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Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae
Pergamon
PII: S0965-1748(97)00059-3
Insect Biochem. Molec. Biol. Vol. 27, No. 12, pp. 1007–1016, 1997 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0965-1748/98 $19.00 + 0.00
Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae KENJI SATO,*† MIWA KOMOTO,†1 TOSHITSUGU SATO,† HITOSHI ENEI,† MICHIHIRO MICHIHIRO KOBAYASHI,‡ TOSHINOBU YAGINUMA‡ Received 21 February 1997; revised and accepted 30 July 1997
It is of interest to understand what kinds of physiological and biochemical changes occur in insects if the homeostasis of trehalose in the hemolymph is disrupted by the infection with a recombinant baculovirus containing a secretory-trehalase gene. For this purpose, two recombinant non-occluded Autographa california multicapsid nucleopolyhedroviruses (AcMNPVs), vTREVL and vERTVL, containing a trehalase cDNA of the mealworm beetle, Tenebrio molitor, were constructed. The trehalase cDNA was inserted in the sense orientation downstream of the polyhedrin promoter for vTREVL, and in the anitsense orientation for vERTVL. The active trehelase of T. molitor was found outside of cells when SF-9 cells or larvae of the cabbage armyworm, Mamestra brassicae, were infected with vTREVL. In the hemolymph of vTREVLinfected larvae, expression of the active trehelase was followed by disappearance of trehalose and appearance of glucose. However, the mortality time of virus-infected 5th instar larvae increased in the following order: AcMNPV C6 (wild-type virus) ⱕ vERTVL ⬍ vTREVL. The symptoms (the browning and liquefying of the host body) of NPV infection were moderated considerably in vTREVL-infected larvae. 1998 Elsevier Science Ltd. All rights reserved Trehelase Tenebrio molitor Baculovirus Autographa californica multicapsid nucleopolyhedrovirus Insect cell line Mamestra brassicae Trehalose Glucose Hemolymph
INTRODUCTION
Trehalose (O-␣-D-glucopyranosyl-[1→1]-␣-D-glucopyranoside) is a main blood sugar of insects and an important energy source for insect tissues (Wyatt, 1967). This trehalose is synthesized mainly by fat body and released rapidly into the hemolymph without its storage within the fat body. To utilize the trehalose, other insect tissues have to have an enzyme trehalase (␣␣-trehalose glucohydrolase, EC 3.2.1.28) which hydrolyzes 1 mole of trehalose to 2 moles of glucose that is finally used in the glycolysis of tissue cells. Therefore, disturbance of *Author for correspondence. Fax: 81-0197-68-3881. E. mail address: [email protected]. †Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024, Japan ‡School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan 1 Present address: Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan.
the homeostasis of trehalose in insect hemolymph is a potential strategy to develop chemical insecticides or genetically enhanced microbial pesticides. Natural products such as validoxylamine A (Takahashi et al., 1995) and trehazolin (Ando et al., 1995) isolated from Streptomyces, are known to be specific inhibitors of insect trehalases. When validoxylamine A is injected into larvae of several species of insects, e.g. the common cutworm, Spodoptera litura, the cabbage armyworm, Mamestra brassicae, and the silkworm, Bombyx mori, it causes the enhanced concentrations of hemolymph trehalose, indicating no utilization of this sugar by insect tissues, and has finally showed insecticidal activities against these insects (Asano et al., 1990; Kono et al., 1993; 1994). However, topical or oral administration of this inhibitor has less of an insecticidal effect. On the other hand, baculoviruses including nucleopolyhedrovirus (NPV) have long been used as microbial insecticides (Benz, 1986), because they have a host range confined to certain arthropods, indicating their safety to
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other animals and plants. However, a fault of baculoviruses is the slow speed of action when the viruses are considered as substitutes of chemical insecticides. This demerit can be improved through genetic engineering (Bonning and Hammock, 1996; Vlak, 1993). For example, recombinant viruses containing hormone (Maeda, 1989), neurotoxin (Chejanovsky et al., 1995; Maeda et al., 1991; McCutchen et al., 1991; Stewart et al., 1991; Tomalski and Miller, 1991), enzyme (Bonning et al., 1995; Gopalakrishnan et al., 1995; Hammock et al., 1990) or a maize mitochondrial protein (Korth and Levings, 1993) genes, and a virus mutant with deletion of a certain gene (O’Reilly and Miller, 1991) have been constructed and shown to have greater insecticidal activities than the wild-type viruses (wt viruses). Recently, trehalase genes have been isolated from two species of insects, e.g. the mealworm beetle, Tenebrio molitor (Takiguchi et al., 1992) and B. mori (Su et al., 1993, 1994). Although many insect trehelases are known to bind to the cell membranes or to be located within the cells, trehelase from male reproductive accessory glands of T. molitor is a secretory type of enzyme which is released outside of the glands (Yaginuma and Happ, 1988). Baculovirus expression of a gene for this trehelase would produce the trehelase in the hemolymph of the virus-infected insects, in which the trehelase directly attacks trehalose as a substrate. Subsequently, infection with the recombinant virus would cause the depletion of trehalose in the hemolymph, leading to lowered physiological and physical abilities of the insects, and is also expected to result in earlier death of the infected insects than that of wt virus-infected insects. Even if the earlier action of the virus is not achieved by the virus-mediated expression of the secretory-trehelase gene, we are interested in the physiological and biochemical changes caused by the recombinant virus-mediated disruption of the homeostasis of trehalose in insects. In this study, we have constructed a recombinant baculovirus vTREVL which is a recombinant non-occluded Autographa californica NPV (AcMNPV) containing a cDNA for secretory trehalase of male T. molitor in the sense orientation under the control of the polyhedrin promoter. Expression of the active trehelase of T. molitor whose leader signal was function, was detected in the culture medium of vTREVL-infected SF-9 cells. The active trehelase of T. molitor was also found in the hemolymph of vTREVL-infected larvae of M. brassicae. Expression of the trehalase was followed by trehalose disappearance and glucose appearance in the hemolymph. However, expression of the trehelase reduced the pathogenicity of the virus. MATERIALS AND METHODS
Cells, baculoviruses and insect larvae The fall armyworm, Spodoptera frugiperda, cell line (SF-9) (Vaughn et al., 1977) was maintained with TNMFH medium (Hink, 1970) supplemented with 10% fetal
bovine serum (FBS). The AcMNPV C-6 clone (Posse, 1986) as a wild-type virus (wt virus) and the recombinant AcMNPVs (see below) containing a male Tenebrio molitor trehelase cDNA (Takiguchi et al., 1992) were propagated on SF-9 cells as described previously (King and Posse, 1992a). Larvae of the cabbage armyworm, Mamestra brassicae, were maintained at 25°C on an artificial diet, INSECTA.LF (Nippon, Nosan Kogyo, Yokohama, Japan), with a 16:8 hour (h) light:dark cycle. Construction of recombinant baculoviruses A 1.84-kb NotI fragment containing the 1.83-kb trehelase cDNA of T. molitor (Takiguchi et al., 1992) was excised from the NotI site of pBluescript KS (+) (Stratagene, La Jolla, Calif.) in which the trehalase cDNA was inserted, and purified by agarose gel electrophoresis. This fragment was inserted into the transfer vector pVL1392 (Invitrogen, San Diego, Calif.), at the NotI site in a polycloning site downstream of the polyhedrin promoter (Ppolh). The recombinant transfer vector in which the trehalase cDNA was ligated in the sense or antisense orientation downstream of Ppolh was designated pVL1492-TRE or pVL1392-ERT, respectively. Recombinant baculoviruses were generated by cotransfecting a linearized and polyhedrin-negative AcMNPV DNA, BaculoGold Baculovirus DNA (PharMingen, San Diego, Calif.) and the recombinant transfer vector DNA into SF-9 cells using lipofectin (GIBCO-BRL, Grand Island, NY) (King and Posse, 1992b), and the recombinant baculoviruses were plaquepurified as described previously (King and Posse, 1992a). The first virus, vTREVL, generated with BaculoGold Baculovirus DNA and pVL1392-TRE is a recombinant in which the trehalase cDNA is expressed under the control of Ppolh. The second virus, vERTVL, generated with BaculoGold Baculovirus DNA and pVL1392-ERT is a recombinant in which the trehelase cDNA is inserted in the antisense orientation downstream of Ppolh. The recombinant baculoviruses were amplified in SF-9 cells. The presence of T. molitr trehelase cDNA within the virus genome was confirmed by amplifying the fragment specific to the trehelase cDNA using PCR method (O’Reilly et al., 1992b) with the sequence specific primers: 5⬘-CGATGATCCCCTTCCTGCTT-3⬘ and 5⬘ACTACCCCGTTCGTCCATCC-3⬘, corresponding to nucleotides 48–67 and 1664–1683 of the trehelase cDNA (Takiguchi et al., 1992), respectively. The culture medium of SF-9 cells infected with vTREVL, vERTVL or wt virus was divded into small aliquots followed by storage at −80°C as virus stocks. The virus stocks prior to freeze were subjected to plaque assay as described previously (King and Posse, 1992a), to determine the titers. Each virus stock was used to infect insect cells or M. brassicae larvae with the virus in the experiments described below. Significant differences were not observed in the plaque assay between samples before and after the storage at −80°C during our experimental period.
