We have produced a monoclonal antibody against human cytomegalovirus (HCMV) which recognizes a structural protein of 28 000 mol. wt. which is present in ...
J. gen. Virol. (1985), 66, 2507-2511. Printed in Great Britain
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Key words : human cytomegalovirus/structural proteins/monoclonal antibody
A 28000 Molecular Weight Human Cytomegalovirus Structural Polypeptide Studied by means of a Specific Monoclonal Antibody By M A R I A C A R L A R E , * M A R I A P A O L A L A N D I N I , P. C O P P O L E C C H I A , G. F U R L I N I AND M. LA P L A C A Institute of Microbiology of the University of Bologna, 9, Via Massarenti, 1-40138 Bologna, Italy (Accepted 2 August 1985)
SUMMARY
We have produced a monoclonal antibody against human cytomegalovirus (HCMV) which recognizes a structural protein of 28 000 mol. wt. which is present in both the cytoplasm of infected cells during the late phase of the viral replication cycle and in the extracellular viral particles. This antigen was detected in all HCMV strains assayed and reacted with human sera having anti-HCMV antibodies. Human cytomegalovirus (HCMV) has been implicated in various diseases in humans. It is the most common agent in congenital infection and also causes significant morbidity and mortality in a variety of immune-compromised hosts (for review, see Ho, 1982). Detailed investigations of the humoral immune response during HCMV infection are necessary both for studying viral pathogenesis and for improving diagnostic reagents. To define better the human immune response induced by HCMV in the host and the immunogenic potential of viral structural antigens, we have recently undertaken a systematic study of the reaction of selected human sera with virus-specific polypeptides. Recent results (Landini et al., 1985) have shown that at least three HCMV structural proteins of molecular weights 155000 (155K), 38.5K and 28K are recognized by all highly positive human sera. The presence of a structural protein of 28K or of a very similar molecular weight in purified HCMV has been detected by several authors (Fiala et al., 1976; Kim et al., 1976; Gupta et al., 1977; Stinski, 1978; Nowak et al., 1984) while Gibson (1983) found a structural polypeptide of corresponding molecular weight (27K) present as an envelope component in simian CMV but not in CMV of human origin. This communication reports the production of a monoclonal antibody (MAb) to the 28K polypeptide and its use in a partial characterization of this viral antigen. Human embryo fibroblasts (HEF) were obtained from the Istituto Zooprofilattico, Brescia, Italy and were grown in Eagle's MEM containing 10% foetal calf serum (FCS). The NS1 Ag4/1 myeloma cell line was obtained through the courtesy of Dr C. Milstein (Cambridge, U.K.). NS1 cells and the hybrids derived by fusion with mouse spleen cells were grown in Dulbecco's MEM with 20% FCS. The Towne strain of HCMV was used in all experiments. Herpes simplex virus type 1, obtained through the courtesy of Dr E. Cassai (Ferrara, Italy) was used as viral control. HCMV was purified from extracellular supernatant when the monotayer exhibited 100% cytopathic effect using a procedure previously described (Landini & Ripalti, 1982). Briefly, extracellular medium was centrifuged at 3500 r.p.m, for 60 min at 4 °C; supernatant was ultracentrifuged at 21 500 r.p.m, for 60 min at 4 °C through a 20% (w/v) sorbitol cushion and the resulting pellet was dissolved in TBS (0.05 M-Tris-HC1 pH 7.4, 0.15 M-NaC1), overlaid on a 20 to 70% sorbitol gradient and ultracentrifuged at 21500 r.p.m, for 60 min; the white band was collected and submitted to a further sedimentation through a discontinuous CsC1 gradient (1.4 and 1-2 g/ml, at 22500 r.p.m, for 60 rain). 0000-6748©1985 SGM
