Biotechnol. Bioprocess Eng. 1997, 2, 59-61

B i o c h e m i c a l Analys i s of B m N P V A t t a c h m e n t to Bombyx Mori BmN-4 Cel ls

T a e Y o n g Kim' , H a n C h u l K a n g ~, J i n O P a r k 1, J o n g H w a P a r k 1, S e o k K w o n K a n g 3 , a n d I n S i k C h u n g 1.

~Institute and Depar tment of Genetic Engineering, Kyung Hee University, Suwon 449-701, Korea 2Rural Development Administrat ion, Suwon 441-100, Korea '~Department of Agricultural Biology, Seoul National University, Suwon 441-744, Korea

Binding characteristics of Bombyx mori nuclear polyhedrosis virus (BmNPV) to B. rnori BmN-4 cells was determined. The cells had a single class of BmNPV-binding sites and dis- played a low binding capacity for BmNPV in the range of 70 sites per cell. The biochemical nature of BmNPV-binding sites on the cell surface was identified. Treatment of BraN-4 cells with protease, wheat germ agglutinin, metabolic inhibitors, or lysosomotropic agents reduc- ed BmNPV binding to BmN-4 cells, whereas treatment with phospholipase C showed no ef- fect on virus binding. These results indicate that the structure of the binding site moiety is a protein-oligosaccharide complex.

Key words: Bombyx rnori nuclear polyhedrosis virus, BmN-4 cells, binding site

INTRODUCTION

The baculovirus, Bornbyx mori nuclear polyhedrosis v i rus (BmNPV) has been used as a vector for the ex- press ion of heterologous proteins in insect cells [1, 2]. I t is a rod-shaped virus with double s t randed and cir- cular DNA genome. BmNPV is capable of infecting B. mori BraN-4 cells. There are two forms of the virus: oc- c luded virus (OV) and non-occluded virus (NOV). In t i ssue culture, the NOV is more infectious t h an OV. The major i ty of the NOVs has been observed to en ter cells in vivo by fusion at the cell surface [3]. However, the infectious ent ry route into t issue cul ture cells has been shown to be pr imar i ly through receptor-medi- a ted endocytosis; a minor i ty has appeared to en ter th rough some other route, possibly by fusion at the cell surface [4].

A t t a c hme n t of virus part icles to specific receptors on p lasma membrane is one of the initial events in the i n t e r a c t i on of baculovi ruses wi th insect cells. The a t t a c h m e n t k ine t i c s of b a c u l o v i r u s e s Lymantria dispar NPV [5] and Autographa californica NPV [3] have been inves t iga ted . However , the a t t a c h m e n t process of a re la ted baculovirus, BmNPV, is ye t to be studied. The BmNPV may follow an a t t achmen t pro- cess different from those of LdNPV and AcNPV since it has re la t ively nar row host specificity both in vivo and in vitro among baculoviruses [6]. Fur the rmore , it is unc lear whe ther the biochemical na tu re of Bm N P V b i n d i n g s i tes is iden t ica l among the va r ious cell species.

The objectives of this s tudy are to verify the binding characteris t ics of BmNPV to BmN-4 cells and to ex- amine the biochemical na ture of binding sites on cell

* Corresponding author Tei:0331-280-2436 Fax:0331-281-4969 e-maih ischung@nms.kynnghee, ac.kr

surface.

MATERIALS AND METHODS

Cells, Media, and Virus Bombyx mori BmN-4 cells were grown in TC-100

(Sigma, St. Louis, MO) medium supplemented with 0.35 g 1 1 sodium bicarbonate, 50 ~g ml ~ gentamycin (Sigma, St. Louis, MO), 2.5 ~g ml fungizone and 10% fetal bovine serum (Gibco, Grand Island, NY). Bombyx mori nuclear polyhedrosis virus was used in all ex- 32 periments. F o r " P-labeled BmNPV, cells were infected wi th B m N P V and the inocu lum was r ep l aced by Grace 's m e d i u m minus phospha te con ta in ing 10% s e r u m a n d 10 pCi ml ' of ' ~2P-o r thophospha t e (Amersham, Buckinghamshire, England). Cells were then incubated at 27~ and virus was harves ted at two days before cell lysis occurred. Virus was concentra ted and purified by centrifugation at 1000• g for 20 min to remove cells and cell debris. The superna tan t was then centr i fuged at 8000• g through a 35% w/w sucrose cushion. The virus pellet was allowed to resuspend overnight in TC-100 medium.