BACULOVIRUS-MEDIATED EXPRESSION OF A MEALWORM BEETLE
Viral infection, TCID50 assay and cell-viability assay in SF-9 cells Viable cells were counted on a Fuchs-Rosenthal hematocytometer. One-million SF-9 cells in 1.5-ml TNM-FH medium containing 10% FBS were seeded into a 35-mmdiameter culture dish, and the cells were attached on the surface of the culture dish for 1 h at room temperature. The culture medium was then removed, and the cells were mock-infected or infected with 100 l of the complete medium containing 5 × 106 plaque forming units (PFU) of vTREVL, vERTVL or wt virus for 1 h at room temperature. After the virus inoculum was removed from the culture dish, 1 ml of fresh TNM-FH medium with 10% FBS was added on the cells followed by culturing the cells at 27°C. Time zero in the experiment was defined as the point at which 1 ml of fresh TNM-FH medium containing 10% FBS was added after the 1-h viral adsorption period. At given times post infection (p.i.), the culture medium were collected from the culture dish and subjected to low-speed centrifugation to remove the cells. After centrifugation, the supernatants were divided into small aliquots. Samples obtained were stored at −80°C until subjected to (1) 50% tissue culture-infective dose (TCID50) assay with serially 10-fold-diluted viral suspensions (O’Reilly et al., 1992a), (2) trehelaseactivity assay by glucose oxidase method, or (3) native polyacrylamide gel electrophoresis followed by staining for active trehalase on the gel. Storage at −80°C did not cause significant decreases in virus titer and in trehalase activity. For cell-viability assay, the cells were dislodged at given times from the surface of the culture dish by gentle pipetting, and the viable cells were counted by using the Trypan blue exclusion method. Insect bioassay of recombinant baculoviruses At approximately 6 h after 4th ecdysis, M. brassicae larvae were injected with 2 × 104 PFU of budded vTREVL, vERTVL or wt virus in 0.5 l of TNM-FH medium containing 10% FBS. Mock-infected or control larvae were injected with 0.5 l of the complete medium. Samples were injected into the larval hemocoel using a glass capillary. The injected larvae were reared individually on an artificial diet in the wells of a 12-well tissue culture plate at 25°C with a 16:8 h light:dark cycle. Mortality was scored at 0.5-day intervals until all viral infected larvae died. Twenty to twenty-four larvae were infected per mortality-time assay for each virus. The larvae that did not respond to gentle stimulation at the head end using a forceps were scored as dead. For examination of the larval growth, the weight and the development status of each larva was monitored at 1-day intervals. Preparation of hemolymph Fifth instar larvae of M. brassicae were infected with vTREVL, vERTVL or wt virus (2 × 104 PFU/larva) or mock-infected, and were reared on an artificial diet at 25°C. Three to five larvae were bled by incising the larval proleg, each larva being bled only once, and then the
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hemolymph of the larvae was pooled in an ice-chilled 1.5-ml microtube containing a small amount of phenylthiourea to prevent the action of tyrosinases in the larval hemolymph. The pooled hemolymph was centrifuged at 5000 g for 5 min at 2°C to remove hemocytes, and the supernatants were divided into small aliquots. Samples obtained were stored at −80°C until subjected to (1) trehalase-activity assay by glucose oxidase method, (2) native polyacrylamide gel electrophoresis followed by staining for trehalase activity on the gel, or (3) sugar determination by glucose oxidase method. Storage at − 80°C had no effects on trehalase activities and sugar levels. Assay of trehalase activity by glucose oxidase method The assay of trehalase activity was performed by modifications of the method described by Yaginuma and Happ (1988). The standard reaction mixture contained 10-l enzyme source (the supernatant of SF-9 cell culture medium or larval hemolymph) and 20-l substrate solution (50 mM trehalose in 100 mM citrate-sodium phosphate, pH 5.6). The reaction was allowed to proceed at 30°C for 20 min and then stopped by heating at 98°C for 10 min with a microtube heater. After cooling, the solution was centrifuged at 14,000 g for 5 min. Aliquots (2 l) of the supernatant were pipetted into the wells of a 96-well microtiter plate, and were subjected to the determination of glucose derived from trehalose by glucose oxidase method (Hugget and Nixon, 1957) using a glucose-determination reagent kit, Iatro-Chrom GLU-LQ (Iatron Laboratories, Tokyo, Japan). Enzyme specific activity was expressed as moles of glucose produced per min per mg of protein. Protein determination was performed by the method of Bradford (Bradford, 1976) using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, Calif.) with bovine serum albumin (BIO-RAD) as a standard. Native polyacrylamide gel electrophoresis (PAGE) and staining for trehalase activity on the gel Samples were electrophoresed at pH 8.3 with molecular weight standards for native proteins (Nondenatured protein molecular weight marker kit; Sigma, St. Louis, MO.) on a ready-made gradient polyacrylamide slab gel (5–20%), PAGEL NPG-520L (ATTO, Tokyo, Japan). After electrophoresis, the gels were incubated in substrate solution (33 mM trehalose in 100 mM citratesodium phosphate, pH 5.6) at 37°C for 30 min, soaked in glucose-fixation solution (100 mM iodoacetamide) at room temperature for 5 min, and then boiled in colorproducing solution (0.1%, 2,3,5-triphenyltetrazolium chloride in 0.5 N NaOH) for 2–3 min until trehalase bands appeared (Sumida and Yamashita, 1983). After the staining for trehalase activities, proteins on the gels were stained with Coomassie brilliant blue R-250-staining solution, PAGE Blue 83 (Daiichi Pure Chemicals, Tokyo, Japan).
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Treatment of virus-infected SF-9 cells with tunicamycin
RESULTS
T. molitor trehalase has been shown to have 5 potential N-glycosylation sites in its amino-acid sequence (Takiguchi et al., 1992). This fact led us to investigate whether the trehalase was N-glycosylated. SF-9 cells were treated with tunicamycin by the method of O’Reilly et al. (1992c) with some modifications as follows. Onemillion SF-9 cells were attached on a 35-mm-diameter culture dish, and infected with 100 l of the complete medium containing 5 × 106 PFU of vTREVL for 1 h at room temperature. After the viral inoculum was removed, the cells were cultured in 1 ml of TNM-FH medium containing 10% FBS for 12 h at 27°C. Tunicamycin (Sigma) was added at the concentration of 10 g per ml into the cell culture at 12 h p.i., and the culture was incubated at 27°C for an additional 36 h. As the control, the infected cells were cultured in the complete medium without tunicamycin. After the additional 36-h incubation, the culture dish was shaken gently to dislodge the cells from the surface of the culture dish, and then the culture medium was centrifuged at 1000 g for 2–3 min to precipitate floating cells. The cells were suspended in 100 l of TNM-FH medium plus 10% FBS, and lysed completely by sonication with a sonicator, Bio-Disrupter BD1 (Nippon Seiki, Tokyo, Japan). The extent of cell lysis was checked by observing a small volume of the sonicated sample with an inverted microscope. The cell lysate was centrifuged at 14,000 g for 2–3 min to remove cell debris. Aliquots of the supernatant of the culture medium or cell lysate were used for native PAGE.
Construction of recombinant baculoviruses Two recombinant baculoviruses, vTREVL and vERTVL, in which T. molitor trehalase cDNA was inserted in the sense and antisense orientations, respectively, downstream of the AcMNPV Ppolh, were constructed. The recombinant viruses were isolated by plaque purification and amplified up to high titers (1–3 × 108 PFU/ml). The presence of the trehalase cDNA within the virus genome was confirmed by amplifying the fragment specific to the trehalase cDNA using PCR method and then sequencing the product (data not shown). The presence or absence of trehalase in the culture medium of vTREVL- or vERTVL-infected SF-9 cells was monitored by the assay of trehalase activity. Expression of T. molitor trehalase in SF-9 cells and larvae of M. brassicae Monolayers of SF-9 cells (1 × 106 cells/35-mm-diameter culture dish) were infected with viruses, and the supernatants of culture media were collected at intervals of 24 h. Although almost negligible levels of trehalase activity were detected in the supernatants of culture media of vERTVL-infected, wt virus-infected and mockinfected cells, extremely high levels of trehalase activity were found in the supernatants of vTREVL-infected cell culture media along with incubation times (Fig. 1). Trehalase activity in the supernatant of vTREVL-infected cell culture medium first occurred between 12 and 24 h p.i. and reached a maximum level at 72 h p.i. Following native PAGE for the supernatants of culture media of
Determination of trehalose and glucose in hemolymph Trehalose in hemolymph was determined by the method of Yaginuma and Happ (1988) with some modifications. The supernatant of hemolymph was diluted 4 times with 100 mM citrate-sodium phosphate (pH 5.6) and heated at 98°C for 10 min with a microtube heater. After cooling, the heated supernatant was suspended by stirring with a micro pipet. Four l of porcine kidneytrehelase (1.47 U/ml; Sigma) was added to 20 l of the suspension, and the mixture was incubated at 37°C for 16 h. For the control reaction, only enzyme-storage solution (50% glycerol containing 1% Triton X-100 and 25 mM potassium phosphate, pH 6.5) (Sigma Chemical Co., 1996) was added instead of the trehalase. The reaction mixture was heated at 98°C for 10 min for the termination of trehalase reaction and were centrifuged at 14,000 g for 5 min. Two l of the supernatant was used for the determination of glucose by glucose oxidase method. As control, 2 l of the hemolymph supernatant which had not received trehalase-treatment was used for the determination of glucose by glucose oxidase method as described above.
