Int. J . Cancer: 15, 799-805 (1975)

EPSTEIN-BARR VIRUS-INDUCED CAP FORMATION IN HUMAN LYMPHOBLASTOID CELLS

Yorio H I N U M A ,Masahiro SUZUKIand Takeshi SAIRENJI Department of Microbiology, Kumamoto University Medical School, Kumamoto 860, Japan

Redistribution and consequent cap formation of Epstein-Barr virus adsorbed to human lymphoblastoid cells were studied by indirect membrane immunopuorescence carried out at 0" C. When EBV was adsorbed on cells at 0" C, the cellsurjacepuorescence had a mostly ring-like pattern. However, the ring cells could be transjormed into cap cells when warmed at 37" C. This cap formation could be induced by EBV alone without participation of antibodies involved in the immunofluorescence procedure. The cap formation was temperature- and pa-dependent, and was reversibly inhibited by sodium azide or some sugars. Thus the EBV-induced cap formation was analogous to that induced by antibodies or ligands on other lymphoid cells.

Recent studies have shown that immunoglobulin (Ig) molecules on the surface of lymphoid cells can be readily displaced and become aggregated at one pole of the cell by interaction with antibody. Taylor et al. (1971) termed this phenomenon " cap " formation. The phenomenon of capping in cells has been observed not only with Ig but also with other receptors or antigens on the cell surface, such as the concanavalin A receptor (Yahara and Edelman, 1972), complement receptor (Theofilopoulos et al., 1974) and histocompatibility antigen (Unanue et al., 1972). The capping consists of the segregation of membrane components, specifically crosslinked by antibody or other ligand, at one pole of the cells. This is interpreted as resulting from a countercurrent flow of membrane components, in which the cross-linked patches are transported towards the centrosome region by interaction with cytoplasmic structures (Taylor et al., 1971 ; de Petris and Raff, 1972). During studies on the adsorption of EpsteinBarr virus (EBV) to human lymphoblastoid cell lines by means of membrane immunofluorescence (Klein et al., 1966; Gergely et al., 1971), we have

observed the clustering of EBV antigen on the cell surface. We conclude that this is an EBVinduced cap formation analogous to that induced by other antibodies or ligands. MATERIAL A N D M E T H O D S

Virus

EBV produced from B95-8 marmoset cell line (Miller and Lipman, 1973) was used. The cells were subcultured every 4 or 5 days at 37" C in RPMI-1640 medium supplemented with 10% fetal calf serum, 100pg/ml of streptomycin and 100 IU/ml penicillin. A virus preparation was obtained from medium cultured for 7 days at 37" C. After removal of cells by centrifugation at 9 0 0 x g for IOmin, the supernatant was centrifuged at 53,000 x g for 60 min. The pellet was resuspended in a small volume of RPMI-1640 medium plus 20% fetal calf serum to give an approximately 100-times concentration. The viral concentrate was dispersed with a Vortex Jr. Mixer (Scientific Instruments Inc., Queens Village, N.Y., USA), centrifuged at 900 x g for 10 min, and then the supernatant was passed

Received: January 3, 1975.

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HINUMA ET AL.

through 0.45 o r 0.8 pm of Millipore membrane and stored at -80" C .

covered with a cover slip and then examined with a Chiyoda microscope equipped with a vertical Ploem-type illuminator.

Cells

The C-6 line (Amano et al., 1973), a clonal subline derived from the NC-37 human lymphoblastoid cell line established from peripheral leukocytes of a normal adult (Durr et a/.. 1970) was used. The C-6 line carries EBV-associated nuclear antigen (EBNA) (Suzuki and Hinuma, 1974) but no other EBV-determined antigens o r cytoplasmic a n d membrane-associated IgG at a significant level. The cells were subcultured every 3 o r 4 days in the same medium as the B95-8 cell line at 37" C . The cells, cultured for 3 days-which gave a viability of over 95%were sedimented once and then resuspended in phosphate-buffered saline (PBS) at PH 7.2 before use. It?itnunofiuorL,scencr

An indirect membrane immunofluorescence procedure was adopted. The procedures for detection of EBV-induced cap formation were as follows, unless otherwise indicated. First, EBV was adsorbed on the cells. An aliquot of cell suspension having about 4 x 10" cells/ml was mixed with an equal volume of virus suspension and then incubated in an ice bath (OoC) for 3 h. The cells were washed twice with ice-cold PBS at 600xg for 5 min, resuspended in PBS and then incubated again a t 37" C for 1 h. Next, the cells were reacted with an anti-virus serum. For this, a 1 :10 dilution of VO-7 serum from a healthy adult was used. This had titers of I :160 of both antibodies to EBV capsid antigen (VCA) a n d to EBV-associated membrane antigen (MA) (Takahashi and Hinuma, 1970), of 1:40 o f anti-EBNA and

Epstein-barr virus-induced cap formation in human lymphoblastoid cells.

Int. J . Cancer: 15, 799-805 (1975) EPSTEIN-BARR VIRUS-INDUCED CAP FORMATION IN HUMAN LYMPHOBLASTOID CELLS Yorio H I N U M A ,Masahiro SUZUKIand Tak...
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