Studies on herpes simplex virus type 1 glycoproteins using monoclonal antibodies

VIROLOGY 151,385-389

(1986)

Studies on Herpes Simplex Virus Type 1 Glycoproteins J. KOGA, Department

Using Monoclonal Antibodies

S. CHATTERJEE,’ AND R. J. WHITLEY

of Pediatrics,

University

Received November

of Alabama, Birmingham,

8, 1985; accepted February

Alabama

35294

24, 1986

Monoclonal antibodies against herpes simplex virus type 1 glycoproteins were isolated and utilized to study the synthesis and processing of glycoproteins B, C, and D (gB, gC, gD, respectively). Monoclonal antibodies against both gB and gD had higher virus-neutralizing activity when compared to that of gC. Differences among these glycoproteins were observed in their time of appearance in the virus-infected cells. The presence of gD was detected at a very early stage of infection when compared to gB and gC. The localization of these glycoproteins during their synthesis and processing was studied. o 19% Academic Press, Inc.

Herpes simplex virus (HSV) infections range from the totally asymptomatic to those which result in life-threatening disease. More than 50 polypeptides have been identified in HSV-infected cells including several major cell surface glycoproteins, such as gB, gC, gD, gE, and gG (1-4). The specific biologic functions of these glycoproteins are not well defined, although gB has been shown to be important for cell fusion activity (5-7) and gC has CSb-binding activity (8). In addition, their exact intracellular localization during the processing pathway has not been demonstrated. Monoclonal antibodies have been shown to be useful tools for characterizing different viral proteins with more antigenic specificity; herpesviruses have been no exception. In this communication, we describe the isolation and characterization of several monoclonal antibodies against herpes simplex virus type 1 (HSV-1) glycoproteins, B, C, and D. Utilizing these monoclonal antibodies, we studied their localization during the synthesis and processing pathway in HSV-l-infected human fibroblast cells. r AH correspondence and requests for reprints should be sent to Dr. Chatterjee, University of Alabama at Birmingham, Suite #653/Children’s Hospital Tower, 1600 Seventh Avenue South, University Station, Birmingham, Ala. 35294.

Monoclonal antibodies against HSV-1 glycoproteins were isolated and characterized as described by Balachandran et al. (9). In brief, BALB/c mice were immunized with purified HSV-1 (F strain). Three days after final injection, spleens were removed and splenocytes were fused with myeloma P3-X63-Ag 8.653 (10; kindly provided by J. F. Kearney, University of Alabama at Birmingham) by PEG-4000. Hybrids were selected in HAT medium, and the culture supernatants obtained from growing colonies were screened by indirect immunofluorescence assay (IFA) as described previously (11). Figure 1A shows the radioimmune precipitation (RIP) pattern obtained with different monoclonal antibodies using [35S]methionine-laheled, HSV-l-infected cell lysates. Distinct bands corresponding to the molecular weights of glycoproteins B, C, and D (126K, 130K and 60K, respectively) and their precursors (llOK, 105K, and 52K, respectively) could be observed. Radioimmune precipitation with [3H]glucosamine-labeled cell lysates was also carried out and similar patterns of protein profile were observed (Fig. lB), which confirmed that these monoclonal antibodies were against glycosylated proteins. Table 1 shows neutralizing activity, subtype of immunoglobulin and type specificity of each monoclonal antibody. Many of the anti-gB and anti-gD monoclonal an385

0042-6822/86 $3.00 Copyright Q 1986 by Academic Press. Inc. All rights of reproduction in any form reserved

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B 1

A

123

4

5

6

789

gc* PN’

96

- 9c

P96 (P9C

2

3

4 gB 4wB

+

+ gD* p@-

gD P9D

FIG. 1. Immunoprecipitation of HSV-1 glycoproteins with monoclonal antibodies. (A) HEp-2 cells (5 X lo6 cell&O-mm petri dish) infected with F strain of HSV-1 (m.o.i. 5) were labeled with [ssS]methionine (1020 Ci/mmol; Amersham, Arlington Heights, Ill.), lysed with lysis buffer (0.5% sodium deoxycholate and 0.5% Nonidet-P40 in phosphate-buffered saline) and then immunopreciphated with culture supernatanta (50 ~1) from each hybridoma and rabbit anti-mouse IgG as secondary antibody. The immunoprecipitates were then dissolved in gel buffer (50 m&f Tris-HCl, pH 7.4, 3% sucrose, 5% 2-mercaptoethanol, and 2% SDS) and finally analyzed by 10.5% polyacrylamide gel electrophoresis and fluorography as described previously (12). Lanes 1-3, immunoprecipitated with supernatants from hybridoma Fd79, Fd24, Fc65, lanes 4-6, Fd35, Fd48, Fd13880, and lanes 7-9, Fd8, Fd15, Fd37. Arrows indicate the positions of the molecular weight standards: 116K, 92K, 66K, and 45K (from top to bottom). “p” denotes precursor. (B) Experimental procedure was same as in (A), except that the cells were labeled with [8HJglucosamine (8.5 Ci/mmol; ICN Pharmaceuticals Inc., Irvine, Calif.). Lane 1, immunoprecipitated with clone Fd8; lane 2, with clone Fd48 and lane 3 with clone Fd18. Arrows indicate the positions of the molecular weight standards: 116K, 92K, 66K, and 45K (from top to bottom). “p” denotes precursor.

