Preparation and characterization of monoclonal antibodies against occluded virions of Bombyx mori nuclear polyhedrosis virus

JOURNAL

OF INVERTEBRATE

PATHOLOGY

57, 31 l-324 (191)

Preparation and Characterization of Monoclonal Antibodies against Occluded Virions of Bombyx mori Nuclear Polyhedrosis Virus TOSHIHIRO NAGAMINE AND MICHIHIRO KOBAYASHI Laboratory of Sericultural Science, Faculty of Agriculture, Chikusa, Nagoya 464-01, Japan

Nagoya University,

AND

SHINSUKE SAGA AND MUNEMITSU HOSHINO’ The Second Department of Pathology, Nagoya University School of Medicine, Showa, Nagoya 466, Japan Received March 27, 1990; accepted July 5, 1990 Six hybridoma cell lines secreting monoclonal antibodies (MAbs) to the structural polypeptides of occluded virions of Bombyx mori nuclear polyhedrosis virus (BmNPV) were established. These MAbs were characterized by immunoblot analysis and classified into three groups on the basis of their specificity to the viral polypeptides of occluded Cons. MAbs classified as group 1 are reactive with occluded virion polypeptides exhibiting a smear pattern ranging in molecular weights (MWs) from 16,000 (16K) to 22K, but show no reactivity with the structural polypeptides of nonoccluded virions. In the BmNPV-infected cells, group 1 MAbs react with a single polypeptide with an apparent MW of 17K, suggesting that group 1 MAbs are directed against the 17K polypeptide or its counterpart present in the occluded virions. Group 2 MAbs react with two occluded virion structural polypeptides with MWs of 66 and 82K that are present in both nonoccluded virions and infected cells, and to a lesser extent, with six additional polypeptides of occluded virions. MAbs in group 3 consisting of four different species recognize the 40K polypeptide and one to five additional polypeptides ranging in MWs from 27 to 39K that are present in the infected cells but not detectable in the nonoccluded virions. In vitro translation of cytoplasmic RNA from infected cells and subsequent immunoprecipitation of translation products showed that polypeptides with MWs similar to those of the polypeptides additionally detected by group 3 MAbs were present among the immunoprecipitates. Immunofluorescence microscopy showed that localization of corresponding antigens in the infected cells differed distinctively among MAbs in the diierent groups. Q test Academic

Press, Inc.

WORDS: Bombyx mori; nuclear polyhedrosis virus; monoclonal antibody; cell culture; occluded viron. KEY

INTRODUCTION Bombyx mori nuclear polyhedrosis virus (BmNPV) is a member of subgroup A baculoviruses in the family Baculoviridae (Matthews, 1982) and pathogenic to several lepidopterous insects including Philosamia Cynthia pryeri, Limantria dispar japonica, and Dendrolimus spectabilis as well as B. mori (Aratake and Kayamura, 1973). The

rod-shaped nucleocapsids of BmNPV are predominantly singly enveloped, although in certain tissues, a number of virions con’ Deceased on May 23, 1988.

taining two to four nucleocapsids per envelope are also detectable (Watanabe, 1975). BmNPV contains double-stranded, circular DNA genomes ranging in molecular size from 115 to 125 kbp (Iatrou et al., 1985; Iizuka et al., 1987) and consists of more than 80 structural polypeptides as identified by high-resolution two-dimensional electrophoretic analysis (Sugimori et al., 1990). BmNPV is one of several well-characterized NPV species, and much information has been collected concerning the events involved in BmNPV multiplication (cf. Benz, 1986). Most of those studies, however, have been conducted in the system 311 0022-2011/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reprduction in any form reserved.

312

NAGAMINE

using whole insects and very few data are available on the events of BmNPV multiplication in cultured cells (Raghow and Grace, 1974). In studies with whole insects, exquisite analysis of the virus multiplication mechanism could not be achieved because of the difficulty in synchronizing the infection processes and subsequent events involved in virus multiplication occurring in whole insects. Furthermore, with whole insects, it is difficult to isolate genetically homogeneous virus stocks which provide the basis for any critical molecular studies of virus multiplication. Because of such disadvantages of whole insects in comparison to cultured cells, the mechanism of BmNPV multiplication is still poorly understood and the understanding of BmNPV lags behind that of several other NPVs which have been studied intensively in cultured cells. Studies using cultured cells have therefore been required for a better understanding of the multiplication mechanism of BmNPV. Recently Maeda (1984) showed that BmNPV was capable of forming plaques in BM-N cells derived from B. mori. This finding facilitated the molecular engineering studies of BmNPV, in which they constructed an expression vector employing the polyhedrin gene and produced interferon-o in Bombyx larvae as well as in cultured cells (Maeda et al., 1984, 1985). However, no systematic studies have appeared on the virus multiplication in this system. Monoclonal antibodies (MAbs) have been found to provide excellent tools for virus research, and a number of hybridoma cell lines that secrete MAbs directed against NPV structural polypeptides have been established (Roberts and Naser, 1982a,b; Hohmann and Faulkner, 1983; Quant et al., 1984). The MAbs raised against NPV polypeptides have been successfully applied to the identification and classification of NPV isolates (Naser and Miltenburger, 1982, 1983; Quant et al., 1984) and the immunocytochemical localization of viral polypeptides in the infected cells (Quant-Russell et al., 1987; Pearson et

ET

AL.

