Antlbody Oihtlon
10-5
Fig. 4. Reactivity of the purified viral antigen envpx (0.2 gg) with antibodies raised in rabbits. The values found with preimmune sera from rabbits were below the cut-off threshold.
12345M
-200
1102 -92
-09
-40 -30 Fig. 5. Radioimmuno~ipi~ti~ of 35S-Iabeled proteins from HSRV-infected (lanes 4 and 5) and uninf~t~ HEL cells (lanes 1 and 2). Lane M shows molecular weight markers in kDa. The arrow in lane 4 marks HSRV em glycoprotein of approximately 135 kDa.
Use of HSRV env recombinant protein for EL.ISA The reactivity of the purified recombinant viral antigen was tested by titrating it against the rabbit envpx antibodies. Fig. 4 presents the optical densities determined for different antibody concentrations. ELISA analyses were performed to select antigen concentrations that would yield the highest possible sensitivity with the lowest background. Diluted human sera that reacted under the assay conditions used were tested in wells of microtiterplates to which different concentrations of either envpx antigen or envxn antigen were absorbed. It was found that the ELISA worked best at an antigen concentration of 0.05 to 1.O fig/well. Different dilutions of human sera were reacted with either the envpx or the envxn antigens. At dilutions ranging from 1:50 to 1: 10 000 most human sera had optical densities that were negligible or below average values. The majority of human sera collected from apparently symptom-free patients or from individuals with completely different diseases therefore do not react as would be expected. However, some human sera had optical densities that were consistently greater than the average values at those same dilutions. This may be due to the fact that selection on a symptom-free or random basis is a relatively poor method for excluding virus-infected subjects. In conclusion, an ELISA system was established based on env protein sequences of HSRV that is specific for human foamy virus. This ELISA will permit large-scale screening of human sera for the presence of HSRV antibodies.
Acknowledgements We thank Jeff Luande, Tanzania Tumor Center for providing sera and for his hospitality; Kunitada Shimotohno, National Cancer Research Institute, Tokyo for advice, helpful discussions and hospitality; and Harald zur Hausen, German Cancer Research Institute for support. The experiments described here were financed by a grant from the Commission of the European Community (TS2*0186-D,AM). We thank Jennifer Reed for reading the manuscript critically.
References Achong, G., Mansell, P.W.A., Epstein, M.A. and Clifford, P. (1971) An unusual virus in cultures from a human nasopharyngeal carcinoma. J. Natl. Cancer Inst. 42, 299-307. Barre-Sinoussi, F., Cherman, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., AxlerBlin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. (1983) Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220, 868-87 1. Clavel, F., Guetard, D., Brun-Vezinet, F., Chamaret, S., Rey, M.A., Santos-Fermira, M.O., Laurent, A.G., Dauguet, C., Katlama, C., Rouzioux, C., Klatzmann, D., Champalimaud, J.L. and Montagnier, L. (1986) Isolation of a new human retmvirus from a West African with AIDS. Science, 233,343-346. Flilgel, R.M., Rethwilm, A., Maurer, B. and Darai, G. (1987) Nucleotide sequence of the env gene and its flanking regions of the human spumaretrovirus reveals two novel genes. Eur. Mol. Biol. Organ. J. 6, 2077-2084. Gessain, A., Barin, F., Vemant, J.C., Gout, 0.. Maurs, L., Calender, A. and de-The, G. (1985) Antibodies
22
to human T-cell lympho~pic virus type-1 in patients with tropical spastic paresis. Lancet ii, 407-409. Kyshe-Andersen, J. (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. B&hem. Biophys. Methods 10, 203-209. Lewe, G. and Fliigel, R.M. (1990) Comparative analysis of the retroviral pal and env protein sequences reveal different evolutionary trees. Virus Genes 3, 195-204. Maurer, B. and Fltlgel, R.M. (1988) Genomic Organization of the human spumaretrovirus and its relatedness to AIDS and other retroviruses. AIDS research and human mtrovirnses 4, 467473. Mauter, B., Bannert, H., Darai, G. and Flilgel, R.M. (1988) Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. J. Vi&. 62, 1590-1597. Poiesz, B.J., Ruscetti, F.W., Gazdar, A.F., Bunn, P.A., Minna, J.D. and Gallo, R.C. (1980) Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with a cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA 77, 7415-7419. Popovic, M., Samgadhamn, M.G., Read, E. and Gallo, R.C. (1984) Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224, 497-500. Rethwilm, A., Darai, G., Rosen, A., Maurer, B. and F&gel, R.M. (1987) Molecular cloning of the genome of the human spumaretrovirus. Gene 59, 19-28. Strebel, K., Beck, E., Strohmaier, K. and Schaller, II. (1986) Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J. Viral. 57, 983-991. Towbin, H., Staehelin, T. and Gordon, J. (1979) El~tropho~tic transfer of proteins from ~lya~l~ide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K. and Uchino, H. (1977) Adult T-Cell leukemia: clinical and hematological features of 16 cases. Blood 50, 481492. Voller, A., Bidwell, D.E. and Bartlett, A. (1976) Enzymes in immunoassays in diagnostic medicine. Theory and practice. Bull. WHO 53, 55-65. Weiss, R.A., Teich, N., Varmus, H. and Coffin, J. (1982) RNA tumor viruses. Ch. 2, 2nd edit., Cold Spring Harbor Laboratory, New York, pp. 157-163. Weiss, R.A. (1988) A virus in search of a disease. Nature (London) 333, 4971198.