tibodies had high neutralizing activity in the absence of complement, although one of the anti-gD monoclonal antibody (Fd48) was totally complement dependent. AntigC monoclonal antibodies, Fd8, Fd15, and Fd30, were found to be type common. This finding is in agreement with recent reports showing HSV-2 gC (previously designated gF) shared common antigenic determinants with HSV-1 gC (~$14) and that the

HSV-2 genome encodes this glycoprotein in a homologous position to the HSV-1 glycoprotein C (15). Some of these monoclonal antibodies, Fd18, Fd79, Fd8, Fd30, and Fd170, were suitable for Western blotting which raises the possibility of their utility in a variety of studies, including the rapid detection of HSV-specific antigens by an immunodot blot procedure. We utilized these monoclonal antibodies

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1

PROPERTIES OF DIFFERENT MONOCLONAL ANTIBODIES AGAINST HSV-1 GLYCOPROTEINS Clone No. Fc5 Fe65 Fc79 Fe154 Fd18 Fd24 Fd79 Fd93 Fd145

Directed to @

I&z subtypes” G2a 2b 2b 1 2b 2b 1 2a 2a

Neutralizing activityb X500 2ooo C-J t-1 C-1 (-) FY (--)

Type specificity” Common Common Common Common Type 1 specific Common Common Type 1 specific Type 1 specific

Fd8 Fd15 Fd30 Fd37 Fd75

2a 2a 2a 2a M

(Y t-1

Common Common Common Type 1 specific Type 1 specific

Fd35 Fd48 Fd138-80 Fd170

2a 2a 2a 2a

1500 (---jd 500 4000

Common Common Common Common

(-) (-)

a Immunoglobulin subtypes were determined by enzyme immunoassay using phosphatase-labeled mouse IgG subtype specific antibodies prepared in goat (Southern Biotechnology Associate Inc., Birmingham, Ala.). b Neutralizing activity was determined by HSV-1 plaque reduction assay on BS-C-1 cells, and is shown as dilution of each hybridoma culture supernatant giving 50% plaque reduction per milliliter. The concentrations of these monoclonal antibodies ranged from 2.5 to 350 pg of mouse IgG/ml of culture supernatants as determined by enzyme immunoassay. ‘Type specificity was determined by indirect immunofluorescence assay using F (type 1) and G (type 2) strain-infected BS-C-1 cells separately. dClone Fd48 was comnlement dependent (3000 with complement). Fresh guinea pig serum was used to determine complement dependent neutralization.

to demonstrate the localization of HSV-1 glycoproteins during their synthesis and processing by IFA. Human foreskin (HFS) fibroblast cells were infected and fixed at different time periods postinfection for the intracellular studies. In parallel, unfixed, infected cells were prepared for the surface antigen expression studies. Cell preparations were reacted with each of the monoclonal antibodies and, subsequently, stained with FITC-conjugated goat antimouse IgG. Glycoprotein D was detected as early as 90 min postinfection while the synthesis of gB and gC could not be demonstrated in this time period (Fig. 2). Thus, this type of monoclonal antibody will be very helpful in detection of HSV early antigen. Figure 2a shows the presence of gD

in the nucleus-associated membrane or rough endoplasmic reticulum (RER). Subsequently, at 4 hr postinfection, fluorescence was observed at the Golgi apparatus and at nuclear membrane of the infected cells treated with anti-gD monoclonal antibody (Fig. 2B). The presence of gB and gC, on the other hand, could be first clearly detected in RER (Figs. 2E, H, respectively) at 4 hr postinfection although in some experiments, specific fluorescence was also observed at 3 hr postinfection. However, none of the anti-gB or anti-gC monoclonal antibodies detected the presence of gB or gC earlier than 3 hr postinfection. At 6-8 hr postinfection, both gB and gC could be detected in Golgi apparatus of HSV-l-infected human fibroblast cells (Figs. 2F, I,

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FIG. 2. Intracellular immunofluorescence pattern of HSV-l-infected human fihrohlast cells. Human foreskin fibroblast cells were infected with F strain of HSV-1 at a m.o.i. of 1. Cells were fixed with a mixture of 95% ethanol and 5% acetic acid 1.5, 4, and 8 hr postinfection and processed for immunofluorescence assay, as described previously (U), using different monoclonal antibodies against HSV-1 glycoproteins. In order to stain the Golgi complex, the fixed cells were treated with rhodamineconjugated wheat germ agglutinin after staining with fluorescein-conjugated mouse IgG. (A, B, C) Stained with monoclonal antibodies against gD, 1.5, 4, and 8 hr postinfection, respectively; (D, E, F) stained with monoclonal antibodies against gB, 1.5, 4, and 8 hr postinfection, respectively; and, (G, H, I) stained with monoclonal antibodies against gC, 1.5, 4, and 8 hr postinfection, respectively. Arrow indicates RER and arrowheads indicate Golgi complex.