al., 1988) and would also be useful for those studies such as analysis of antigenic structures of viral polypeptides, identification of biological functions of antigenic epitopes of viral polypeptides, and examination of relationship among polypeptides in a virus. In an effort to facilitate the study on the mechanism of BmNPV replication, we examined the composition of structural polypeptides (Sugimori et al., 1990) and the property of genomic DNA of a plaquepurified isolate of BmNPV. In addition, we have shown the growth characteristics of the virus (Nagamine et al., 1989). In the present study, we prepared six MAbs directed against structural polypeptides of polyhedron-derived occluded virions of BmNPV. Characterization by some immunological techniques showed that these MAbs fell into three groups that differed in their specificity to viral structural polypeptides and their localization of corresponding antigens in the infected cells. MATERIALS

AND METHODS

Virus and Cell The plaque-purified isolate of BmNPV (Nagamine et al., 1989) was used in these experiments. The working stocks of the cloned virus were prepared by passing the virus two times on BM-N cell cultures at an input multiplicity of infection (MOI) of about 0.1 plaque-forming units (PFU) per cell. The continuous cell line, BM-N, derived from Bombyx (cf. Volkman and Goldsmith, 1982) was maintained at 28°C in BML-TClO medium (Gardiner and Stockdale, 1975) supplemented with 10% fetal calf serum. Virus Propagation

in Bombyx Larvae

Two-day-old frfh instar larvae were each injected with 25 pl of virus suspension at 1 X lo6 PFU/ml and the larvae inoculated were reared at 25°C on mulberry leaves. The infected larvae were collected 5 days

MONOCLONAL

ANTIBODIES

after the inoculation and stored at -20°C until used. The larvae employed for inoculation were reared aseptically at 25°C on an artificial diet (Kyodo Shiryo, Yokohama, Japan) and the nonoccluded virus used for inoculation was derived from the culture medium of BM-N cells infected with BmNPV.

313

OF B. mori NPV

25-55% (w/w) linear sucrose gradient in TE buffer in a Hitachi RPS-40T rotor. Virions were removed from the sucrose gradient, dialyzed overnight against TE buffer, and pelleted by centrifugation at 123,400gfor 1 hr at 4°C. The purified vu-ions were suspended in TE buffer and stored at - 70°C. Immunization

Virus Purification

Occluded virions were purified from the infected larvae as described by Kobayashi and Nakagaki (1984) with some modifications. Briefly, 100 infected larvae were homogenized in 5 vol (v/w) of water and polyhedra were purified from the homogenate by washing three times with water and once with 1 M NaCl with the aid of Polytron, followed by centrifugation on a 40-63% (w/ w) sucrose density gradient (Summers and Smith, 1978). Virions were released from the polyhedra by the treatment with 50 mM Na&Os for 30 min at 0°C and purified by centrifugation at 81,OOOgfor 120min at 4°C on a 30-60% (w/w) linear sucrose gradient in a Hitachi RPS-27 rotor. The purified virions were collected from the gradient, dialyzed against 5 rn~ Na,C03, and pelleted by the centrifugation at 123,400gfor 1 hr at 4°C. The purified vu-ions were suspended in distilled water and stored in aliquots at - 70°C. For the purification of nonoccluded virions, 1.5 x lo7 of BM-N cells were seeded in each 75-cm’ tissue culture flask and infected with BmNPV at an MO1 of 1 PFUI cell at 24 hr after the cell seeding. After incubation for 48 hr at 28”C, culture medium was collected from 15 culture flasks and clarified by centrifugation at 12OOgfor 10 min. The tissue culture supematant was layered onto a cushion of 20% (w/w) sucrose in TE buffer (10 mM Tris-HCl, pH 7.8, 1 mM EDTA), and progeny virions were pelleted by centrifugation at 123,400g for 1hr at 4°C. The pellet was suspended in TE buffer and virions were purified by centrifugation at 198,600gfor 4 hr at 4°C on a

Occluded vu-ions purified from the infected larvae and disrupted in 0.1% sodium dodecyl sulfate (SDS) were used for immunizations of BALB/c mice. The mice were subcutaneously immunized four times with 1 mg BmNPV preparations in Freund’s complete adjuvant at 2- to 4-week intervals and were boosted intravenously 3 days prior to the removal of spleen. Production

and Screening

of Hybridomas

Spleen cells (3 x 10”) from the immunized mice were fused with NS-1 myeloma cells (5 x 10’) using 50% polyethylene glyco1 4000 as the fusing agent (Kohler and M&stein, 1975). After fusion, cells were suspended in HAT medium, distributed into %-well tissue culture plates, and incubated at 37°C for 2-3 weeks until the colonies of hybridoma developed. After an initial screening by an enzyme-linked immunosorbent assay (ELISA) (Kimura-Kuroda and Yasui, 1983), positive colonies were subjected to the second screening by an indirect immunofluorescence assay, and the cells were cloned by the limiting dilution procedure from the colonies producing antibodies which reacted preferentially with BmNPV-infected BM-N cells. Cloned hybridomas were injected into BALB/c mice and ascitic fluids were harvested l-3 weeks later. ELZSA

Occluded virions purified from infected larvae and disrupted in 0.1% SDS were used as the antigen. The disrupted virions

314

NAGAMINE

ET

AL.

at a protein concentration of 10 mg/ml were diluted lOO-fold with 100 mM carbonate buffer, pH 9.6, and individual wells of plastic %-well plates were coated with 50 ~1 of the antigen preparation. The plates were processed exactly as described by KimuraKuroda and Yasui (1983) using peroxidaseconjugated goat anti-mouse IgG.