Scatehard Analysis B m N P V b ind ing was m e a s u r e d by a d d i n g pro-

gressively higher concentrat ions of labelled virus to cells. 32P-labeled B m N P V were incubated wi th con- s tant number (4• 10 '5 cells/plate) of BraN-4 cells at 4~ for 3 h. Heat - t rea ted BmN-4 cells were used as a non- permissive control for non-specific binding. All the ex- per iments were performed at tr iplicate for scatchard analysis. The radioactivi ty was measured by a scin- ti l lation counter (Wallac, 1209 RackBeta, Turku, Fin- land). The n u m b e r of bound/f ree par t ic les aga ins t bound particles were plotted for a Scatchard analysis. TCID~0 was de termined for BmNPV as the procedure described elsewhere [7] and converted into infectious

60 Biotechnol. Bioprocess Eng. 1997, Vol. 2, No. 1

part icle number [8]

BmNPV-binding Studies

All the experiments were performed at triplicate for BmNPV-binding studies. BmN-4 cells were treated with enzymes and lectin as follows. We determined the effective concentrations for enzymes and lectin based on the data obtained form the literature [9, i0]. The cells were t rea ted with 1 mg ml 1 proteinase K (Gibco BRL, Grand Island, NY), 1 mg ml 1 c~-chymotrypsin (Sigma, St. Louis, MO), or 1 mg ml 1 phospholipase C (Sigma, St. Louis, MO) in PBS buffer (pH 6.2) for 1 h at 27~ and then washed with PBS to remove residual ac- tivity. The cells were also t rea ted with 2 mg ml 1WGA (Sigma, St. Louis, MO) in PBS buffer for 1 h at 27~ In the exper iments us ing glycosylation inhibitors, the cells were pre incubated for 48 h with 1 ~g ml I tun- i c amyc in (Sigma, St. Louis , MO) or 4 mM deox- ynojir imycin (Sigma, St. Louis, MO). For analysis of weak bases effects, BmN-4 cells were pre incubated with 30 mM NH4CI (Sigma, St. Louis, MO), or 15 mM methylamine (Sigma, St. Louis, MO) for 30 min at 4~ To study the effect of endocytosis inhibitors, the cells were preincubated with i0 mM sodium azide (Sigma, St. Louis, MO) or 1 mM dinitrophenol (Sigma, St. Louis, MO) for 30 min at 27~ The effective con- centrations for the experiments described above were also determined based on previous data for other AcNPV binding studies [9]. The same amount of radio- labeled BmNPV was added to control and treated cell layer. The cell layer was designed to contain about i- 2• i0 G cells in 35 mm dishes or 4• 105 cells in 24-well plates. After 3 h of incubation at 4~ cells and su- pernatant were separated. The radioactivity was measured in a scintillation counter.

RESULTS AND DISCUSSION

The t ime-course of BmNPV adsorpt ion to BmN-4 cells was studied by virus binding assay at 4~ Heat- t r ea t ed BmN-4 cells were used as a non-permissive con- trol. This non-specific binding value of the control was

0 x--

0 2 ' " 0 c

0 m

0 1(]0 " 260 " 3C)0 " 4()0 ' 5(]0 600

Time (min)

Fig. 1. Time-course of specific BmNPV binding to BmN-4 cells.

0 . 8

o.6 t CL

�9 0 .4

LL

c-

o m 0 .2

0 .0 , ~ , I

20 40 60 80 1 O0

Infectious Particles/Cell Fig. 2. Scatchard plot of specific BmNPV binding to BmN-4 cells.

subtracted from the amount of total binding data. As shown in Fig. 1, BmNPV binding to BmN-4 cells occurs notably between 0 and 50 rain, but the max imum value of virus binding to the cells was obtained after about 300 min.

Specific binding data for BmN-4 cells were obtained with 3~P-labeled BmNPV. The da ta t ransformed into the Scatchard plot showed a s traight line, suggesting tha t a single class of BmNPV-binding sites is present on BraN-4 cells. The abscissa in te rcept of the plot shows tha t there are about 70 binding sites per cell (Fig. 2). The receptor number of BmN-4 cell surface for BmNPV binding de termined in this s tudy was found to be ext remely low in comparison to the repor ted values of 6000 binding sites per Trichoplusia ni 5B1-4 cell for AcNPV and 106 binding sites per Lymantria dispar cells for L. dispar NPV [5, 11]. This resul t suggests tha t BmNPV-BmN-4 in teract ion could be quite dif- ferent from other baculovirus-insect cell interactions.