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Fig. 1. Immunofluorescence staining of HCMV-infected cells with MAb P2G 11. HEF were infected with 1 p.f.u./cell of the Towne strain of HCMV and fixed with methanol :acetone (3 : 1) at (a) 48 or (b) 96 h post-infection. The IIF was performed with 1:200 dilution of P2G11 ascitic fluid. Procedures for producing immunoglobulin-secreting hybrid cell lines were described by Kohler & Milstein (1975). Briefly, BALB/c mice were immunized with purified H C M V disrupted by sonication (40 s at a setting of 3 in a Branson B 12 sonifier) and 4 days after the last injection, antibody levels of mice were tested and spleens removed. Spleen cells were fused with N S 1 Ag4/1 cells in a ratio of 3 : 1 by using polyethylene glycol. Hybrids were selected and clones, which were visible approximately 2 weeks after fusion, were tested by enzyme immunoassay with a commercial preparation of H C M V antigen (Cytomegalisa, M.A. Bioproducts, Md., U.S.A.). After subcloning, by limiting dilution, clones were retested, expanded and injected into BALB/c mice to obtain ascitic fluid. The reactivity of M A b with HCMV-infected cells was determined by indirect immunofluorescence (IIF) as described by Schmitz & Haas (1972). M A b was assayed for its reactivity with (i) uninfected HEF, (ii) HCMV-infected H E F treated with 50 p,g/ml cytosine arabinoside, (iii) HCMV-infected HEF, fixed at 24, 48, 72, 96 and 114 h post-infection, (iv) H E F infected with different H C M V strains and fixed at 96 h post-infection and (v) HSV-infected HEF. Neutralization assay was performed as described by Reynolds et al. (1979). Untreated H C M V was used as control. Purified H C M V and lysate of infected cells at 10, 20, 40, 60 and 96 h post-infection were denatured in the presence of SDS with and without 2-mercaptoethanol, electrophoresed in
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(b)
(a)
1
2
4
5
6
7
8
9
/Viol. wt. x 10-3
Mol. wt. x 10-3
~92-5 4 - 66.2
•-.-- 92.5 -.,- 66-2
--.--45
~31
45
-,--31
~21.5 14.4
Fig. 2. (a) Identificationof HCMV protein recognized by MAb P2G t 1. Infected cells were disrupted by two cycles of freezing and thawing, suspended in phosphate-buffered saline brought to 1.5 M-NaC1, centrifuged at 10000 r.p.m~ for 30 min and the supernatant used immediately after. Cell extracts and purified extracellular virus particles were separated by SDS-PAGE in the presence of 2mercaptoethanol, electrotransferred to nitrocellulose paper and incubated with ascitic fluid of P2G 11 clone. Twenty-five ~tg protein (determined by Bio-Rad protein assay) was run in each lane. Lane 1, uninfected cells; lanes 2, 3, 4, 5 and 6, infected cells 10, 20, 40, 60 and 96 h post-infection respectively; lane 7, purified extracellular HCMV particles; lanes 8 and 9, cytosine arabinoside-treated, uninfected and HCMV-infected (72 h post-infection) cells respectively. (b) Electrophoretic mobility of the 28K protein in the absence of 2-mercaptoethanol. Infected cells 144 h post-infection (lane 1) and purified viral particles (lane 2) were separated by SDS-PAGE, in the absence of 2-mercaptoethanol in the dissociating buffer, transferred to nitrocellulose paper and incubated with P2G11 ascitic fluid. The molecular weight of the HC MV-specific protein reacting with MAb P2G 11 was calculated on the basis of relative mobility with respect to the following proteins of known molecular weight: myosin (200000), /~-galactosidase (116250), phosphorylase B (92500), bovine serum albumin (66200), ovalbumin (45000), carbonic anhydrase (31000), soybean trypsin inhibitor (21500) and lysozyme (14400).
polyacrylamide gels a n d transferred to nitrocellulose sheets as described by B r a u n et al. (1983). Nitrocellulose paper with blotted materials was processed as described in detail elsewhere ( L a n d i n i et al., 1985). A M A b designated P 2 G 11 was identified which reacted with a cytoplasmic a n t i g e n expressed only in the late phase of the viral replication cycle. This was d e t e r m i n e d by I I F a n d the results are shown in Fig. 1. The M A b reacted with cytoplasm of infected cells starting from 48 h postinfection a n d the intensity of the fluorescence increased thereafter, with a completely different d i s t r i b u t i o n of the fluorescence to that observed with a non-specific ( H C M V - n e g a t i v e ) ascitic fluid which produced only scant fluorescence in a limited p a r a n u c l e a r area ( F u r u k a w a et al., 1975). N o reaction was observed with u n i n f e c t e d cells or cells infected a n d kept in the presence of cytosine arabinoside a n d n o cross-reaction was detected with herpes simplex virus-infected cells (data not shown). By the double i m m u n o d i f f u s i o n test (Miles Laboratories) M A b was identified as IgG1.