were processed for different MAb preparations as described previously (Saga et al., 1985), and antigens reacted with each MAb were visualized either by the peroxidaseconjugated goat anti-mouse IgG or by autoradiography using 1251-labeled goat antimouse IgG (Amersham International, Amersham, England)

Immunoj7uorescence

Identification of Viral Antigens by MAbs in the BmNPV-Infected Cell Lysates

Staining

BM-N cells seeded on coverslips in 35 mm plastic Petri dishes were incubated at 28°C for 24 hr to semiconfluency and were infected with BmNPV N9 at an MO1 of 10 PFU/cell. At 12, 24, and 48 hr postinfection (pi), coverslips were removed from the dishes and cells were fixed for 30 min at room temperature in 4% paraformaldehyde in PBS+ (50 mM phosphate buffer, pH 7.4, 140 mM NaCl, 0.5 mM CaCl,, 0.2 mM MgCl,) containing 5% sucrose. After being permeabilized in 0.2% Triton X-100 for 4 min and blocked with 10% nonimmune goat serum in PBS+, fixed cells were successively incubated at room temperature for 30 min with individual MAbs and rhodamineconjugated goat anti-mouse IgG. Following incubation, stained cells were mounted in 80% glycerol containing 1 mg/ml p-phenylendiamine in 40 mM Tris-HCl, pH 8, and examined under an Olympus BH-RFL fluorescence microscope. For the screening of hybridomas producing MAbs specific to infected cells, uninfected and BmNPV-infected BM-N cells incubated in microtest tissue culture plates (Falcon, No. 3034) were fixed at 21 hr pi and processed for immunofluorescence microscopy. Immunoblot

Analysis

Polypeptides resolved on the SDSpolyacrylamide gels were transferred electrophoretically onto nitrocellulose membranes as described previously (Choi et al., 1989) according to the method of Towbin et al. (1979). The nitrocellulose membranes

Monolayer cultures consisting of 1.7 x IO6 cells in 35-mm plastic Petri dishes were infected with BmNPV N9 at an MO1 of 5 PFU/cell. At 3-hr intervals until 36 hr pi, cells were scraped into the gel-loading buffer (62.5 mM Tris-HCI, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol) and resolved by SDS-PAGE according to the method of Laemmli (1970). Polypeptides on the gels were then subjected to immunoblot analysis using six different MAbs, and viral antigens were visualized by autoradiography using 1251-labeled goat anti-mouse IgG. RNA Extraction

and In Vitro Translation

Monolayer cultures consisting of 5 x IO6 cells in 60-mm plastic Petri dishes were infected with BmNPV at an MO1 of 5 PFU/ cell. At 12-hr intervals pi, the cultures were washed twice with ice-cold PBS (50 mM phosphate buffer, pH 7.4, 140 mM NaCl) and the cells were scraped from the Petri dishes. After washing with PBS, the cells were suspended in 10 mM Tris-HCl, pH 8.5, containing 1.5 mM MgCl, and 140 mM NaCl, to which Nonidet-P40 was added to 0.1%. The cell suspension was vortexed for several times and centrifuged at 2000g for 8 min at 4°C to precipitate nuclear materials. The supematants were removed into 10 rnrvr Tris-HCl, pH 8, containing 2 mM EDTA, 2 mM NaCl, and 0.1% SDS and extracted with phenol (saturated with 100 mM TrisHCl, pH 9):chloroform:isoamylalcohol (25:24: 1). Cytoplasmic RNA extracted was

MONOCLONAL

ANTIBODIES

pelleted by the addition of 0.1 vol of 3 M CH,COON,, pH 5, and 2.5 vol of cold ethanol and kept at -20°C overnight. The RNA was dissolved in sterile water and employed for in vitro translation experiments. In vitro translation of the RNA was performed in a rabbit reticulocyte lysate pretreated with micrococcal nuclease (Wako Pure Chemicals, Osaka, Japan) in the presence of [35S]methionine (800-1200 Ci/ mmol, Amersham International, Amersham, England) as described previously (Kobayashi et al., 1988). Immunoprecipitation of in Vitro Translation Products

Thirty microliters of translation products was mixed successively with each 3 ~1 of 10% SDS and 20% Triton X-100. Following incubation at 30°C for 1 hr with 10 ~1 of ascitic fluids containing different species of MAbs, the mixtures were incubated at 30°C for another 1 hr with 25 pJ of Staphylococcal protein A-Cellulofine (Seikagaku Kogyo, Osaka, Japan) pretreated with 25 ~1 of 0.1% (w/v) rabbit anti-mouse IgG (ZYMED Laboratories, California). The immunoreaction mixtures were transferred to small columns made of l-ml micropipette tips, and antigen-antibody complexes on the protein A beads were eluted in the gelloading buffer as described previously (Kobayashi et al., 1988). The eluants were analyzed by SDS-PAGE and fluorography. Fluorography

After electrophoresis, gels were stained with Coomassie brilliant blue and processed for fluorography according to the method of Skinner and Griswold (1983). Dried gels were exposed to a preflashed Fuji X-ray fdm at -70°C. Zsotyping of MAbs

The isotype of MAbs produced by hybridomas was determined by the doublediffusion method employing the class-

OF

B. mori NPV

315

specific anti-mouse immunoglobulin sera (ZYMED Laboratories) and the hybridoma culture supematants as the antigens. The hybridoma culture supematants were concentrated lo-fold by a microconcentrator (Amicon, Lexington, Massachusetts) before use. RESULTS Production of Monoclonal Antibodies