FIGURE 1. Trehalase activities in supernatants of the culture media of vTREVL-, vERTVL-, wt virus- or mock-infected SF-9 cells. Cells (1 × 106 cells/35-mm-diameter culture dish) were infected with each virus (5 PFU/cell) or mock-infected, and then cultured in 1 ml of TNM-FH medium with 10% FBS at 27°C. Supernatants of the culture media of vTREVL-, vERTVL-, wt virus- or mock-infected cells were collected at 1, 12, 24, 48, 72 and 96 h p.i., and used for assays of trehalase activities. Each point represents the mean trehalase activity ± range (n = 2; vertical bars). Symbol only means that range is within the symbol.
BACULOVIRUS-MEDIATED EXPRESSION OF A MEALWORM BEETLE
FIGURE 2. Detection of trehalase activities on the gel after native PAGE of supernatants of the culture media of vTREVL-infected SF9 cells. Supernatants (6 l) of the culture media from vTREVLinfected cells in Fig. 1 were analyzed by native PAGE, and trehalase activities were detected on the gel. Lanes 1–6: supernatants of the culture media collected at 1, 12, 24, 48, 72 and 96 h p.i., respectively. The locations of the molecular weight standards are indicated on the right margin.
vTREVL-infected cells, staining for trehalase activity on the gel was carried out. An active trehalase with an apparent molecular weight of approximately 85 kDa was detected (Fig. 2). Expression of active trehalase in vTREVL-infected SF-9 cells, from 12 to 48 h p.i., in the culture medium with or without tunicamycin is shown in Fig. 3. Although in the extracts of the cells incubated without tunicamycin, two active trehalases of 85 kDa and 62 kDa were detected (lane 1), an active trehalase of 85 kDa was detected in the culture medium (lane 2). In contrast, in the presence of tunicamycin, cells expressed only an active trehalase with an apparent molecular weight of approximately 62 kDa (lane 3), and any active trehalases were not found in the culture medium (lane 4). These results indicated that the 85-kDa trehalase extracted from the cells without tunicamycin treatment was N-glycosylated.
FIGURE 3. Effects of tunicamycin on the expression of T. molitor active trehalase in SF-9 cells. Cells were infected and then cultured in the same condition as Fig. 1. Tunicamycin was added into the cell culture at 12 h p.i., and the culture was incubated for an additional 36 h. As the control, the infected cells were cultured in the medium without tunicamycin. After the additional 36-h incubation, the infected cells and culture medium were separated. The infected cells were suspended in 100 l of the complete medium and then lysed. Supernatants (6 l) of the culture medium and cell lysate were analyzed by native PAGE followed by staining for trehalase activity on the gel. Lanes 1 and 2: cell lysate and culture medium without tunicamycin treatment, respectively; lanes 3 and 4: cell lysate and culture medium with tunicamycin treatment, respectively. The locations of the molecular weight standards are indicated on the right margin.
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An active trehalase of 85 kDa was also detected in the hemolymph of vTREVL-infected M. brassicae larvae at 3 days p.i., and the activity reached a peak at 4 days p.i., while in the hemolymph of vERTVL-, wt virus- or mockinfected larvae no active trehalases occurred (Figs 4 and 5). Trehalose level in the hemolymph of vTREVLinfected larvae decreased markedly at 3 days p.i., at the same time as the active trehalase became detectable in the hemolymph, and trehalose disappeared from the hemolymph by 4 days p.i [Fig. 6(A)], accompanied by the occurrence of a significant level of glucose derived from trehalose in the hemolymph. However, glucose also disappeared from the hemolymph of vTREVL-infected larvae by 6 days p.i., namely shortly before death (Fig. 9). On the other hand, there were substantial levels of trehalose and almost negligible levels of glucose in the hemolymph of vERTVL-, wt virus- or mock-infected larvae throughout the experimental period [Fig. 6(B, C and D)]. Effects of recombinant baculovirus-infection on SF-9 cells and larvae of M. brassicae No significant differences in the growth of extracellular virus on SF-9 cells were found among vTREVL, vERTVL and wt virus (Fig. 7). The growth of each extracellular virus reached a maximum titer at 72 h p.i. at about 6 × 108 TCID50 per ml. Few cytopathological differences were seen in the virus-infected cells among the recombinant viruses and wt virus, except that polyhedra were formed in the wt virus-infected cells (data not shown). Growth of the virus-infected cells was suppressed markedly, irrespective of the recombinant viruses or wt virus (Fig. 8). To examine whether the insertion of T. molitor trehalase gene into the AcMNPV genome has any effect on the insecticidal activity of the virus, the mortality of M. brassicae larvae infected with vTREVL, vERTVL or wt virus in the early stage of 5th instar period was monitored (Fig. 9). At 6 days p.i., more than 95% of the larvae infected with wt virus died. On the same day, 72% of the larvae infected with vERTVL died. At 6.5 days p.i., all the larvae infected with wt virus died. Although at 7 days p.i. all the larvae infected with vERTVL died, 65% of the larvae infected with vTREVL still survived. At 8.5 days p.i., all the larvae infected with vTREVL died (Fig. 9). However, significant differences in the weight gains of virus-infected larvae were not observed among the recombinant viruses and wt virus at any time point p.i. (Fig. 10). The mock-infected larvae ecdysed into 6th instar (last instar) by 2 days p.i., and continued to gain weight up to 4 days p.i. From 4–5 days p.i., they experienced a retardation of weight gain, coincident with the onset of gut-purge prior to pupation. All of the mockinfected larvae metamorphosed into pupae by 8 days p.i. Regardless of the recombinant viruses or wt virus, all of the virus-infected larvae showed the signs of illness, such as retardation of growth, swelling of body and blocking of molt into 6th instar, by 3 days p.i., and died during 5th instar period. At 4 days p.i., the titer of extra-
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FIGURE 4. Trehalase activities in the hemolymph of vTREVL-, vERTVL-, wt virus- or mock-infected M. brassicae larvae. Fifth instar larvae were infected with each virus (2 × 104 PFU/larva) or mock-infected, and then reared on an artificial diet at 25°C. Hemolymph from 3 to 5 larvae was pooled at 0.25, 1, 2, 3, 4, 5 and 6 days p.i., and trehalase activities in supernatants of the hemolymph were assayed. Each point represents the mean of activities ± range (n = 2).
FIGURE 5. Detection of trehalase activities on the gel after native PAGE of the hemolymph of vTREVL-infected M. brassicae larvae. Supernatants (6 l) of the hemolymph collected in Fig. 4 were analyzed by native PAGE followed by staining for trehalase activity on the gel. Lanes 1–7: supernatants of the hemolymph collected from vTREVL-infected larvae at 0.25, 1, 2, 3, 4, 5 and 6 days p.i., respectively. The locations of the molecular weight standards are indicated on the right margin.
cellular virus (TCID50 values ± SEM; n = 2) in the hemolymph of virus-infected larvae was 7.2 × 108 ± 5.8 × 107 for vTREVL, 7.3 × 108 ± 2.0 × 107 for vERTVL and 2.8 × 109 ± 2.8 × 108 for wt virus. There were about 4-fold differences between the recombinant viruses and wt virus. Figure 11 shows the virus-infected 5th instar larvae shortly after death. Significant differences in the symptoms of virus-infected larvae were detected among the recombinant viruses and wt virus. The larvae infected
with wt virus exhibited the typical symptoms of NPV infection, namely the browning and liquefying of the host body prior to death or shortly thereafter. Unlike wt virusinfected larvae, the body color of dead vTREVL-infected larvae remained yellow-green similar to that of the healthy larvae during the molting period. In addition, the dead body of a vTREVL-infected larva was soft, but did not liquefy. The symptoms of vERTVL-infected larvae were intermediate between those of wt virus-infected and vTREVL-infected larvae. The abdominal color of a dead
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FIGURE 6. Levels of trehalose and glucose in the hemolymph of M. brassicae larvae infected with vTREVL, vERTVL or wt virus. Concentrations of trehalose and glucose in supernatants of the hemolymph from vTREVL-infected (A), vERTVLinfected (B), wt virus-infected (C) and mock-infected (D) larvae collected in Fig. 4, were determined. Each point represents the mean ± range (n = 2).