The biochemical na ture of the BmNPV-binding sites on the cell surface was examined by incubation of BraN- 4 cells wi th p ro t e in a se K, a - c h y m o t r y p s i n , phos- pholipase C, or wheat germ agglutinin. As shown in Table 1, t r e a t m e n t wi th c~-chymotrypsin and pro- t e inase K r e s u l t e d in 38% and 45% reduc t ion of BmNPV binding to BmN-4 cells, respectively. s pholipase C, which affects lipid components of cell membranes , had no effect on virus binding. These would indicate tha t an essent ia l component of the BmNPV-binding moiety on BmN-4 is a protein struc- ture ra the r than lipid components in the cell mem-

Table 1. Effect of enzymes and lectin on BmNPV binding to BmN-4 cells

Treatment Concentration % Virus binding

None 100 Enzyme

Proteinase K 1 mg ml 1 55(+3) a-Chymotrypsin 1 mg m l ~ 62(+_ 3) Phospholipase C 1 mg m l ~ 94(_+3)

Lectin WGA 1 2 mg mV1 69(_+4)

1WGA, wheat germ agglutinin

Biotechnol. Bioprocess Eng. 1997, Vol. 2, No. 1

Table 2. Effect of metabolic inhibitors and lysosomotropic agents on BmNPV binding to BmN-4 cells

Treatment Concentration (%) Virus binding

None 100 Glycosylation inhibitors

Tunicamycin 1 gg ml 1 64(_+ 13) Deoxynojirimycin 4 mM 81(_+2)

Lysosomotropic agents Methylamine 15 mM 68(_+ 17) NH4C1 30 mM 86(_+ 9)

Endocytosis inhibitors 2,4-Dinitrophenol 1 mM 40(_+ 8) Sodium azide 10 mM 40(_+9)

brane. The pretreatment of BmN-4 cells with wheat germ agglut inin, which specifically binds to oli- gosaccharides, resulted in 30% reduction of BmNPV binding. This suggests that oligosaccharide is another necessary moiety for the attachment of BmNPV.

As shown in Table 2, BmNPV binding to the cells was investigated using metabolic inhibitors (tunica- mycin and deoxynojirimycin). Tunicamycin and deox- ynojirimycin were employed as specific inhibitors to N- l inked glycosylation and (~-glucosidases I and II. BmNPV binding was reduced by 36% and 19% after t r e a t m e n t of cells with tunicamycin and deoxyno- jirimycin. The fact that t reatment of cells with glycosy- la t ion inhibi tors such as tun icamycin and deox- ynojirimycin reduced BmNPV binding further sup- ports that the protein receptor for BmNPV contains oli- gosaccharide structure. This result is contrary to the findings of Wickham et al. [12] that oligosaccharide is not a major component in AcNPV binding on insect cells. The effects of lysosomotropic agents on BmNPV binding are also summarized in Table 2. Inhibition ef- fects were observed with lysosomotropic agents such as NH4C1 and methylamine. The negative effect of NH4C1 and methylamine on BmNPV binding is due to the fact that BmNPV entry via an endocytic pathway is blocked by the increased pH of the endosome with weak bases such as NH4C1 and methylamine. Sodium azide and dinitrophenol (endocytosis inhibitors) ex- hibited stronger inhibition effects on BmNPV binding. Data on endocytosis inhibitors reaffirms that BmNPV attachment to BmN-4 cells is closely linked to the virus entry via receptor-mediated endocytosis. This result is in agreement with previous report on alphavirus entry mechanism of recepor-mediated endocytosis in mos- quito cell [13].

This study has provided an insight into the nature of BmN-4 cell surface receptor in BmNPV-insect cell in- teraction and our findings can be used as fundamental data for bioinsecticide development research. However, the entire mechanisms of BmNPV host specificity are still unclear. Further research is needed to resolve the controversy surrounding the nature of insect cell re- ceptor in baculovirus-insect cell interation and to test whether the host range of baculovirus is restricted to the level of initial attachment to insect cell receptors.

A c k n o w l e d g m e n t The present studies were sup- por ted by the Basic Science Research Inst i tu te Pro- g ram, Min i s t ry of Educat ion , 1995, Project No.

61

BSRI 95-4411. T.Y. Kim thanks the Research Cent- er for New Bio-Materials in Agriculture for the fel- lowship.

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