BALB/c mice were immunized by occluded virions of BmNPV pretreated with 0.1% SDS, and immunized spleen cells were fused with NS-1 myeloma cells. Viable hybridomas derived from the fusion reactions were initially screened for their anti-BmNPV activity by ELISA using occluded virion polypeptides and peroxidase-conjugated anti-mouse IgG; thereby 40 hybridoma colonies were selected. After secondary screening by indirect immunofluorescence microscopy, 22 of 40 hybridoma colonies tested positive by the ELISA assay were found to produce antibodies that reacted with the infected BM-N cells at 21 hr pi. Further experiments by indirect immunofluorescence microscopy revealed that 14 out of 22 hybridoma colonies secreted antibodies that preferentially stain infected BM-N cells, and the remaining 8 hybridoma colonies produced antibodies positive to both infected and uninfected BM-N cells. Finally, 6 hybridoma cell lines with antibodies specifically positive to the infected cells were cloned by the limiting dilution procedure and were designated B9, F17, L39, R23,524, and D31. These hybridoma clones were each injected into BALB/ c mouse cavities and produced ascitic fluids that had ELISA titers of 1 X 105, 1.2 X 104, 4 x 105, 1.2 x 104, and 3.2 x lo3 for B9, F17, L39, R23, and 524, respectively (Table I). Isotyping experiments showed that MAbs secreted by B9, F17, 524, and D31 clones were IgGl while IgG2a was produced by L39 and R23 clones (Table 1). The ELISA titer of MAb D31 was not determined.

316

NAGAMINE

ET AL.

TABLE 1 CHARACTERIZATION OF MON~CLONAL ANTIBODIES TO B. mori NUCLEAR POLYHEDROSIS VIRUS OCCLUDED VlRlON POLYPEPTIDES Poiypeptide specificity’ Group No.”

Clone No.

w class

1 2 3

B9 F17 L39 K23 524 D31

1 1 2a 2a 1 1

ELISA titeP 1 x 105 1.2 x 104 4 x 16 1.2 x 104 3.2 x ld NW

Occluded virion

Nonoccluded virion

Cell lysated

16-2T 66,82 35-40’ 35-40 35-40 354

2 f&82 -

17 66,82 3740 35,37,39,40 21,29,35,37,39,40 3940

a MAbs were divided into three groups on the basis of their specificity to occluded virion polypeptides. b Individual wells of plastic %-well plates were coated with 5 pg of occluded virion polypeptides and used for the titration as described in the text using peroxidase-conjugated goat anti-mouse IgG as the second antibody. c Polypeptide specificity was determined by immunobbt analysis, and apparent MWs (X lo-‘) of the polypeptides reacted with respective MAbs were shown. d Cell lysates prepared at 36 hr pi were used for the determination of polypeptide specificity. e MAbs were reacted with polypeptides whose MWs were not well defined but were in the range from 16,000 (16K) to 22K or from 35 to 4OK. f No detectable reactivity was observed. B Not determined.

Speci&ity of MAbs to Virion Structural Polypeptides

showed that only MAb F17 reacted with nonoccluded virion structural polypeptides and gave clear bands of 66 and 82K polypeptides (Fig. 2, lane 6), as in the case when occluded virions were subjected to the immunoblot analysis (Fig. 1, lane 3). On the other hand, when polyclonal antiserum conventionally raised against occluded virions in a mouse was used, the immunoblot analysis showed that at least 15viral polypeptides were detectable in the occluded virions (Fig. 2, lane 4), whereas in the nonoccluded virions, only 8 of 21 viral polypeptides detectable on the Coomassie brilliant blue-stained gel reacted with the specific antiserum (Fig. 2, lane 5). The 66 and 82K polypeptides identified by F17 and the major structural polypeptide with an approximate MW of 39K are included among these 8 polypeptides (Fig. 2, lane 5).

Structural polypeptides of purified occluded virions were resolved on 10% SDSpolyacrylamide gels and subjected to the immunoblot analysis using respective MAbs (Fig. 1, Table 1). The results showed that six MAbs fell into three groups on the basis of their specificity to BmNPVoccluded virion polypeptides. MAb B9 (group 1) reacted with relatively low MW polypeptides (Fig. 1, lane 2) ranging in apparent MWs from 16to 22K as estimated on a 12.5% SDS-polyacrylamide gel (data not shown). MAb F17 (group 2) gave strong bands of 66 and 82K polypeptides and at least six additional faint bands (Fig. 1, lane 3). MAbs L39, R23, J24, and D31 (group 3) mainly reacted with polypeptides ranging in molecular weights from 35 to 40K (Fig. 1, Immunoblot identification of Viral lanes 4-7). Polypeptides Expressed in the Infected These six MAbs were also examined for Cells by MAbs their specificity to the structural polypeptides of nonoccluded virions. The results The MAbs were analyzed for their spec-