FIGURE 7. Growth curves of vTREVL, vERTVL and wt virus on SF9 cells. Cells were infected with each virus and then cultured in the same condition as Fig. 1. Supernatants of the culture media were collected at 1, 12, 24, 48, 72 and 96 h p.i., and subjected to TCID50 assay. Each point represents the mean of TCID50 values ± range (n = 2; vertical bars).
FIGURE 8. Viability of SF-9 cells infected with vTREVL, vERTVL or wt virus. The cells used in Fig. 7 were monitored. At the times indicated, viable cells were counted by using the Trypan blue exclusion method. Each point represents the mean density of viable cells ± range (n = 2; vertical bars).
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FIGURE 9. Mortality of M. brassicae larvae infected with vTREVL, vERTVL or wt virus. Twenty to twenty-four fifth instar larvae were infected with each virus or mock-infected and then reared in the same condition as Fig. 4. The cumulative mortality observed in two separate series different from Figs 4–6, was monitored at 0.5-day intervals. Each point represents the mean value ± range (n = 2).
vERTVL-infected larva became brown, while the remainder of the dead body retained yellow-green color. However, dead vERTVL-infected larvae did not liquefy. DISCUSSION
This is the first report of a catalytically active trehalase produced in baculovirus-infected insect cells and insect tissues. Active T. molitor trehalase was detected in the culture medium of vTREVL-infected SF-9 cells (Fig. 1) or in the hemolymph of vTREVL-infected larvae of M. brassicae (Fig. 4), and the active trehalase was shown to be an apparent molecular weight of approximately 85 kDa as determined by native PAGE and subsequent staining for trehalase activity (Figs 2 and 5). This size of molecular weight was larger than the calculated molecular weight (62.7 kDa) of the putative protein encoded by an open reading frame of the trehalase cDNA (Takiguchi et al., 1992). This finding that the 85-kDa protein is synthesized and released from the insect cells infected with vTREVL, indicates that the T. molitor trehalase undergoes post-translational modifications through the secretion. Consistent with this idea are the following observations: 1) the trehelase cDNA has a putative signal sequence and 5 potential N-glycosylation sites, Asn-XaaSer/Thr (Takiguchi et al., 1992), and 2) the 85-kDa active trehalase was not detected, in the presence of tunicamycin, in the culture medium or the cells (Fig. 3). Since only active trehalase of 62 kDa was detected in the tunicamycin-treated cells (Fig. 3), the active trehalase of 62 kDa appeared to be the non-glycosylated precursor of the trehalase. The results have led us to the idea that the 62-kDa enzyme-precursor with no sugar moieties is Nglycosylated and subsequently secreted as the mature trehalase of 85 kDa into the culture medium, because inhi-
bition of N-glycosylation by tunicamycin prevented the trehalase from being secreted, as such cases described for certain proteins (Gopalakrishnan et al., 1995; Jarvis and Summers, 1989; Jarvis et al., 1990). In the hemolymph of larvae infected with vTREVL, the time in increase of trehalase activity coincided with that in decrease of trehalose [Fig. 4 and Fig. 6(A)]. In general, in insects, glucose in the hemolymph is transferred to the fat body and then converted to trehalose, which is rapidly released from the fat body into the hemolymph to maintain almost constant levels of trehalose (Wyatt, 1967). Therefore, no significant accumulation of glucose has usually been observed in insect hemolymph (Wyatt, 1967), as in the hemolymph of normal larvae shown in Fig. 6(D). The observations in Fig. 6(A), such as a decrease of trehalose and an increase of glucose in the hemolymph of vTREVL-infected larvae, indicate that the total activity of trehalase expressed in the hemolymph was well over the synthetic activity of trehalose from glucose in the fat body, subsequently leading to an accumulation of glucose which was derived from trehalose and finally used by the other tissues until the stage just before larval death. This imbalance in trehalose metabolism may have resulted from the production of the active trehalase in the hemolymph and the lowered synthetic activity due to the damage of the fat-body tissue infected with the virus. In the larvae infected with vERTVL or wt virus, the fat body and the other tissues that can utilize the hemolymph trehalose may have simultaneously been damaged, subsequently resulting in a substantial levels of trehalose in the hemolymph [Fig. 6(B and C)]. As described above, in spite of the essentiality of trehalase in the utilization of hemolymph trehalose by insect tissues, molecular relationship between the enzyme structure and the activity remains unclear. The potential of this baculovirus system for producing a large amount of active trehalase should permit the following studies: (1) structure-function analysis by site-directed mutagenesis as utilized for the investigation of the catalytic mechanism of certain enzymes (Funk et al., 1989; McKegney et al., 1996; Ward et al., 1992), (2) crystallization and subsequent tertiary structure analysis, (3) in vitro elucidation of direct effects of various inhibitors on the trehalase, and (4) tertiary structure analysis of the enzymeinhibitor complex. These studies should pave the way to determine such functional domains as substrate- and inhibitor-binding sites of this enzyme, and to develop trehalase inhibitors effective as insecticides. We first assumed that baculovirus-mediated expression of trehalase in insects would cause the depletion of trehalose in the hemolymph, and then result in the earlier effects with respect to killing these infected insects. Contrary to this assumption, the mortality time of virusinfected 5th instar larvae of M. brassicae increased in the following order: wt virus ⱕ vERTVL ⬍ vTREVL (Fig. 9). Nevertheless, both the recombinant viruses, vTREVL and vERTVL, showed a tendency of slower
BACULOVIRUS-MEDIATED EXPRESSION OF A MEALWORM BEETLE
FIGURE 10. Weights of M. brassicae larvae infected with vTREVL, vERTVL or wt virus. Fifth instar larvae used in Fig. 9 were weighed at 1-day intervals. Each point represents the mean weight of 20 to 24 larvae ± range (n = 2).
speed of viral growth in the larvae than wt virus, because there was about a 4-fold difference in the titer (TCID50) of extracellular virus in the hemolymph of virus-infected larvae between the recombinant viruses and wt virus at 4 days p.i. However, the weight gains of virus-infected larvae were scarcely affected by such differences in virus-type as vTREVL, vERTVL and wt virus (Fig. 10). Further, significant differences in the symptoms of virusinfected larvae of M. brassicae were observed among vTREVL, vERTVL and wt virus (Fig. 11). The typical symptoms of NPV infection such as the browning and liquefying of the host body were observed in wt virusinfected larvae. The symptoms of NPV infection were significantly moderated in vTREVL-infected larvae. The symptoms of vERTVL-infected larvae were intermediate between those of vTREVL-infected and wt virus-infected
FIGURE 11. vTREVL-, vERTVL- or wt virus-infected M. brassicae larva shortly after death. Fifth instar larvae used in Fig. 9 were monitored until death. (A): vTREVL-infected larva; (B) vERTVL-infected larva; and (C): wt virus-infected larva.