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I
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Mol, wt. x 10-3
3 i
i
--
155
E
110 99.5 82,5
--
74.5 67 62
--
57
--
55
--
51
49 46.5 45 38.5 d
28
~ ii~ i~i:~il~
Fig. 3. Immune reaction of MAb P2GII (lane 1) and two human sera, PF and GR (lanes 2 and 3 respectively), positive (>1: 160) in the complement fixation test with commercial (Behringwerke) HCMV antigen, with HCMV structural polypeptides separated in SDS PAGE and electrotransferred to nitrocellulose. The immune reaction was performed as previously describ~edin detail (Landini et el., 1985). Numbers on the right represent approximate molecular weights of HCMV structural proteins calculated as described in the legend to Fig. 2. To assess the size of the antigen reacting with P2G 11 MAb, infected cell lysates at different times post-infection as well as purified extracellutar virions were subjected to S D S - P A G E , Western blotting and immune reaction. The results obtained are shown in Fig. 2. Starting from 60 h post-infection, P2G 11 M A b reacted with an antigen of 28K approximate tool. wt. which is also present in mature extracellular virions. N o reaction was observed with uninfected or infected and cytosine arabinoside-treated cell extracts (Fig. 2a) and the reaction was abolished by pretreatment of the blots for 15 rain at 37 °C with 25 gg/ml trypsin, or 100 p_g/ml Pronase or 100 gg/ml proteinase K (data not shown). The electrophoretic mobility of this structural protein was not significantly affected by the lack of 2-mercaptoethanol in the dissociating buffer either in the cell lysate or in the extracellular virus, suggesting that it is not a part of a larger disulphide-linked complex (Fig. 2b).
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P 2 G l l M A b did not exhibit any neutralizing activity either with or without complement (data not shown), indicating that the 28K structural protein, or at least the epitope reacting with this MAb, is not responsible for induction of neutralizing antibody. The 28K structural protein was recognized by two h u m a n sera, P F and G R , possessing antibody to H C M V (Fig. 3), indicating that antibodies to this antigen are produced during natural infection. This was further substantiated by the finding that cells infected by other H C M V strains, such as AD169, Mira, and five fresh isolates, gave a positive cytoplasmic reaction when stained with M A b P 2 G l l (data not shown). The results we have obtained with M A b P 2 G l l demonstrate that (i) a 28K protein is produced as a late virus-specific product in HCMV-infected cells, (ii) the 28K protein is present in extensively purified viral particles and (iii) it can be detected in all wild H C M V strains tested. These results are in agreement with the finding that a 28K antigen is detected by the great majority of h u m a n Sera possessing a n t i - H C M V antibody when tested by immunoblotting assay (Landini et al., 1985). The exact distribution of the 28K structural antigen in infected cells and its localization in the viral particle are currently under investigation as well as the possible use of P2G11 M A b as a diagnostic tool in H C M V infection. The possible relationships of the 28K structural protein with the 25K glycoprotein detected by Pereira et al. (1983) in immunoprecipitates of several sera from congenitally infected infants and with the 29K phosphoprotein detected by Nowak et al. (1984) deserve further study. We thank Dr M. Ferrari (Brescia, Italy) for providing cultured HEF and Mrs Franca Balboni for typing the manuscript. This work was partially supported by C. N. R., P.F. 'Controllo delle Malattie da Infezione' Grant. No. 84.02007.52 and by the Italian Ministry of Education (40 and 60~). REFERENCES (1983). Application of denatured, electrophoretically separated and immobilized lysates of herpes simplex virus infected cells for detection of monoclonal antibodies and for_the studies of the properties of viral proteins. Journal of Virology 46, 103-112. FIALA, M., HONESS, R. W., HEINER, D. C., HEINE, J. W., JR, MUNRANE, J., WALLACE, R. & GUSE, L. B. (1976). Cytomegalovirus proteins. I. Polypeptides of virions and dense bodies. Journal of Virology 19, 243-254. FURUKAWA, T., HORNBERGER, E., SAKUMA, S. & PLOTKIN, S. A. (1975). Demonstration of immunoglobulin G receptors induced by human cytomegalovirus. Journal o/'C/inical Microbiology 2, 332 336. GIBSON,W. (1983). Protein counterparts of human and simian cytomegaloviruses. Virology 128, 391-406. GUPTA, P., ST. JEOR, S. & RAPP, F. (1977). Comparison of the polypeptides of several strains of human cytomegalovirus. Journal of General Virology 34, 447 454. HO, M. (1982). In Cytomegalovirus. Biology and lnJbction, pp. 119-204. Edited by W. B. Greenough III& T. Merigan. New York: Plenum Press. RIM, K. S., SAVlENZA,V., CARV,R. 8LMOON,n. M. (1976). Analysis of structural polypeptides of purified human cytomegalovirus. Journal of Virology 20, 604-611. KOHLER, 6. ~ M~LSTEIN,C. (1975). Continuous cultures of fused cells secreting antibody of predetermined specificity. Nature, London 256, 495-497. LANDINI, M. P. & RIPALTI,A. (i982). A DNA-nicking activity associated with the nucleocapsid of human cytomegalovirus. Archives of Virology 73, 351-356. LANDINI, M. P., RE, M. C., MIROLO, G., BALDASSARRI, B. & LA PLACA, M. (1985). Human immune response to cytomegalovirusstructural polypeptides studied by immunoblotting. JournalofMedical Virology 12 (in press). BRAUN, D. J., PEREIRA, L., NORRILD, B. & ROIZMAN, B.
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(Received 14 June 1985)