MONOCLONAL 1

ANTIBODIES

1

23456769

317

OF B. mori NPV 2

3

4

I

6

ov

n7

-n2-66--c

-39-

/

F-

FIG. 1. Immunoblot identification of BmNPV occluded virion structural polypeptides by MAbs. About 1 mg of structural polypeptides was applied to a sample well (35~mm width) and electrophoresed on a 10% SDS-polyacrylamide gel. Polypeptides in lanes 2-9 were transferred to a nylon membrane, cut into strips (3-mm width), and reacted with a L/l00dilution of ascitic fluids containing B9 (lane 2), F17 (lane 3), L39 (lane 4), B23 (lane 5), 524 (lane 6), or D31 (lane 7) MAb. The polypeptides were also reacted with a polyclonal antibody conventionally raised in the mouse against occluded virion structural polypeptides (lane 8) and nonimmune mouse serum (lane 9). Peroxidase-conjugated goat anti-mouse IgG at a dilution of l/zoo was used as the second antibody. Lane 1 represents occluded virion structural polypeptides analyzed on a 10% SDSpolyacrylamide gel and stained with Coomassie brilliant blue. The numbers alongside the gel indicate MWs (X 10m3). F, front of the gels.

ificity to the viral polypeptides being expressed in the infected cells. BM-N cells were infected with BmNPV at an input MO1 of 5 PFU per cell, and cell lysates harvested at various times pi were resolved by SDS-PAGE followed by the immunoblot analysis using six different MAbs. The results showed that antigens specific to these MAbs were first detected between 15 and 21 hr pi and increased with time pi until 36 hr pi (Fig. 3). The time pi when the virusspecific antigens became detectable coincided with when the extracellular nonoceluded virions started to be released into the culture medium (cf. Nagamine et al.,

AD--ov

2. Immunoblot identification of BmNPV nonoccluded virion structural polypeptides by MAbs. Nonoccluded (lanes 3, 5, and 6) and occluded virion polypeptides (lanes 2 and 4) were resolved on 12.5% SDS-polyacrylamide gels and stained with Coomassie brilliant blue (lanes 2 and 3) or transferred to nitrocellulose membranes (lanes 4,5, and 6). The polypeptides transferred to nitrocellulose membranes were reacted with a YIO,OOO dilution of anti-occluded virion serum conventionally raised in the mouse (lanes 4 and 5) or a I/COOdilution of ascitic fluid containing F17 MAb (lane 6). 1251-labeled goat anti-mouse IgG was used as the second antibody. The immunoblots were exposed to X-ray films for 24 (lanes 4 and 5) or 48 hr (lane 6) at -70°C. MWs (X lo-‘) of the marker polypeptides are indicated on the left. FIG.

1989). MAb B9, which gave several indistinct bands in the region between 16 and 22K on the immunoblot of occluded virions, reacted with a single polypeptide of 17K (Fig. 3A). MAb F17 recognized 66 and 82K polypeptides that appeared at 21 and 30 hr pi, respectively, and corresponded to those detected on the immunoblot of the polypeptides of occluded and nonoccluded virions (Fig. 3B). MAbs in group 3, L39, R23, J24, and D31 reacted with the 40K polypeptide appearing between 15 and 18 hr pi, and each MAb recognized one to five additional polypeptides with apparent MWs of 27, 29, 35, 37, and 39K (Figs. 3C-F).

FIG. 3. Immunoblot identification of viral polypeptides in BmNPV-infected BM-N cells by MAbs. BM-N cells were infected with BmNPV at an MO1 of 5 PFUkell and cell lysates were harvested at 3-hr intervals until 36 hr pi. Polypeptides were resolved on 12% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and then reacted with a YIOOO dihttion of ascitic fluids containing B9 (A), F17 (B), L39 (C), B23 (D), 524 (E), or D23 MAb (F). ‘251-labeled goat anti-mouse IgG was used for the second antibody and immunoblots were processed as in Figure 2. Approximate MWs (X 10T3) of the polypeptides identified by MAbs are indicated on the right. Lane c represents uninfected cells. 318

MONOCLONAL

Zmmunoprecipitation of in Vitro Translation Products of Infected RNA by MAbs

ANTIBODIES

OF E. mori NPV

319

Cell

To examine whether or not the translatable mRNAs encoding the polypeptides with MWs smaller than 40K that are detectable by group 3 MAbs are present in the infected cells, cytoplasmic RNAs from infected cells at 12, 24, and 48 hr pi were translated in vitro in a rabbit reticulocyte lysate in the presence of [“Slmethionine, and the translation products were immunoprecipitated with MAb R23. Analysis of the immunoprecipitates by fluorography showed that not only the 40K polypeptide but also 30, 37, and 39K polypeptides were reactive to MAb R23 (Fig. 4). Detection of Virus-Specijic Antigens the Infected Cells by Zmmunofluorescence Microscopy with MAbs

in

BM-N cells were infected with BmNPV and fixed with paraformaldehyde at 12,24, and 48 hr pi. The fixed cell preparations were then stained with rhodamineconjugated goat anti-mouse IgG in conjunction with either of six MAbs from the three different groups. Preliminary observations under the fluorescence microscope showed that there was no detectable staining in the infected culture at 12 hr pi as well as in the uninfected culture, and clear staining in the infected cells was first observed at 24 hr pi irrespective of the MAb used. The intensity of the staining at 48 hr pi was similar to that at 24 hr pi except when MAb B9 was used. With MAb B9, a slightly less intense staining was observed at 48 hr pi (data not shown). Further observations of the infected cells at 24 hr pi revealed that the staining patterns in the infected cells differed considerably among MAbs in the different groups. With MAb B9, staining was observed in the cytoplasm and in the periphery of the nucleus (Fig. 5B). When stained with MAb F17, the nuclei of infected cells were uni-