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larvae. These results appear to be consistent with those of recent reports (O’Reilly et al., 1995; Wood et al., 1993) have shown that the lack of polyhedrin production and the further expression of another gene reduce the insecticidal activity of the virus. The expression of the trehalase gene may be involved in the increased mortality time and moderated symptom of infected larvae (Fig. 9 and Fig. 11), in addition to the lack of polyhedrin protein. However, a recombinant virus expressing both polyhedrin gene and inserted foreign gene has been shown to increase the mortality time and reduce the weight gain of infected larvae, compared to the wt virus as a control (Bonning et al., 1992). Therefore, to understand the relationship between gene expressions of trehalase and polyhedrin, and the increased mortality time and moderated symptom of infected larvae, it is further required to examine whether a recombinant virus that can express both the polyhedrin and secretory-trehalase genes, expresses much higher or lower insecticidal activity than wt virus. REFERENCES Ando O., Nakajima M., Kifune M., Fang H. and Tanzawa K. (1995) Trehazolin, a slow, tight-binding inhibitor of silkworm trehalase. Biochim. Biophys. Acta 1244, 295–302. Asano N., Takeuchi M., Kameda Y., Matsui K. and Kono Y. (1990) Trehalase inhibitors, validoxylamine A and related compounds as insecticides. . J. Antibiot. 43, 722–726. Benz, G.A. (1986) Introduction: historical perspectives. In The Biology of Baculoviruses (Edited by Granados R. R. and Federici B. A.), Vol. I, pp. 1–35. CRC Press, Boca Raton, Fla. Bonning B.C., Hirst M., Posse R.D. and Hammock B.D. (1992) Further development of a recombinant baculovirus insecticide expressing the enzyme juvenile hormone esterase from Heliothis virescens. Insect Biochem. Molec. Biol. 22, 453–458. Bonning B.C., Hoover K., Booth T.F., Duffey S. and Hammock B.D. (1995) Development of a recombinant baculovirus expressing a modified juvenile hormone esterase with potential for insect control. Arch. Insect. Biochem. Physiol. 30, 177–194. Bonning B.C. and Hammock B.D. (1996) Development of recombinant baculoviruses for insect control. Annu. Rev. Entomol. 41, 191–210. Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248–254. Chejanovsky N., Zilberberg N., Rivkin H., Zlotkin E. and Gurevitz M. (1995) Functional expression of an alpha anti-insect scorpion neurotoxin in insect cells and lepidopterous larvae. FEBS Lett. 376, 181–184. Funk C.D., Gunne H., Steiner H., Izumi T. and Samuelssen B. (1989) Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system. Proc. Natl. Acad. Sci U.S.A. 86, 2592–2596. Gopalakrishnan B., Muthukrishnan S. and Kramer K.J. (1995) Baculovirus-mediated expression of a Manduca sexta chitinase gene: properties of the recombinant protein. Insect Biochem. Molec. Biol. 25, 255–265. Hammock B.D., Bonning B.C., Posse R.D., Hanzlik T.N. and Maeda S. (1990) Expression and effects of the juvenile hormone esterase in a baculovirus vector. Nature 344, 458–461. Hink W.F. (1970) Established insect cell line from the cabbage looper Trichoplusia ni.. Nature 226, 466–467. Hugget A.S.G. and Nixon D.A. (1957) Enzymatic determination of blood glucose. Biochem J. 66, 12. Jarvis D.L. and Summers M.D. (1989) Glycosylation and secretion of
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human tissue plasminogen activator in recombinant baculovirusinfected insect cells. Mol. Cell. Biol. 9, 214–223. Jarvis D.L., Oker-Blom C. and Summers M.D. (1990) Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells. J. Cell. Biol. 42, 181–191. King, L.A., Posse, R.D. (1992a) The Baculovirus Expression System, pp. 106–119. Chapman and Hall, London. King, L.A., Posse, R.D. (1992b) The Baculovirus Expression System, pp. 132–133. Chapman and Hall, London. Kono Y., Takeda S., Kameda Y., Takahashi M., Matsushita K., Nishina M. and Hori E. (1993) Lethal activity of a trehalase inhibitor, validoxylamine A, and its influence on the blood sugar level in Bombyx mori (Lepidoptera: Bombycidae). Appl. Ent. Zool. 28, 379–386. Kono Y., Takeda S. and Kameda Y. (1994) Lethal activity of a trehalase inhibitor, validoxylamine A, against Mamestra brassicae and Spodoptera litura. J. Pesticide Sci. 19, 39–42. Korth K.L. and Levings C.S. (1993) Baculovirus expression of the maize mitrochondrial protein URF13 confers insecticidal activity in cell cultures and larvae. Proc. Natl. Acad. Sci. U.S.A. 90, 3388–3392. Maeda S. (1989) Increased insecticidal effect by a recombinant baculovirus carrying a synthetic diuretic hormone gene. Biochem. Biophys. Res. Commun. 165, 1177–1183. Maeda S., Volrath S.L., Hanzlik T.N., Harper S.A., Majima K., Maddox D.W., Hammock B.D. and Fowler E. (1991) Insecticidal effects of an insect-specific neurotoxin expressed by a recombinant baculovirus. Virology 184, 777–780. McCutchen B.F., Choudary P.V., Crenshaw R., Maddox D., Kamita S.G., Palekar N., Volrath S., Fowler E., Hammock B.D. and Maeda S. (1991) Development of a recombinant baculovirus expressing an insect-selective neurotoxin: potential for pest control. Bio/Technology 9, 848–852. McKegney G.R., Butland S.L., Theilmann D. and Ellis B.E. (1996) Expression of poplar phenylalanine ammonia-lyase in insect cell cultures. Phytochemistry 41, 1259–1263. O’Reilly D.R. and Miller L.K. (1991) Improvement of a baculovirus pesticide by deletion of the egt gene. Bio/Technology 9, 1086– 1089. O’Reilly, D.R., Miller, L.K., Luckow, V.A. (1992a) Baculovirus Expression Vectors, pp. 132–134. W. H. Freeman and Co., N. Y. O’Reilly, D.R., Miller, L.K., Luckow, V.A. (1992b) Baculovirus Expression Vectors, pp. 161–165. W. H. Freeman and Co., N. Y. O’Reilly, D.R., Miller, L.K., Luckow, V.A. (1992c) Baculovirus Expression Vectors, pp. 231–232. W. H. Freeman and Co., N. Y. O’Reilly D.R., Kelly T.J., Masler E.P., Thyagaraja B.S., Robson R.M., Shaw T.C. and Miller L.K. (1995) Overexpression of Bombyx mori prothoracicotropic hormone using baculovirus vectors. Insect Biochem. Molec. Biol. 25, 475–485. Posse R.D. (1986) Cell-surface expression of influenza virus haemag-
glutinin in insect cells using a baculovirus vector. Virus Res. 5, 43–59. Sigma Chemical Co. (1996) Sigma General Catalog, p. 1009. Sigma Chemical Co, St. Louis, Mo. Stewart L.M.D., Hirst M., Ferber M.L., Merryweather A.T., Cayley P.J. and Posse R.D. (1991) Construction of an improved baculovirus insecticide containing an insect-specific toxin gene. Nature 352, 85–88. Su H.-Z., Sato Y. and Yamashita O. (1993) Purification, cDNA cloning and northern blot analysis of trehalase of pupal midgut of the silkworm. Bombyx mori. Biochim. Biophys. Acta 1173, 217–224. Su H.-Z., Ikeda M., Sato Y., Saito H., Imai K., Isobe M. and Yamashita O. (1994) Molecular characterization of ovary trehalase of the silkworm, Bombyx mori, and its transcriptional activation by diapause hormone. Biochim. Biophys. Acta 1218, 366–374. Sumida M. and Yamashita O. (1983) Purification and some properties of soluble trehalase from midgut of pharate adult of the silkworm Bombyx mori. Insect Biochem. 13, 257–265. Takahashi M., Kono Y., Kurahashi H., Matsushita K., Nishina M. and Kameda Y. (1995) Effect of a trehalase inhibitor, validoxylamine A, on three species of flies. Appl. Entomol. Zool. 30, 231–239. Takiguchi M., Niimi T., Su H.-Z. and Yaginuma T. (1992) Trehalase from male accessory gland of an insect Tenebrio molitor. . Biochem. J. 288, 19–22. Tomalski M.D. and Miller L.K. (1991) Insect paralysis by baculovirusmediated expression of a mite neurotoxin gene. Nature 352, 82–85. Vaughn J.L., Goodwin R.H., Tomkins G.J. and McCawley P. (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213–217. Vlak, J.M. (1993) Genetic engineering of baculoviruses for insect control. In Molecular Approaches to Fundamental and Applied Entomology (Edited by Oakeshott J. and Whitten M. J.), pp. 90-127. Springer, NY. Ward V.K., Bonning B.C., Huang T., Shiotsuki T., Griffeth V.N. and Hammock B.D. (1992) Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis. Int. J. Biochem. 24, 1933–1941. Wood H.A., Trotter K.M., Davis T.R. and Hughes P.R. (1993) Per os infectivity of preoccluded virions from polyhedrin-minus recombinant baculoviruses. J. Invertebr. Pathol. 62, 64–67. Wyatt, G.R. (1967) The biochemistry of sugars and polysaccharides in insects. In Advances in Insect Physiology (Edited by Beamont J. W. L., Treherne J. E. and Wigglesworth V. B.), Vol. 4, pp. 287– 360. Academic Press, London. Yaginuma T. and Happ G.M. (1988) Trehalase from the bean-shaped accessory glands and the spermatophore of the male mealworm beetle Tenebrio molitor. J. Comp. Physiol. B 157, 765–770.
Acknowledgements—The authors thank T. Uchiyama and Y. Sato (Hokko Chemical Industry Co., Atsugi, Kanagawa, Japan) for their kind supplies of Mamestra brassicae eggs.
PII: S0965-1748(97)00059-3
Insect Biochem. Molec. Biol. Vol. 27, No. 12, pp. 1007–1016, 1997 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0965-1748/98 $19.00 + 0.00
Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae KENJI SATO,*† MIWA KOMOTO,†1 TOSHITSUGU SATO,† HITOSHI ENEI,† MICHIHIRO MICHIHIRO KOBAYASHI,‡ TOSHINOBU YAGINUMA‡ Received 21 February 1997; revised and accepted 30 July 1997
It is of interest to understand what kinds of physiological and biochemical changes occur in insects if the homeostasis of trehalose in the hemolymph is disrupted by the infection with a recombinant baculovirus containing a secretory-trehalase gene. For this purpose, two recombinant non-occluded Autographa california multicapsid nucleopolyhedroviruses (AcMNPVs), vTREVL and vERTVL, containing a trehalase cDNA of the mealworm beetle, Tenebrio molitor, were constructed. The trehalase cDNA was inserted in the sense orientation downstream of the polyhedrin promoter for vTREVL, and in the anitsense orientation for vERTVL. The active trehelase of T. molitor was found outside of cells when SF-9 cells or larvae of the cabbage armyworm, Mamestra brassicae, were infected with vTREVL. In the hemolymph of vTREVLinfected larvae, expression of the active trehelase was followed by disappearance of trehalose and appearance of glucose. However, the mortality time of virus-infected 5th instar larvae increased in the following order: AcMNPV C6 (wild-type virus) ⱕ vERTVL ⬍ vTREVL. The symptoms (the browning and liquefying of the host body) of NPV infection were moderated considerably in vTREVL-infected larvae. 1998 Elsevier Science Ltd. All rights reserved Trehelase Tenebrio molitor Baculovirus Autographa californica multicapsid nucleopolyhedrovirus Insect cell line Mamestra brassicae Trehalose Glucose Hemolymph
INTRODUCTION
Trehalose (O-␣-D-glucopyranosyl-[1→1]-␣-D-glucopyranoside) is a main blood sugar of insects and an important energy source for insect tissues (Wyatt, 1967). This trehalose is synthesized mainly by fat body and released rapidly into the hemolymph without its storage within the fat body. To utilize the trehalose, other insect tissues have to have an enzyme trehalase (␣␣-trehalose glucohydrolase, EC 3.2.1.28) which hydrolyzes 1 mole of trehalose to 2 moles of glucose that is finally used in the glycolysis of tissue cells. Therefore, disturbance of *Author for correspondence. Fax: 81-0197-68-3881. E. mail address: [email protected]. †Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024, Japan ‡School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan 1 Present address: Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan.
