Accumulation of Herpes Simplex Virus Type 1 Glycoprotein D in Adhesion Areas of Infected Cells

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1 J. gen. Virol. (1983), 64, Printed in Great Britain 2499 Key words: HSV/gD/focal adhesion/vinculin Accumulation of Herpes Simplex Virus Type 1 Glycoprotein D in Adhesion Areas of Infected Cells By B. NORRILD, 1. I. VIRTANEN, 2 V.-P. LEHTO 2 AND B. PEDERSEN 1 l Institute of Medical Microbiology, University of Copenhagen, 22 Juliane Mariesvej, DK-2100 Copenhagen, Denmark and 2Department of Pathology, University of HelsinkL Finland (Accepted 12 July 1983) SUMMARY By indirect immunofluorescence microscopy (IIF) we have localized glycoprotein D (gd) of herpes simplex virus-infected cells to the vinculin-containing junctional areas and to the focal adhesion sites on the ventral surface of the cells. Double-staining IIF showed that gd and vinculin co-distributed in the infected cells and treatment of the infected cells 4 h post-infection with rabbit antibodies to gd prevented attachment of the cells to the growth substratum. Our results therefore support the hypothesis that gd plays a central role in the social behaviour of infected cells. Herpes simplex virus type I (HSV-1)-infected cultured cells synthesize five well-characterized glycoproteins: ga, gb, gc, gd and ge (Spear, 1976; Bauke & Spear, 1979; Eisenberg et al., 1979; Pereira et al., 1982), which can be found in the plasma membrane of the cells 4 h post-infection (Norrild et al., 1980). The glycosylation of the various proteins takes place in discrete steps (Honess & Roizman, 1975; Eberle & Courtney, 1980; Wenske et al., 1982), and the majority of the carbohydrate chains are linked to the peptide backbone through N-glycosidic bonds, although the HSV-1 type-specific gc in particular also contains O-glycosidically linked sugar chains (Norrild & Pedersen, 1982; Olofsson et al., 1983; Johnson & Spear, 1983). The detailed route of transport of the glycoproteins from the site of synthesis to the plasma membrane and the function of the membrane-inserted glycoproteins are unknown. We describe studies which show that gd is specifically transported to the focal adhesion areas on the ventral surface of infected Vero cells. Veto cells were grown as monolayer cultures on glass coverslips and infected with HSV-1 strain F at a multiplicity of infection of 10 p.f.u./cell (Norrild et al., 1983). At 9 h post-infection, cells were fixed and stained by the indirect immunofluorescence method (IIF). In sparse cultures the IIF technique allowed identification of plaque-like patches in the cells which accumulated gd (Fig. 1). These patches were mainly concentrated in the areas of the cells mediating adhesion to the growth substratum, and the morphology of the patches was similar to that of the focal adhesion plaques identified also in uninfected cells and known to contain the cytoskeletal protein, vinculin (Geiger, 1979; Lehto et al., 1982; Virtanen et al., 1982). Vinculincontaining adhesion areas are known to mediate adhesion of cultured cells to the growth substratum and to be sites at which bundles of microfilaments terminate close to the cell surface membrane (Geiger, 1979). Similar sites can be found at junctional areas of epithelial cells, corresponding to adherence junctions (Geiger et al., 1983). Double-staining of HSV-l-infected cells with antibodies to both gd (Pereira et al., 1980) and vinculin showed co-distribution of the two proteins both at the ventral plaques and at the junctions between adjacent cells (Fig. 2a to d). In order to confirm that gd had accumulated at the focal adhesion plaques, HSV-l-infected cells were removed from the growth substratum and treated with rabbit antibodies reactive only with gd (Vestergaard & Norrild, 1979) or with preimmune rabbit serum. The infected cells treated with antibodies to gd could not re-attach to the growth substratum, whereas the cells treated with pre-immune serum did attach and spread /83/ $ SGM