FIG. 4. Immunoprecipitation by MAbs of in vitro translation products of cytoplasmic RNA from BmNPV-infected BM-N cells. BM-N cells were mockinfected (lane C) or infected with BmNPV at an MO1 of 5 PFU/cell and cytoplasmic RNAs were extracted at 12 (lane 12), 24 (lane 24), and 48 (lane 48) hr pi. Cytoplasmic RNAs were translated in vitro in a rabbit reticulocyte lysate in the presence of [35S]methionine, and the translation products were reacted with a ascitic fluid containing R23 MAb. The immunoprecipitates were reacted with rabbit anti-mouse IgG and then recovered by using protein A-Cellulofine. The immunoprecipitates were resolved on a 12% SDSpolyacrylamide gel and processed for fluorography. Approximate MWs (X 10m3) of the polypeptides identified are indicated on the right.

formly stained (Fig. 5D). The four MAbs in group 3 gave a similar staining pattern (Fig. 5F). In the infected cells with little cytopathic effects, stain was associated with plasma membranes, while in the infected cells with cytopathic effects of rounding, stain was distributed mainly in the perinuclear regions. In some infected cells, both plasma and nuclear membranes were uniformly stained with group 3 MAbs. DISCUSSION In the present study, we established six hybridoma cell lines that secreted MAbs directed against structural polypeptides of

FIG. 5. Immunocytochemical localization of viral polypeptides by MAbs in three different groups. BmNPV-infected BM-N cells were fixed at 24 hr pi, reacted with B9 (A and B), F17 (C and D), and R23 (E and F) MAbs, respectively, and stained with rhodamine-conjugated goat anti-mouse IgG. The stained cells were examined under a phase-contrast microscope (A, C, and E) or under an immunofluorescent microscope (B, D, and F).

320

MONOCLONAL

ANTIBODIES

BmNPV-occluded virions . These hybridoma cell lines were screened for the production of anti-occluded virion activity by immunofluorescence microscopy after an initial screening by ELISA and were found to be reactive with SDS-denatured viral structural polypeptides, suggesting that their corresponding antigenic determinants might be conformation independent. These facts raise the possibility that the MAbs produced in the present study may serve as useful immunological reagents for a variety of studies to correlate the viral polypeptides with their biological functions by using immunocytochemical techniques and SDS-PAGE followed by immunoblot analysis. The MAbs produced in the present study are classified into three groups on the basis of their specificity to the structural polypeptides of occluded virions (Fig. 1, Table 1). MAbs classified as group 1 (B9) are reactive with polypeptides exhibiting a smear pattern ranging in MWs from 15 to 22K on the immunoblot of occluded virion polypeptides, but show no reactivity with the structural polypeptides of nonoccluded virions. Group 2 MAbs (F17) react with two structural polypeptides with MWs of 66 and 82K that are present in both the occluded and nonoccluded virions and, to a lesser extent, with six additional polypeptides of the occluded virions (Fig. 1). Group 3 MAbs consisting of four different species (L39, R23, 524, and D31) are reactive mainly with the 40K polypeptide, and none of them react with nonoccluded virion structural polypeptides. These MAbs in the different groups also differ in their localization in the infected BM-N cells (Fig. 5). In the BM-N cells infected with BmNPV, group 1 MAbs recognized only a single polypeptide with an apparent MW of 17K (Fig. 3), indicating that the polypeptides detected by group 1 MAbs as a smear pattern on the immunoblot of occluded virion polypeptides (Fig. 1, lane 2) were absent in the BmNPV-infected cultured cells. This result suggests that group 1 MAbs are directed

OF B. mri

NPV

321

against the 17K polypeptide or its counterpart that is present in the occluded virions but cannot be detected unambiguously on the immunoblot of occluded virion polypeptides. It is likely that the polypeptides exhibiting the smear on the immunoblot might be contaminants or artifacts of virus purification from polyhedra from insect larvae since they are not detectable in the nonoccluded virions . Analysis on Coomassie brilliant bluestained gels has shown that a polypeptide analogous to the 17K polypeptide is hardly present, if at all, in the BmNPV-occluded virions (Sugimori et al., 1990), and the present immunoblot analysis using a polyclonal antibody reactive with lfX2K polypeptides on the immunoblot of occluded virion polypeptides showed that no polypeptide correspondii to the 17K polypep tide was detectable in the nonoccluded virions (Fig. 2, lane 5). On the other hand, analysis of the lysates of [35Sjmethioninelabeled cells and in vitro translation products of cytoplasmic RNA has shown that a polypeptide analogous to the 17K polypep tide is abundantly synthesized and accumulates in a large amount in the BmNPVinfected BM-N cells (H. Sugimori et al., unpubl.). Similar results have also been obtained in Bombyx pupae infected with BmNPV (Kobayashi et al., 1990). In addition, immunofluorescence microscopy has shown that the 17K polypeptide locates in the nucleus of the infected cells (Fig. SB). The results described above suggest that the 17K polypeptide does not represent a major structural component of BmNPV virions but is responsible for the events concerning BmNPV multiplication. The 66 and 82K polypeptides detected in the infected cells by group 2 MAbs appear to correspond to the 66 and 82K polypep tides present in both occluded and nonoceluded vii-ions. This fact excludes the possibility that one or both of these polypeptides might be artificial products produced during the virus purification or the preparation of cell lysates for SDS-PAGE. On the