the homeostasis of trehalose in insect hemolymph is a potential strategy to develop chemical insecticides or genetically enhanced microbial pesticides. Natural products such as validoxylamine A (Takahashi et al., 1995) and trehazolin (Ando et al., 1995) isolated from Streptomyces, are known to be specific inhibitors of insect trehalases. When validoxylamine A is injected into larvae of several species of insects, e.g. the common cutworm, Spodoptera litura, the cabbage armyworm, Mamestra brassicae, and the silkworm, Bombyx mori, it causes the enhanced concentrations of hemolymph trehalose, indicating no utilization of this sugar by insect tissues, and has finally showed insecticidal activities against these insects (Asano et al., 1990; Kono et al., 1993; 1994). However, topical or oral administration of this inhibitor has less of an insecticidal effect. On the other hand, baculoviruses including nucleopolyhedrovirus (NPV) have long been used as microbial insecticides (Benz, 1986), because they have a host range confined to certain arthropods, indicating their safety to
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other animals and plants. However, a fault of baculoviruses is the slow speed of action when the viruses are considered as substitutes of chemical insecticides. This demerit can be improved through genetic engineering (Bonning and Hammock, 1996; Vlak, 1993). For example, recombinant viruses containing hormone (Maeda, 1989), neurotoxin (Chejanovsky et al., 1995; Maeda et al., 1991; McCutchen et al., 1991; Stewart et al., 1991; Tomalski and Miller, 1991), enzyme (Bonning et al., 1995; Gopalakrishnan et al., 1995; Hammock et al., 1990) or a maize mitochondrial protein (Korth and Levings, 1993) genes, and a virus mutant with deletion of a certain gene (O’Reilly and Miller, 1991) have been constructed and shown to have greater insecticidal activities than the wild-type viruses (wt viruses). Recently, trehalase genes have been isolated from two species of insects, e.g. the mealworm beetle, Tenebrio molitor (Takiguchi et al., 1992) and B. mori (Su et al., 1993, 1994). Although many insect trehelases are known to bind to the cell membranes or to be located within the cells, trehelase from male reproductive accessory glands of T. molitor is a secretory type of enzyme which is released outside of the glands (Yaginuma and Happ, 1988). Baculovirus expression of a gene for this trehelase would produce the trehelase in the hemolymph of the virus-infected insects, in which the trehelase directly attacks trehalose as a substrate. Subsequently, infection with the recombinant virus would cause the depletion of trehalose in the hemolymph, leading to lowered physiological and physical abilities of the insects, and is also expected to result in earlier death of the infected insects than that of wt virus-infected insects. Even if the earlier action of the virus is not achieved by the virus-mediated expression of the secretory-trehelase gene, we are interested in the physiological and biochemical changes caused by the recombinant virus-mediated disruption of the homeostasis of trehalose in insects. In this study, we have constructed a recombinant baculovirus vTREVL which is a recombinant non-occluded Autographa californica NPV (AcMNPV) containing a cDNA for secretory trehalase of male T. molitor in the sense orientation under the control of the polyhedrin promoter. Expression of the active trehelase of T. molitor whose leader signal was function, was detected in the culture medium of vTREVL-infected SF-9 cells. The active trehelase of T. molitor was also found in the hemolymph of vTREVL-infected larvae of M. brassicae. Expression of the trehalase was followed by trehalose disappearance and glucose appearance in the hemolymph. However, expression of the trehelase reduced the pathogenicity of the virus. MATERIALS AND METHODS
Cells, baculoviruses and insect larvae The fall armyworm, Spodoptera frugiperda, cell line (SF-9) (Vaughn et al., 1977) was maintained with TNMFH medium (Hink, 1970) supplemented with 10% fetal
bovine serum (FBS). The AcMNPV C-6 clone (Posse, 1986) as a wild-type virus (wt virus) and the recombinant AcMNPVs (see below) containing a male Tenebrio molitor trehelase cDNA (Takiguchi et al., 1992) were propagated on SF-9 cells as described previously (King and Posse, 1992a). Larvae of the cabbage armyworm, Mamestra brassicae, were maintained at 25°C on an artificial diet, INSECTA.LF (Nippon, Nosan Kogyo, Yokohama, Japan), with a 16:8 hour (h) light:dark cycle. Construction of recombinant baculoviruses A 1.84-kb NotI fragment containing the 1.83-kb trehelase cDNA of T. molitor (Takiguchi et al., 1992) was excised from the NotI site of pBluescript KS (+) (Stratagene, La Jolla, Calif.) in which the trehalase cDNA was inserted, and purified by agarose gel electrophoresis. This fragment was inserted into the transfer vector pVL1392 (Invitrogen, San Diego, Calif.), at the NotI site in a polycloning site downstream of the polyhedrin promoter (Ppolh). The recombinant transfer vector in which the trehalase cDNA was ligated in the sense or antisense orientation downstream of Ppolh was designated pVL1492-TRE or pVL1392-ERT, respectively. Recombinant baculoviruses were generated by cotransfecting a linearized and polyhedrin-negative AcMNPV DNA, BaculoGold Baculovirus DNA (PharMingen, San Diego, Calif.) and the recombinant transfer vector DNA into SF-9 cells using lipofectin (GIBCO-BRL, Grand Island, NY) (King and Posse, 1992b), and the recombinant baculoviruses were plaquepurified as described previously (King and Posse, 1992a). The first virus, vTREVL, generated with BaculoGold Baculovirus DNA and pVL1392-TRE is a recombinant in which the trehalase cDNA is expressed under the control of Ppolh. The second virus, vERTVL, generated with BaculoGold Baculovirus DNA and pVL1392-ERT is a recombinant in which the trehelase cDNA is inserted in the antisense orientation downstream of Ppolh. The recombinant baculoviruses were amplified in SF-9 cells. The presence of T. molitr trehelase cDNA within the virus genome was confirmed by amplifying the fragment specific to the trehelase cDNA using PCR method (O’Reilly et al., 1992b) with the sequence specific primers: 5⬘-CGATGATCCCCTTCCTGCTT-3⬘ and 5⬘ACTACCCCGTTCGTCCATCC-3⬘, corresponding to nucleotides 48–67 and 1664–1683 of the trehelase cDNA (Takiguchi et al., 1992), respectively. The culture medium of SF-9 cells infected with vTREVL, vERTVL or wt virus was divded into small aliquots followed by storage at −80°C as virus stocks. The virus stocks prior to freeze were subjected to plaque assay as described previously (King and Posse, 1992a), to determine the titers. Each virus stock was used to infect insect cells or M. brassicae larvae with the virus in the experiments described below. Significant differences were not observed in the plaque assay between samples before and after the storage at −80°C during our experimental period.