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respectively). At this time, fluorescence tibodies will be utilized in rapid diagnosis could also be demonstrated at nuclear of HSV-specific antigens by various techmembrane of the infected cells. Subse- niques. quently (10 to 12 hr postinfection) all three ACKNOWLEDGMENTS glycoproteins were detected at the surface of the infected HFS cells (data not shown). The authors thank Sheila Foitz for her help in the Four major species of HSV-l-specified preparation of this manuscript and Dr. E. Hunter for glycoproteins designated gB, gC, gD, and his critical review. This work was supported under gE have been identified (1-3). The biologic Contract NO-l-AI-12667 from the Developmental and functions of these glycoproteins are not yet Applications Branch, National Institute of Allergy and Infectious Diseases, and by Japan Chemical Research clearly understood (16). The biosynthetic pathway of HSV glycoproteins in general, Company, Ltd., Kobe, Japan. follows that of cellular or viral glycosylated REFERENCES polypeptides. However, the processing of 1. SPEAR, P. G., J. viral. 17,991-1998 (1976). HSV-specified glycoproteins from imma2. BAUCKE, R. B., and SPEAR, P. G., J. Fir01 32,779ture to mature products probably occurs in 789 (1979). a multistep fashion. Newly synthesized a EBERLE, R., and COURTNEY, R. J., J. fir01 36,665glycoproteins, in most cases, migrate from 675 (1980). RER to the Golgi cisternae and, trans4 ROIZMAN, B., NORRILD, B., CHAN, C., and PEREIRA, ported to their final destination, the cell L., virology 133,242-247 (1984). surface where budding occurs. In this 5. MANSERVIGI, R., SPEAR, P. G., and BUCHAN, A., report utilizing monoclonal antibodies Proc Natl. Acad Sci USA 74,3913-3917 (1977). 6. SARMIENTO, M., HAFFEY, M., and SPEAR, P. G., J. against HSV-glycoproteins, we have demViral. 29,1149-1158 (1979). onstrated the presence of mature glyco7. KOUSO~LAS, K. G., BZIK, D. J., and PERSON, S., Inproteins on the cell surface as well as on teruirology 20,56-60 (1983). nuclear membrane where virus particles 8. FRIEDMAN, H. M., COHEN, G. H., EISENBERG, R. J., bud into the perinuclear region (16). Thus, SEIDEL, C. A., and CINES, P. B., Nature (London) HSV glycoproteins could play a pleiotropic 309,633-635 (1984). role, i.e., induction of cell fusion through 9. BALACHANDRAN, N., HARNISH, D., KILLINGTON, the cell surface molecules (in addition to R. A., BACCHET~I, S., and RAWLS, W. E., J. vird other biological functions) and budding of 39,438-446 (1981). virus particles through those located on the 10. KEARNEY, J. F., RADBRUCH, A., LIESEGANG, B., and 123, X48-1550 RAJEWSKY, K., .I. Immwwl inner nuclear membrane. (1979). The three major glycoproteins described here vary in respect to their time of ap- 11. CHAT~ERJEE, S., and HUNTER, E., virdogy 95,421433(1979). pearance in the infected cells. Appearance 12. CHATTERJEE, S., BRADAC, J., and HUNTER, E., J. of gD could be detected earlier than gB and firol. 44,1903-1012 (1982). gC in the HSV-infected cell. This obser- 1.9.ZWEIG, M., SHOWALTER, S. D., BLADEN, S. V., vation supports the data reported by BalHEILMAN, C. J., JR., and HAMPAR, B., J. vird achandran et al. (17’). However, at the cell 47,185-192 (1983). surface, both gD and gB can be demon- 14 ZEZIJLAK, K. M., and SPEAR, P. G., J. Fir01 49,741747 (1984). strated almost at the same time postinfection, suggesting that gD and gB share a 15. SWAIN, M. A., PEET, R. W., and GALLOWAY, D. A., J Viral 53,561-569 (1985). common biological role at the cell surface. ROIZMAN, B., and BATTERSON, W., In “Virology” 16. Indeed, recent data from this laboratory (B. N. Fields, et al, eds.), pp. 497-526. Raven indicate that probably gD and gB are both Press, New York, 1985. involved in cell-to-cell fusion activity of 17: BALACHANDRAN, N., HARNISH, D., RAWLS, W. E., this virus (18,19). Studies are now in progand BACCHETTI, S., J. I%oE 44,344-355 (1982). ress to investigate the exact biological roles 18. CHATTERJEE, S., LAKEMAN, A. D., WHITLEY, R. J., of these HSV-glycoproteins in viral pathoand HUNTER, E., virus Res. 1,81-87 (1984). genesis utilizing different monoclonal an- 19. CHAITERJEE, S., HUNTER, E., and WHITLEY, R. J., J. Viral 56.419-425 (1985). tibodies. In addition, these monoclonal an-