322

NAGAMINE

basis of their MWs, it is also excluded that the 82K polypeptide is a doublet of the 66K polypeptide. It remains to be elucidated whether these two polypeptides represent a precursor-product relationship or whether they exhibit distinctly different primary structures that share a common antigenic determinant (cf. Zweig et al., 1980). The MW of the 40K polypeptide recognized by group 3 MAbs on the immunoblot of purified occluded virions and cell lysates of the infected culture is comparable to those of major structural polypeptides present in both occluded and nonoccluded virions. However, group 3 MAbs failed to detect any structural polypeptides of nonoccluded virions, while the antioccluded virion serum from the mouse used for the preparation of MAbs was able to react with the major structural polypeptides of nonoccluded virions with an approximate MW of 39K. Whether the major structural polypeptides of approximately 39K are localized in the nucleocapsid or in the envelope has not been established because of the difficulty to remove selectively the envelope from BmNPV virions (Sugimori et al., 1990), it appears probable from their MWs and abundance in the purified virions as compared to other baculovirus that these major structural polypeptides are the components of nucleocapsid that would be common in both occluded and nonoccluded virions (cf. Summers and Smith, 1978; Stiles et al., 1983; Monroe and McCarthy, 1984; Pearson et al., 1988). It is therefore inconclusive whether or not the 40K polypeptide identified by group 3 MAbs is one of the major structural polypeptides of BmNPV virions . In addition to the 40K polypeptide, several polypeptides ranging in MWs from 27 to 39K were identified in the infected cells at later times pi by the immunoblot analysis using group 3 MAbs (Figs. 3C-F). The reasons for the detection of these smaller MW polypeptides by group 3 MAbs are unclear. However, it is unlikely that these polypeptides represent the polypeptides reacted

ET AL.

nonspecifically with the MAbs since numbers of the smaller MW polypeptides detected differ with different MAbs used. It also appears unlikely that these smaller MW polypeptides, at least those of 37 and 39K, are the artifacts of sample preparation for SDS-PAGE since the in vitro translation experiment showed that the polypeptides of 37 and 39K, as well as the 40K polypeptide, were immunoprecipitated by one of the group 3 MAbs. To characterize these viral antigens, we have constructed the hgtll library of BmNPV DNA and isolated several clones reactive with group 3 MAbs. Further experiments are now in progress. ACKNOWLEDGMENTS T.N. and M.K. thank Drs. S. Kawase, 0. Yamashita, and T. Yaginuma of their laboratory for helpful discussions during this study. This work was supported in part by grants-in-aid for scientific research (No. 61480052 and No. 63440011) from the Ministry of Education, Science and Culture of Japan.

REFERENCES Y., AND KAYAMURA, T. 1973. Pathogenecity of a nuclear-polyhedrosis virus of the silkworm, Bombyx mori, for a number of lepidopterous insects. Japan. J. Appl. Entomol. Zool., 17, 121126. BENZ, G. T. 1986. Introduction: Historical prospective. In “Biology of Baculoviruses” (R.R. Granados and B.A. Federici, Eds.), Vol. 1, pp. l-35. CRC Press, Boca Raton, FL. BLISSARD, G. W., QUANT-RUSSELL, R. L., ROHRMANN, G. F., AND BEAUDREAU, G. S. 1989. Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding ~39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyiapseudotsugata. Virology, 168, 354-362. CHOI, H. K., KOBAYASHI, M., AND KAWASE, S. 1989. Inhibition of the accumulation of virus-specific translatable mRNA and structural polypeptides by guanidine hydrochloride in the midgut of the silkworm, Bombyx mori, infected with infectious flacherie virus. J. Znvertebr. Pathol., 53, 392400. GARDINER, G. R., AND STOCKDALE, H. 1975. Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J. Znvertebr. Pathol., 25, 363-370. ARATAKE,

MONOCLONAL

ANTIBODIES

HOHMANN, A. W., AND FAULKNER, P. 1983. Monoclonal antibodies to baculovirus structural proteins: Determination of specificities by Western blot analysis. Virology, 125, 432-444. IATROU, K., ITO, K., AND WITKIEWICA, H. 1985. Polyhedrin gene of Bombyx man’ nuclear polyhedrosis virus. .I. Viral., 54, 43W5. IIZUKA, T., AOKI, S., AND GOTO, C. 1987. Identitication of two nuclear polyhedrosis virus from the spotted cut-worm, Xestia c-nigrum (Lepidoptera, Noctuidae). J. Fat. Agricult. Hokkaido Univ., 63, 104113. KIMURA-KURODA, J., AND YASUI, K. 1983. Topographical analysis of antigenic determinants on envelope glycoprotein V3 (E) of Japanese encephalitis virus, using monoclonal antibodies. J. Virol., 45, 124-132. KOBAYASHI, M., AND NAKAGAKI, M. 1984. Effect of dissolution conditions of polyhedral inclusion bodies in sodium carbonate solution on the yield of nuclear polyhedrosis virus of the silkworm, Bombyx mori, (Lepidoptera: Bombycidae). Appl. Entomol. Zool., 19, 280-287. KOBAYASHI, M., HASHIMOTO, Y., SEKI, H., AND WATANABE, H. 1988. In vitro translation of RNA from the midgut of the silkworra, Bombyx mori, infected with a densonucleosis virus. J. Znvertebr. Pathol., 52, 259-267. KOBAYASHI, M., KOTAKE, M., SUGIMORI, H., NAGAMINE, T., AND KAJIURA, Z. 1990. Identification of virus-specific polypeptides and translatable mRNAs in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus. J. Znvertebr.

Pathol.,

55, 52-60.

KOHLAR, G., AND MILSTEIN, C. 1975. Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature (London), 256, 49W97. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature

(London),

227, 680-685.