13
Etsevier VDRMET 01031
Specific enzyme-linked immunosorbent assay for the detection of antibodies to the human spumavirus Christoph Mahnke, Martin L&helt, Helmut Bannert and Rolf M. F%igel institut f@r~rusforschu~g,
Deutsches Krebsforschungszentruna,
(Accepted
Heidelberg,
F.R.G.
20 March 1990)
Summary Recombinant plasmid clones were constructed harbouring the central domains of the outer membrane protein and the transmembrane protein of the env gene of human spumaretrovirus (HSRV), The corresponding fusion proteins were expressed in E. cdi, purified and used sub~quendy to produce antibodies against the HSRV env proteins in rabbits. The a~~enticity of the bacte~~ly produced domain of the HSRV env proteins was shown by radioimmunoprecipitation of the viral env glycoprotein from HSRV-infected human cells with rabbit antibodies raised against the recombinant antigens. The recombinant viral antigens were used to establish a sensitive and spumavirus-specific enzyme-linked immunosorbent assay (ELISA). This anti HSRV antibody ELISA makes it possible to screen human sera for the presence of spumavirus infections. Foamy virus; ELISA screening test, env; Recombinant antigen
Retroviruses have been implicated in the etiology of a number of diseases of man. Such viruses include the human immunodeficiency viruses HIV-l and HIV-2 that have been shown to be the etiological agents of the acquired immunodeficiency syn~ome, AIDS (Be-S~oussi et al., 1983; Popovic et al., 1984; Clavel et al., 1986). In addition, the human T-cell lymphotropic viruses HTLV-I and HTLV-II are considered to be responsible for adult T-cell leukemia and, furthermore, for a Correspondence
to: Rolf M. Fliigel, projektgruppe Humane Retroviren, Institut fiir Virusforschung, DKFZ, Im Neuenheimer Feld 280, 6900 Heidelberg, F.R.G. 01~0934/90/?§03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
14
rare form of myopa~y, tropic spastic paresis (U~hiy~a et al., 1977; Poiesz et al., 1980; Gessain et al., 1985). The corresponding virus-specific ELISAs have proved essential for establishing the prevalence of viral infections in man and in correlating the presence of a given virus with clinically defined groups of patients. A third subfamily or group of retroviruses consists of spumaviruses also called foamy viruses (Weiss et al., 1982; Maurer and Fhigel, 1988; Lewe and Fliigel, 1989). Previously, we have cloned molecularly and characterized extensively the genome of one member of this group of viruses, the human spumaretrovirus, HSRV (Fliigel et al., 1987; Maurer et al., 1988; Rethwilm et al., 1987) that had originally been isolated from a patient with a nasopharyngeal carcinoma (Achong et al., 1972). Since this is a virus in search of a disease (Weiss, 1988), so to speak, it will be necessary to screen a relatively large number of human sera. HSRV-specific ELBA was, therefore, developed.