2 2500 S h o r t communication Fig. 1. Vero cells cultured in minimal essential medium (MEM) with 10~o foetal calf serum. The cells were stained for IIF 9 h after infection with HSV-1. For that purpose the cells cultured on glass coverslips were fixed with 4 ~ paraformaldehyde and permeabilized with 0-1~ Nonidet P40. Thereafter, the specimens were exposed to hybridoma antibodies against gd (Pereira et al., 1980) followed by fluorescein isothiocyanate (FITC)-coupled goat antimouse IgG antiserum (Cappel Laboratories, Cochranville, Pa., U.S.A.). Similar results were obtained with rabbit antibodies against gd. In sparse cultures of Veto cells especially, gd is seen to be located in plaque-like patches on the ventral surface of cells (marked with triangles) close to the growth substratum. Note also the staining at the junctional areas (arrow). The specimens were examined in a Zeiss Universal microscope equipped with an epiilluminator III RS and filters for FITC and tetramethylrhodamine isothiocyanate (TRITC) fluorescence. Magnifications: x 400; inset x 700. Fig. 2. Double HF staining of HSV-l-infected Vero cells for gd (a, c) and vinculin (b, d) after 9 h of infection. For double IIF, the specimens were fixed in methanol at - 20 C and were first exposed to rabbit antibodies to vinculin (Lehto et al., 1982; Virtanen et al, 1982) followed by TRITC-coupled swine anti-rabbit IgG antiserum (Dakopatts Ltd.), hybridoma antibodies to gd and FITC-coupled goat anti-mouse IgG (Cappel Laboratories). Note the co-distribution ofgd (a) and vinculin (b) at the ventral plaques in sparse cultures of infected cells and at junctional areas in dense cultures (c, gd; d, vinculin). Magnifications: (a, b) 525; (c, d) x 300.

3 Short communication 2501 (b) ; ~M~ ) ~ tp Fig. 3. Vero cells grown in 25 cm 2 Nunc flasks infected with HSV-1 at an m.o.i, of 5. Four h postinfection, infected (a, b) and uninfected (c, d) cells were removed from the surface of tissue culture flasks by trypsin treatment and washed extensively in phosphate-buffered saline. The cells were resuspended in MEM with 1 ~ foetal calf serum supplemented with either 5 ~ rabbit serum to gd (Vestergaard & Norrild, 1979) (b, d) or with pre-immune rabbit serum (a, c) and seeded in 25 cm 2 tissue culture flasks. The rabbit sera were adsorbed with uninfected cells before use. The specificity of antiserum to gd was tested by immunoprecipitation, and only gd was precipitated when the serum was added to an extract of radioactively labelled HSV-1 proteins from infected ceils. Rabbit antiserum to gd did not stain uninfected cells when used in the IIF test. Note that HSV-l-infected cells seeded in the presence of preimmune serum attach and spread on the growth substratum (a), whereas infected cells treated with antibodies to gd do not attach but form loose aggregates which either float or might be attached at one spot to the growth substratum as demonstrated in (b). The arrow indicates the point of attachment of the large aggregate. The cells were photographed 5 h after seeding in medium with antibodies. Magnifications x 150. w i t h i n 5 h a f t e r s e e d i n g (Fig. 3). S t a i n i n g o f i n f e c t e d cells w i t h m o n o c l o n a l a n t i b o d i e s to g C d i d not identify a similar accumulation of gc whereas ga/gb occasionally co-distributed with v i n c u l i n (results n o t shown). F r o m t h e a b o v e, we c o n c l u d e t h a t o u r results d e m o n s t r a t e t h a t g D is t r a n s p o r t e d specifically to t h e a d h e r e n c e j u n c t i o n s a n d to t h e v i n c u l i n - c o n t a i n i n g focal a d h e s i o n p l a q u e s. T h e f u n c t i o n o f g D is largely u n k n o w n, b u t c o - d i s t r i b u t i o n o f g D a n d v i n c u l i n a n d l a c k o f a d h e s i o n o f i n f e c t e d cells in t h e p r e s e n c e o f a n t i b o d i e s to g D s u g g e s t t h a t o n e f u n c t i o n o f g D c o u l d b e to affect t h e