MAEDA, S. 1984. A plaque assay and cloning of Bombyx mori nuclear polyhedrosis virus. J. Sericult. Sci. Japan,

53, 547-548.

MAEDA, S., KAWAI, T., OBINATA, M., CHIKA, T., HORIUCHI, T.. MAEKAWA, K., NAKASUJI, K., SAEKI, SAWA,

Y.,

SATO,

Y.,

YAMADA,

K.,

AND

FURU-

M. 1984. Characteristics of human interferon-a produced by a gene transferred by a baculovims vector in the silkwortn, Bomyx mori. Proc. MAEDA,

Japan

Acad.

Ser. B, 60, 423-426.

S., KAWAI, T., OBINATA, M., FUJIWARA, H., HORIUCHI, T., SAEKI, Y., SATO, Y., AND FuRUSAWA, 1985. Production of human a-interferon in silkworm using a baculovirus vector. Nature (London),

315, 592-594.

MATTHEWS, R. E. F. 1982. Classification and nomenclature of viruses; Fourth Report of the Intema-

OF B. mori

323

NPV

tional Committee on Taxonomy of Viruses. Zntervi17, 1-199. MONROE, J. E., AND MCCARTHY, W. J. 1984. Polypeptide analysis of genotypic variants of occluded Heliothis spp. baculoviruses. J. Znvertebr. Pathol., 43, 32-40. NAGAMINE, T., SHIMOMURA, M., SUGIMORI, H., AND KOBAYASHI, M. 1989. Titration of Bombyx mori (Lepidoptera: Bombycidae) nuclear polyhedrosis viNS in a Bombyx mori cell line. Appl. Entomol. rology,

Zool.,

24, 235-237.

NASER, W. L., AND MILTENBURGER, H. G. 1982. A rapid method for the selective identification of Autographa califirnica nuclear polyhedrosis virus using a monoclonal antibody. FEMS Microbial. Lett., 15, 261-264.

NASER, W. L., AND MILTENBURGER, H. G. 1983. Rapid baculovirus detection, identification, and serological classification by Western blotting-ELISA using a monoclonal antibody. J. Cert. Virol., 64, 639647. PEARSON, M. N., RUSSELL, R. Q., ROHRMANN, G. F., AND BEAUDREAU, G. S. 1988. ~39, a major baculovirus structural protein: Immunocytochemical characterization and genetic location. Virology, 167, 407413.

QUANT, R. L., PEARSON, M. N., ROHRMANN, G. F., AND BEAUDREAU, G. S. 1984. Production of polyhedrin monoclonal antibodies for distinguishing two Orgyia pseudotsugata baculoviruses. Appl. Environ.

Microbial.,

48, 732-736.

QUANT-RUSSELL, R. L., PEARSON, MANN, G. F., AND BEAUDREAU,

M.

N.,

ROHR-

G. S. 1987. Charp10 synthesis using

acterization of baculovirus monoclonal antibodies. Virology, 160, 9-19. RAGHOW, R., AND GRACE, T. D. C. 1974. Studies on a nuclear polyhedrosis virus in Bombyx mori cells in vitro. 1. Multiplication kinetics and ultrastructural studies. J. Ultrastruct. Res., 47, 384-399. ROBERTS, P. L., AND NASER, W. 1982a. Preparation of monoclonal antibodies to a baculovirus. FEMS Microbial.

Lett.,

14, 79-83.

ROBERTS, P. L., AND NASER, W. 1982b. Characterization of monoclonal antibodies to the Autographa californica nuclear polyhedrosis virus. Virology, 122, 424-430.

S., HAMAGUCHI, M. HOSHINO, M., AND KoK. 1985. Expression of meta-vinculin associated with differentiation of chicken embryonal muscle cells. Exp. Cell Res., 156, 45-56.

SAGA, JIMA,

SKINNER,

M. K., AND GRISWOLD,

M. D. 1983. Fluo-

rographic detection of radioactivity in polyacrylamide gels with 2,Sdiphenyloxazole in acetic acid and its comparison with existing procedures. Biothem.

J., 209.281-284.

STILES, B., BURAND, J. P., AND WOOD, H. A. 1983.

324

NAGAMINE

Characterization of gypsy moth (Lymuntriu nuclear polyhedrosis virus. Appl. Environ.

dispar) Micro-

biol., 46, 297-303. SUGIMORI, H., NAGAMINE, T., AND KOBAYASHI, M. 1990. Analysis of structural polypeptides of Bombyx mori (Lepidoptera: Bombycidae) nuclear polyhedrosis virus. Appl. Entomol. Zool., 25, 67-77. SUMMERS, M. D., AND SMITH, G. E. 1978. Baculovirus structural polypeptides. Virology, 84,390-402. TOWB~N, H., STAEHELIN, R., AND GODON, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA, 76,

4350-4354.

ET

AL.

VOLKMAN, eralized nuclear

L. E., AND GOLDSMITH, P. A. 1982. Genimmunoassay for Autographa californica polyhedrosis virus infectivity in vitro. Appl.

Environ. Microbial.,

44, 227-233.

WATANABE, H. 1975. Variation in the number of nucleocapsid within the envelope of nuclearpolyhedrosis virus multiplied in different tissues of the silkworm, Bombyx mori. .I. Sericult. Sci. Japan,

44,497-498. ZWEIG, M., HEILMAN, JR., C. J., RAABIN, H., AND HAMPAR, B. 1980. Shared antigenic determinants between two distinct classes of proteins in cells infected with herpes simplex virus. J. Viral., 35, 644-

652.