Materials and Methods Construction of recombinant plasmids expressing HSRV env gene regions Plasmid clones that harbor foamy viral inserts including the env gene have been described previously (Fliigel et al., 1987; Maurer et al., 1988; Rethwilm et al., 1987). To construct a recombinant clone that contained the central region of the env protein, pHSRV-HH-C55 (F&gel et al., 1987) was digested with PstI and XbaI and the resultant DNA fragment was inserted into the co~esponding sites of the expression vector pEx34 with T4 DNA ligase (Strebel et al., 1986). (The only difference between vector pEx34 and pEx31 is that the PstI site in the ampicillin resistance gene was deleted). The resulting recombinant plasmid, pEx34PX was characterized by restriction enzyme analysis and nucleotide sequencing of the vector/insert border. The results showed uneqivocally that the viral insert DNA had the expected size of 825 bp and was oriented in the proper direction for expression. Moreover, the nucleotide sequence analysis proved that the HSRV env domain was in reading frame ‘a’, the first of the three possible frames, so that it was fused to the MS2 polymerase protein (Strebel et al., 1986). Another recombinant plasmid was constructed that contained the central part of the coding sequences of the tr~smembr~e protein of the HSRV env region. Plasmid clone pHSRV-HH-C55 was digested with NIzeI and the ends filled up with four dNTPs by the Klenow DNA polymerase thereby creating blunt ends. Subsequently, the plasmid was digested with XbaI. The plasmid vector pEx34 was separately digested with Hindi and the Hind111 site filled up as described above to generate blunt ends, followed by a digestion with XbaI. Subsequently purified vector and insert DNAs were ligated with T4 DNA ligase. The resulting recombinant, pEx34XN, was characterized as described above and shown to ha&our a viral insert of 801 bp as expected.
15
Expression of recombinant antigen in E. coli and purification offusion protein envpx Expression and purification of fusion proteins was carried out according to Strebel et al. (1986) with the following modification. To visualize the protein bands after preparative SDS-polyacrylamide gel electrophoresis, precipitation of protein with 4 M sodium acetate was used instead of staining with Coomassie blue. After eluting the fusion proteins from the gel, the extracted proteins were rerun on 18% polyacrylamide gels and eluted from the gels through a Centricon microconcentrator (Amicon Co.). The purity of the recombinant antigens was finally reevaluated on analytical gels. Production of antisera against envpx and envxn Rabbits were initially immunized with about 100 pg of the purified fusion proteins suspended in Freund’s complete adjuvant and further stimulated by immunizing five times at intervals of 2 to 4 weeks with the same amount of recombinant antigen in Freund’s incomplete adjuvant. Sera were titrated by ELISA and immunoblotting with the purified recombinant antigen that was used for immunization. ELISA test for spumaviral env antibodies The assays were performed according to Voller et al. (1976) but omitting the fixation with glutaraldehyde. Recombinant envpx antigen (0.05-0.1 &well) were pipetted onto 96-well microtiter plates (Flow Labs. and Greiner Co.). After incubation at room temperature for 1 h the wells were washed three times with 0.15 ml of 0.1% Tween 20 in PBS. After drying, 100 ~1 of 3% bovine serum albumin in 0.1% Tween/PBS solution were added to each well followed by an incubation at 4°C overnight. Subsequently, the plates were dried and washed as before and test serum samples or rabbit antibodies against envpx were added at increasing dilutions in 0.1% tieen 2O/PBS. The microtiterplates were tightly sealed and incubated at 37°C for 1 h. After decanting and washing with 0.1% Tween 2O/PBS, the plates were dried and subsequently 50 ,~l protein A-peroxidase solution (1 mg/ml) diluted 1:250 were added in PBS. Thereafter the microtiterplates were sealed in plastic bags and incubated at 37°C for 60 min. After washing and drying 100 ~1 of color solution were added to each well. To prepare the color solution 28 mg of o-phenylenediamine dihydrochloride (Sigma Co., Deisenhofen) was dissolved in 10 ml of 100 mM citric acid, pH 5.0; 5 ~1 30% hydrogen peroxide were added shortly before use. After an incubation for 15 min at 25°C the color reaction was stopped by adding 100 ~1 2 M sulphuric acid Optical densities were determined by an automated ELISA reader (Biotec Model EL310). As negative controls for the ELISA, human sera from symptom-free individuals were used. Additionally, human sera that reacted with the envpx antigen under the assay conditions used gave negative results when the bacterially expressed fusion protein was omitted. Diluted rabbit hyperimmune sera against the HSRV env fusion proteins served as positive controls. Routinely, test sera from individuals were diluted 1: 150.