BACULOVIRUS-MEDIATED EXPRESSION OF A MEALWORM BEETLE
Viral infection, TCID50 assay and cell-viability assay in SF-9 cells Viable cells were counted on a Fuchs-Rosenthal hematocytometer. One-million SF-9 cells in 1.5-ml TNM-FH medium containing 10% FBS were seeded into a 35-mmdiameter culture dish, and the cells were attached on the surface of the culture dish for 1 h at room temperature. The culture medium was then removed, and the cells were mock-infected or infected with 100 l of the complete medium containing 5 × 106 plaque forming units (PFU) of vTREVL, vERTVL or wt virus for 1 h at room temperature. After the virus inoculum was removed from the culture dish, 1 ml of fresh TNM-FH medium with 10% FBS was added on the cells followed by culturing the cells at 27°C. Time zero in the experiment was defined as the point at which 1 ml of fresh TNM-FH medium containing 10% FBS was added after the 1-h viral adsorption period. At given times post infection (p.i.), the culture medium were collected from the culture dish and subjected to low-speed centrifugation to remove the cells. After centrifugation, the supernatants were divided into small aliquots. Samples obtained were stored at −80°C until subjected to (1) 50% tissue culture-infective dose (TCID50) assay with serially 10-fold-diluted viral suspensions (O’Reilly et al., 1992a), (2) trehelaseactivity assay by glucose oxidase method, or (3) native polyacrylamide gel electrophoresis followed by staining for active trehalase on the gel. Storage at −80°C did not cause significant decreases in virus titer and in trehalase activity. For cell-viability assay, the cells were dislodged at given times from the surface of the culture dish by gentle pipetting, and the viable cells were counted by using the Trypan blue exclusion method. Insect bioassay of recombinant baculoviruses At approximately 6 h after 4th ecdysis, M. brassicae larvae were injected with 2 × 104 PFU of budded vTREVL, vERTVL or wt virus in 0.5 l of TNM-FH medium containing 10% FBS. Mock-infected or control larvae were injected with 0.5 l of the complete medium. Samples were injected into the larval hemocoel using a glass capillary. The injected larvae were reared individually on an artificial diet in the wells of a 12-well tissue culture plate at 25°C with a 16:8 h light:dark cycle. Mortality was scored at 0.5-day intervals until all viral infected larvae died. Twenty to twenty-four larvae were infected per mortality-time assay for each virus. The larvae that did not respond to gentle stimulation at the head end using a forceps were scored as dead. For examination of the larval growth, the weight and the development status of each larva was monitored at 1-day intervals. Preparation of hemolymph Fifth instar larvae of M. brassicae were infected with vTREVL, vERTVL or wt virus (2 × 104 PFU/larva) or mock-infected, and were reared on an artificial diet at 25°C. Three to five larvae were bled by incising the larval proleg, each larva being bled only once, and then the
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hemolymph of the larvae was pooled in an ice-chilled 1.5-ml microtube containing a small amount of phenylthiourea to prevent the action of tyrosinases in the larval hemolymph. The pooled hemolymph was centrifuged at 5000 g for 5 min at 2°C to remove hemocytes, and the supernatants were divided into small aliquots. Samples obtained were stored at −80°C until subjected to (1) trehalase-activity assay by glucose oxidase method, (2) native polyacrylamide gel electrophoresis followed by staining for trehalase activity on the gel, or (3) sugar determination by glucose oxidase method. Storage at − 80°C had no effects on trehalase activities and sugar levels. Assay of trehalase activity by glucose oxidase method The assay of trehalase activity was performed by modifications of the method described by Yaginuma and Happ (1988). The standard reaction mixture contained 10-l enzyme source (the supernatant of SF-9 cell culture medium or larval hemolymph) and 20-l substrate solution (50 mM trehalose in 100 mM citrate-sodium phosphate, pH 5.6). The reaction was allowed to proceed at 30°C for 20 min and then stopped by heating at 98°C for 10 min with a microtube heater. After cooling, the solution was centrifuged at 14,000 g for 5 min. Aliquots (2 l) of the supernatant were pipetted into the wells of a 96-well microtiter plate, and were subjected to the determination of glucose derived from trehalose by glucose oxidase method (Hugget and Nixon, 1957) using a glucose-determination reagent kit, Iatro-Chrom GLU-LQ (Iatron Laboratories, Tokyo, Japan). Enzyme specific activity was expressed as moles of glucose produced per min per mg of protein. Protein determination was performed by the method of Bradford (Bradford, 1976) using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, Calif.) with bovine serum albumin (BIO-RAD) as a standard. Native polyacrylamide gel electrophoresis (PAGE) and staining for trehalase activity on the gel Samples were electrophoresed at pH 8.3 with molecular weight standards for native proteins (Nondenatured protein molecular weight marker kit; Sigma, St. Louis, MO.) on a ready-made gradient polyacrylamide slab gel (5–20%), PAGEL NPG-520L (ATTO, Tokyo, Japan). After electrophoresis, the gels were incubated in substrate solution (33 mM trehalose in 100 mM citratesodium phosphate, pH 5.6) at 37°C for 30 min, soaked in glucose-fixation solution (100 mM iodoacetamide) at room temperature for 5 min, and then boiled in colorproducing solution (0.1%, 2,3,5-triphenyltetrazolium chloride in 0.5 N NaOH) for 2–3 min until trehalase bands appeared (Sumida and Yamashita, 1983). After the staining for trehalase activities, proteins on the gels were stained with Coomassie brilliant blue R-250-staining solution, PAGE Blue 83 (Daiichi Pure Chemicals, Tokyo, Japan).
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Treatment of virus-infected SF-9 cells with tunicamycin
RESULTS
T. molitor trehalase has been shown to have 5 potential N-glycosylation sites in its amino-acid sequence (Takiguchi et al., 1992). This fact led us to investigate whether the trehalase was N-glycosylated. SF-9 cells were treated with tunicamycin by the method of O’Reilly et al. (1992c) with some modifications as follows. Onemillion SF-9 cells were attached on a 35-mm-diameter culture dish, and infected with 100 l of the complete medium containing 5 × 106 PFU of vTREVL for 1 h at room temperature. After the viral inoculum was removed, the cells were cultured in 1 ml of TNM-FH medium containing 10% FBS for 12 h at 27°C. Tunicamycin (Sigma) was added at the concentration of 10 g per ml into the cell culture at 12 h p.i., and the culture was incubated at 27°C for an additional 36 h. As the control, the infected cells were cultured in the complete medium without tunicamycin. After the additional 36-h incubation, the culture dish was shaken gently to dislodge the cells from the surface of the culture dish, and then the culture medium was centrifuged at 1000 g for 2–3 min to precipitate floating cells. The cells were suspended in 100 l of TNM-FH medium plus 10% FBS, and lysed completely by sonication with a sonicator, Bio-Disrupter BD1 (Nippon Seiki, Tokyo, Japan). The extent of cell lysis was checked by observing a small volume of the sonicated sample with an inverted microscope. The cell lysate was centrifuged at 14,000 g for 2–3 min to remove cell debris. Aliquots of the supernatant of the culture medium or cell lysate were used for native PAGE.
Construction of recombinant baculoviruses Two recombinant baculoviruses, vTREVL and vERTVL, in which T. molitor trehalase cDNA was inserted in the sense and antisense orientations, respectively, downstream of the AcMNPV Ppolh, were constructed. The recombinant viruses were isolated by plaque purification and amplified up to high titers (1–3 × 108 PFU/ml). The presence of the trehalase cDNA within the virus genome was confirmed by amplifying the fragment specific to the trehalase cDNA using PCR method and then sequencing the product (data not shown). The presence or absence of trehalase in the culture medium of vTREVL- or vERTVL-infected SF-9 cells was monitored by the assay of trehalase activity. Expression of T. molitor trehalase in SF-9 cells and larvae of M. brassicae Monolayers of SF-9 cells (1 × 106 cells/35-mm-diameter culture dish) were infected with viruses, and the supernatants of culture media were collected at intervals of 24 h. Although almost negligible levels of trehalase activity were detected in the supernatants of culture media of vERTVL-infected, wt virus-infected and mockinfected cells, extremely high levels of trehalase activity were found in the supernatants of vTREVL-infected cell culture media along with incubation times (Fig. 1). Trehalase activity in the supernatant of vTREVL-infected cell culture medium first occurred between 12 and 24 h p.i. and reached a maximum level at 72 h p.i. Following native PAGE for the supernatants of culture media of
Determination of trehalose and glucose in hemolymph Trehalose in hemolymph was determined by the method of Yaginuma and Happ (1988) with some modifications. The supernatant of hemolymph was diluted 4 times with 100 mM citrate-sodium phosphate (pH 5.6) and heated at 98°C for 10 min with a microtube heater. After cooling, the heated supernatant was suspended by stirring with a micro pipet. Four l of porcine kidneytrehelase (1.47 U/ml; Sigma) was added to 20 l of the suspension, and the mixture was incubated at 37°C for 16 h. For the control reaction, only enzyme-storage solution (50% glycerol containing 1% Triton X-100 and 25 mM potassium phosphate, pH 6.5) (Sigma Chemical Co., 1996) was added instead of the trehalase. The reaction mixture was heated at 98°C for 10 min for the termination of trehalase reaction and were centrifuged at 14,000 g for 5 min. Two l of the supernatant was used for the determination of glucose by glucose oxidase method. As control, 2 l of the hemolymph supernatant which had not received trehalase-treatment was used for the determination of glucose by glucose oxidase method as described above.
FIGURE 1. Trehalase activities in supernatants of the culture media of vTREVL-, vERTVL-, wt virus- or mock-infected SF-9 cells. Cells (1 × 106 cells/35-mm-diameter culture dish) were infected with each virus (5 PFU/cell) or mock-infected, and then cultured in 1 ml of TNM-FH medium with 10% FBS at 27°C. Supernatants of the culture media of vTREVL-, vERTVL-, wt virus- or mock-infected cells were collected at 1, 12, 24, 48, 72 and 96 h p.i., and used for assays of trehalase activities. Each point represents the mean trehalase activity ± range (n = 2; vertical bars). Symbol only means that range is within the symbol.