4 2502 Short communication integrity of the vinculin-containing adhesion and cell-cell junctional areas of infected cells. This proposal is also supported by the finding that adhesion sites contain specific glycoproteins not found elsewhere at the cell surface (Oesch & Birchmeier, 1982). It remains to be elucidated how gd functions in the behaviour of cells but the present results suggest that gd could affect actincontaining microfilaments anchored at the focal adhesion plaques, eventually leading to rounding-up of the infected cells (Roizman, 1971). It should be noted that gd is apparently an essential structural component of the virus, as no mutants have been obtained so far that are deficient in synthesis of gd. Contrary to this, the other well-characterized glycoproteins of HSV-1, A, B and C, are less essential to the virus structure, and mutants that are defective in the synthesis or processing of ga/gb and gc are available (Manservigi et al., 1977; Sarmiento et al., 1979; Hoggan et al., 1960). The HSV particles made under conditions where ga/gb synthesis is impaired are not infectious, but viral particles that lack gc are infectious, although they change what has been called the 'social behaviour' of the infected cells (Ruyechan et al., 1979; Roizman, 1971). Based on the observation that the gc does not appear in the focal adhesion plaques and that ga/gb may occasionally be located in these structures, it is likely that the maturation of the various glycoproteins and their transport to the cell surface followed specialized and different routes (see also Norrild et al., 1983). Additional work is necessary, however, to substantiate this hypothesis. The skilful technical assistance of Ms Raili Taavela and Mr Carsten Juhl is kindly acknowledged. The work was supported by the NOVO Foundation, the Carlsberg Foundation, the Finnish Medical Research Council, the Sigrid Jus~lius Foundation and the Association of Finnish Life Insurance Companies. Bente Pedersen was supported by a grant from the Danish Cancer Society. REFERENCES BAUKE, R. B. & SPEAR, P. G. (1979). Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein. Journal of Virology 32, EBERLE, R. & COURTNEY, R. J. (1980). ga and gb glycoproteins of herpes simplex virus type 1 : two forms of a single polypeptide. Journal of Virology 36, 665~75. EISENBERG, R. J., HYDRAN-STERN, E. & COHEN, G. H. (1979). Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1. Journal of Virology 31, GEIGER, B. (1979). A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell 18, GEIGER, B., SCHMID, E. & FRANKE, W. W. (1983). Spatial distribution of proteins specific for desmosomes and adherence junctions in epithelial cells demonstrated by double immunofluorescence microscopy. Differentiation 23, HOGGAN, M. D., ROIZMAN, B. & TURNER, T. B. (1960). The effect of the temperature of incubation on the spread of herpes simplex virus in an immune environment in cell culture. Journal oflmmunology 84, HONESS, R. W. & ROlZMAN, B. (1975). Proteins specified by herpes simplex virus. XIII. Glycosylation of viral polypeptides. Journal of Virology 16, JOHNSON, D. C. & SPEAR, P. G. (1983). O-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the Golgi apparatus. Cell 32, LEHTO, V.-P., HOVI, T., VARTIO, T., BADLEY, R. A. & VIRTANEN, I. (1982). Reorganization of cytoskeletal and contractile elements during transition of human monocytes into adherent macrophages. Laboratory Investigation 47, MANSERVIGI, R., SPEAR, P. G. & BUCHAN, A. (1977). Cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins. Proceedings of the National Academy of Sciences, U.S.A. 74, NORRILD, B. & PEDERSEN, B. (1982). The effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and on their expression on the cell surface. Journal of Virology 43, 395~,02. NORRILD, B., SHORE, S. L., CROMEANS, T. L. & NAHMIAS, A. J. (1980). Participation of three major glycoprotein antigens of herpes simplex virus type 1 early in the infectious cycle as determined by antibody-dependent cellmediated cytotoxicity. Infection and Immunity 28, NORRILD, B., VIRTANEN, 1., PEDERSEN, B. & PEREIRA, L. (1983). Processing of HSV-1 glycoproteins in human fibroblasts and Vero-cells. Archives of Virology (in press). OESCH, B. & mrcrlmeier, W. (1982). New surface component of fibroblasts' focal contacts identified by a monoclonal antibody. Cell 31,

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