16
Labeling and immunoprecipitation of viral proteins in HSRV-infected human embryonic lung (HEL) cells
Monolayers of HEL cells grown in 25cm* tissue culture flasks were infected as described previously (Fltigel et al., 1987) and washed with medium free of methionine or cysteine. L-[35S]methionine (spec. activity 1129 Ci/mmol, NEN, Dreieich, F.R.G.) or cysteine (or both) were added 1 h after infection to a final concentration of 10 &i/ml each. Labeling was stopped by lysing cells eight days post-infection in 0.5 ml of RIPA-buffer (150 mM NaCl, 10 mM sodium phosphate, pH 7.5, 1% Nonidet P-40, 0.5% sodium desoxycholate, and 0.1% SDS). To avoid protein degradation, 10 ,@ml aprotinin were added. Extracts were incubated for 10 min at 65°C to denature virions. To immunoprecipitate viral proteins, the method of Strebel et al. was used (1986). Briefly, a suspension of Staphylococcus protein A (Sigma Co.) was incubated with antiserum for 1 h at room temperature in RIPA-buffer (200 ~1). After washing to remove unbound antibodies, 100 ,ul of the radioactive viral antigen was incubated in 500 ~1 for 45 min at 25°C. Unbound material was removed by washing with RIPA-buffer. Immunoprecipitated proteins were treated by heating to 95°C for 5 min in an equal volume of sample buffer and detected by fluorography after SDS-polyacrylamide gel electrophoresis.
Results and Discussion Construction of recombinant plasmids expressing and expression of fusion proteins in E. coli
HSRV env domains
Since in natural retroviral infections antibodies are produced predominantly against the env and gag proteins, defined regions of the HSRV env gene were chosen to construct recombinant clones. The recombinant protein envpx was derived from the central domain of the HSRV env gene products as shown in Fig. 1. The region of the HSRV env gene that encompassed 815 bp was inserted into the PstI and XbaI sites of the expression vector pEX34 (Strebel et al., 1986). One of the resulting recombinant clones (pExPX) was established and characterized by restriction enzyme analysis. PstI-XbaI double-digestion resulted in DNA fragments of 3.3 kbp (vector) and 815 bp (viral insert) as expected. To ascertain that the reading frame of the HSRV env protein was in the same direction and correctly linked to the vector encoded MS2 polymerase protein, the vector/insert boundary of the clone pExPX was sequenced. The nucleotide sequence revealed that the viral insert was indeed in the proper reading frame fused to the MS2 polymerase peptide (data not shown). The recombinant plasmid pExPX was expressed as a fusion protein consisting of 98 amino acid residues that were derived from the MS2 phage polymerase (Strebel et al., 1986) plus 273 amino acids of the central domain of the HSRV env protein. The two combined domains would lead to a molecular mass of about 41 kDa. To determine the size of the fusion peptide, the envpx protein was separated from soluble bacterial proteins by two cycles of differential centrifugation and precipitation followed by polyacrylamide gel electrophoresis under denaturing
17
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,/’ ,’ 1
I-h&’
. ,’
,
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/
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/
’
It I
I’ 3
f’
\ \
\
\\
,I
I
Xbal
Rtl
\
‘\\
kbp
Hind III
t
Fig. 1. Outline of strategy for molecular cloning of recombinant plasmid pEx34envPX. The arrow marks the position of the putative proteolytic cleavage site of the HSRV env precursor protein. For further details see &rebel et al. (1986).
conditions. Fig. 2A presents the migration pattern of the envpx protein (lane 7, marked by arrow) together with analyses of E. coli proteins without the vector pEx34 or transfected in alternative reading frames as controls. It is evident from Fig. 2A that only E. coli cells transfected with pEx34a (reading frame ‘a’ is the first of the three possible frames) expressed, an envpx fusion protein of molecular mass of approximately 42 kDa in agreement with the expected value of 41 kDa calculated above. A second recombinant plasmid that contained 267 amino acid residues of the HSRV transmembrane protein (‘IMP) as insert was separately constructed and established. Fig. 3 outlines the strategy for cloning the central part of the TMP of HSRV encompassing 267 residues out of the total 417. The recombinant plasmid, pEx34XN was characterized by restriction enzyme analysis and nucleotide sequencing of the vector/insert boundary, It was found that the HSRV env sequence was in the proper direction fused to the phage MS2 polymerase reading frame (data not shown). The resulting fusion protein, envxn, was expressed in E. culi and subsequently purified and used to raise antibodies in rabbits as described above for the recombinant antigen envpx. Characterization
of the fusion proteins
The recombinant fusion proteins were mainly found in the particulate fraction of the bacterial cell extracts and only partially soluble in 7 M urea. The E. coli
18
MLMl
2
34
56
76HM
kd
-66 -43 -25 -18 -15
Fig. 2. (A) SDS-polyactylamide gel electrophoresis of viral fusion proteins expressed in E. coli W537 after subsequent staining with Coomassie blue. Lanes were loaded with cell extracts as follows: E. co/i W537 only (lane I), E. coli W537 after transformation with the vector pEX34a but without any spumavira1 insert (lane Z), E. cooli W537 cells transformed with pEX34 in different reading frames that harbored a 2 kbp viral DNA fragment (lanes 3 to 6), E. coli W537 cells after ~~sfo~ation with
19
HSRV
i
\
/’ ,’ /’
xx’ ,/’ ,’
/’
1
II I’
\ I
\
I
\
\ \
!
‘\,kbp HindIII
Fig. 3. Schematic outline of the strategy for the molecular cloning of recombinant plasmid pEx34envXN. The arrow marks the site of the putative proteolytic cleavage of the env precursor glycoprotein into the outer membrane and transmembrane proteins.
proteins that copurified with the HSRV env protein were eliminated by repeated preparative SDS-polyacrylamide gel electrophoresis (Fig. 2B). The HSRV-specific envxn fusion protein was eluted from the gel and used to raise antibodies in rabbits. To demonstrate that rabbit antibodies against the recombinant antigen envpx reacted specifically with native HSRV env proteins, total extracts from HSRV-infected HEL cells that had been labeled with radioactive methionine were immunoprecipitated with S. aureu~ protein A-bound anti-envpx antibodies. The result of the subsequent electrophoretic analysis on polyacrylamide gels under denaturing conditions is shown in Fig. 5. The appearance of an env glycoprotein band of about 135 kDa clearly documents that the rabbit antiserum directed against the recombinant viral protein envpx was immunoreactive with the authentic HSRV env glycoprotein of the expected size. the vector pEX34PX that contained the spumaviral env insert in the correct reading frame ‘a’ (lane 7). the alternative reading frame ‘c’ with the same viral envpx insert was not expressed (lane 8). M, LM, and HM are marker proteins as indicated on the right hand side in kDa. (B) SDS-polyacrylamide gel electrophoresis of recombinant proteins and purhlcation of meombinant antigen envpx by repeated runs of SDS-polyacrylamide gel electmphomsis. Conditions were as described under Fig. 2A. The lanes were loaded as follows: E. coli W537 only (lane 1). E. coli W ‘537 after transformation with pEx34a, pEx34b, and pEx34c without any viral insert (lanes 2.4, and 6)and with the HSRV envpx insert (lanes 3,5 and 7). The reextracted recombinant envpx after repeated rerun (lanes 8 and 9). As control for the precise mobility of the envpx fusion protein lane 10 was loaded with E. coli 537 extracts transformed with pExPX as in lane 3. M. low and high marker proteins.
20
10-3
10-L
Antlbody Oihtlon
10-5
Fig. 4. Reactivity of the purified viral antigen envpx (0.2 gg) with antibodies raised in rabbits. The values found with preimmune sera from rabbits were below the cut-off threshold.
12345M
-200
1102 -92
-09
-40 -30 Fig. 5. Radioimmuno~ipi~ti~ of 35S-Iabeled proteins from HSRV-infected (lanes 4 and 5) and uninf~t~ HEL cells (lanes 1 and 2). Lane M shows molecular weight markers in kDa. The arrow in lane 4 marks HSRV em glycoprotein of approximately 135 kDa.
Use of HSRV env recombinant protein for EL.ISA The reactivity of the purified recombinant viral antigen was tested by titrating it against the rabbit envpx antibodies. Fig. 4 presents the optical densities determined for different antibody concentrations. ELISA analyses were performed to select antigen concentrations that would yield the highest possible sensitivity with the lowest background. Diluted human sera that reacted under the assay conditions used were tested in wells of microtiterplates to which different concentrations of either envpx antigen or envxn antigen were absorbed. It was found that the ELISA worked best at an antigen concentration of 0.05 to 1.O fig/well. Different dilutions of human sera were reacted with either the envpx or the envxn antigens. At dilutions ranging from 1:50 to 1: 10 000 most human sera had optical densities that were negligible or below average values. The majority of human sera collected from apparently symptom-free patients or from individuals with completely different diseases therefore do not react as would be expected. However, some human sera had optical densities that were consistently greater than the average values at those same dilutions. This may be due to the fact that selection on a symptom-free or random basis is a relatively poor method for excluding virus-infected subjects. In conclusion, an ELISA system was established based on env protein sequences of HSRV that is specific for human foamy virus. This ELISA will permit large-scale screening of human sera for the presence of HSRV antibodies.
Acknowledgements We thank Jeff Luande, Tanzania Tumor Center for providing sera and for his hospitality; Kunitada Shimotohno, National Cancer Research Institute, Tokyo for advice, helpful discussions and hospitality; and Harald zur Hausen, German Cancer Research Institute for support. The experiments described here were financed by a grant from the Commission of the European Community (TS2*0186-D,AM). We thank Jennifer Reed for reading the manuscript critically.
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