BACULOVIRUS-MEDIATED EXPRESSION OF A MEALWORM BEETLE
FIGURE 2. Detection of trehalase activities on the gel after native PAGE of supernatants of the culture media of vTREVL-infected SF9 cells. Supernatants (6 l) of the culture media from vTREVLinfected cells in Fig. 1 were analyzed by native PAGE, and trehalase activities were detected on the gel. Lanes 1–6: supernatants of the culture media collected at 1, 12, 24, 48, 72 and 96 h p.i., respectively. The locations of the molecular weight standards are indicated on the right margin.
vTREVL-infected cells, staining for trehalase activity on the gel was carried out. An active trehalase with an apparent molecular weight of approximately 85 kDa was detected (Fig. 2). Expression of active trehalase in vTREVL-infected SF-9 cells, from 12 to 48 h p.i., in the culture medium with or without tunicamycin is shown in Fig. 3. Although in the extracts of the cells incubated without tunicamycin, two active trehalases of 85 kDa and 62 kDa were detected (lane 1), an active trehalase of 85 kDa was detected in the culture medium (lane 2). In contrast, in the presence of tunicamycin, cells expressed only an active trehalase with an apparent molecular weight of approximately 62 kDa (lane 3), and any active trehalases were not found in the culture medium (lane 4). These results indicated that the 85-kDa trehalase extracted from the cells without tunicamycin treatment was N-glycosylated.
FIGURE 3. Effects of tunicamycin on the expression of T. molitor active trehalase in SF-9 cells. Cells were infected and then cultured in the same condition as Fig. 1. Tunicamycin was added into the cell culture at 12 h p.i., and the culture was incubated for an additional 36 h. As the control, the infected cells were cultured in the medium without tunicamycin. After the additional 36-h incubation, the infected cells and culture medium were separated. The infected cells were suspended in 100 l of the complete medium and then lysed. Supernatants (6 l) of the culture medium and cell lysate were analyzed by native PAGE followed by staining for trehalase activity on the gel. Lanes 1 and 2: cell lysate and culture medium without tunicamycin treatment, respectively; lanes 3 and 4: cell lysate and culture medium with tunicamycin treatment, respectively. The locations of the molecular weight standards are indicated on the right margin.
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An active trehalase of 85 kDa was also detected in the hemolymph of vTREVL-infected M. brassicae larvae at 3 days p.i., and the activity reached a peak at 4 days p.i., while in the hemolymph of vERTVL-, wt virus- or mockinfected larvae no active trehalases occurred (Figs 4 and 5). Trehalose level in the hemolymph of vTREVLinfected larvae decreased markedly at 3 days p.i., at the same time as the active trehalase became detectable in the hemolymph, and trehalose disappeared from the hemolymph by 4 days p.i [Fig. 6(A)], accompanied by the occurrence of a significant level of glucose derived from trehalose in the hemolymph. However, glucose also disappeared from the hemolymph of vTREVL-infected larvae by 6 days p.i., namely shortly before death (Fig. 9). On the other hand, there were substantial levels of trehalose and almost negligible levels of glucose in the hemolymph of vERTVL-, wt virus- or mock-infected larvae throughout the experimental period [Fig. 6(B, C and D)]. Effects of recombinant baculovirus-infection on SF-9 cells and larvae of M. brassicae No significant differences in the growth of extracellular virus on SF-9 cells were found among vTREVL, vERTVL and wt virus (Fig. 7). The growth of each extracellular virus reached a maximum titer at 72 h p.i. at about 6 × 108 TCID50 per ml. Few cytopathological differences were seen in the virus-infected cells among the recombinant viruses and wt virus, except that polyhedra were formed in the wt virus-infected cells (data not shown). Growth of the virus-infected cells was suppressed markedly, irrespective of the recombinant viruses or wt virus (Fig. 8). To examine whether the insertion of T. molitor trehalase gene into the AcMNPV genome has any effect on the insecticidal activity of the virus, the mortality of M. brassicae larvae infected with vTREVL, vERTVL or wt virus in the early stage of 5th instar period was monitored (Fig. 9). At 6 days p.i., more than 95% of the larvae infected with wt virus died. On the same day, 72% of the larvae infected with vERTVL died. At 6.5 days p.i., all the larvae infected with wt virus died. Although at 7 days p.i. all the larvae infected with vERTVL died, 65% of the larvae infected with vTREVL still survived. At 8.5 days p.i., all the larvae infected with vTREVL died (Fig. 9). However, significant differences in the weight gains of virus-infected larvae were not observed among the recombinant viruses and wt virus at any time point p.i. (Fig. 10). The mock-infected larvae ecdysed into 6th instar (last instar) by 2 days p.i., and continued to gain weight up to 4 days p.i. From 4–5 days p.i., they experienced a retardation of weight gain, coincident with the onset of gut-purge prior to pupation. All of the mockinfected larvae metamorphosed into pupae by 8 days p.i. Regardless of the recombinant viruses or wt virus, all of the virus-infected larvae showed the signs of illness, such as retardation of growth, swelling of body and blocking of molt into 6th instar, by 3 days p.i., and died during 5th instar period. At 4 days p.i., the titer of extra-
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FIGURE 4. Trehalase activities in the hemolymph of vTREVL-, vERTVL-, wt virus- or mock-infected M. brassicae larvae. Fifth instar larvae were infected with each virus (2 × 104 PFU/larva) or mock-infected, and then reared on an artificial diet at 25°C. Hemolymph from 3 to 5 larvae was pooled at 0.25, 1, 2, 3, 4, 5 and 6 days p.i., and trehalase activities in supernatants of the hemolymph were assayed. Each point represents the mean of activities ± range (n = 2).
FIGURE 5. Detection of trehalase activities on the gel after native PAGE of the hemolymph of vTREVL-infected M. brassicae larvae. Supernatants (6 l) of the hemolymph collected in Fig. 4 were analyzed by native PAGE followed by staining for trehalase activity on the gel. Lanes 1–7: supernatants of the hemolymph collected from vTREVL-infected larvae at 0.25, 1, 2, 3, 4, 5 and 6 days p.i., respectively. The locations of the molecular weight standards are indicated on the right margin.
cellular virus (TCID50 values ± SEM; n = 2) in the hemolymph of virus-infected larvae was 7.2 × 108 ± 5.8 × 107 for vTREVL, 7.3 × 108 ± 2.0 × 107 for vERTVL and 2.8 × 109 ± 2.8 × 108 for wt virus. There were about 4-fold differences between the recombinant viruses and wt virus. Figure 11 shows the virus-infected 5th instar larvae shortly after death. Significant differences in the symptoms of virus-infected larvae were detected among the recombinant viruses and wt virus. The larvae infected
with wt virus exhibited the typical symptoms of NPV infection, namely the browning and liquefying of the host body prior to death or shortly thereafter. Unlike wt virusinfected larvae, the body color of dead vTREVL-infected larvae remained yellow-green similar to that of the healthy larvae during the molting period. In addition, the dead body of a vTREVL-infected larva was soft, but did not liquefy. The symptoms of vERTVL-infected larvae were intermediate between those of wt virus-infected and vTREVL-infected larvae. The abdominal color of a dead
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FIGURE 6. Levels of trehalose and glucose in the hemolymph of M. brassicae larvae infected with vTREVL, vERTVL or wt virus. Concentrations of trehalose and glucose in supernatants of the hemolymph from vTREVL-infected (A), vERTVLinfected (B), wt virus-infected (C) and mock-infected (D) larvae collected in Fig. 4, were determined. Each point represents the mean ± range (n = 2).
FIGURE 7. Growth curves of vTREVL, vERTVL and wt virus on SF9 cells. Cells were infected with each virus and then cultured in the same condition as Fig. 1. Supernatants of the culture media were collected at 1, 12, 24, 48, 72 and 96 h p.i., and subjected to TCID50 assay. Each point represents the mean of TCID50 values ± range (n = 2; vertical bars).
FIGURE 8. Viability of SF-9 cells infected with vTREVL, vERTVL or wt virus. The cells used in Fig. 7 were monitored. At the times indicated, viable cells were counted by using the Trypan blue exclusion method. Each point represents the mean density of viable cells ± range (n = 2; vertical bars).
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FIGURE 9. Mortality of M. brassicae larvae infected with vTREVL, vERTVL or wt virus. Twenty to twenty-four fifth instar larvae were infected with each virus or mock-infected and then reared in the same condition as Fig. 4. The cumulative mortality observed in two separate series different from Figs 4–6, was monitored at 0.5-day intervals. Each point represents the mean value ± range (n = 2).
vERTVL-infected larva became brown, while the remainder of the dead body retained yellow-green color. However, dead vERTVL-infected larvae did not liquefy. DISCUSSION
This is the first report of a catalytically active trehalase produced in baculovirus-infected insect cells and insect tissues. Active T. molitor trehalase was detected in the culture medium of vTREVL-infected SF-9 cells (Fig. 1) or in the hemolymph of vTREVL-infected larvae of M. brassicae (Fig. 4), and the active trehalase was shown to be an apparent molecular weight of approximately 85 kDa as determined by native PAGE and subsequent staining for trehalase activity (Figs 2 and 5). This size of molecular weight was larger than the calculated molecular weight (62.7 kDa) of the putative protein encoded by an open reading frame of the trehalase cDNA (Takiguchi et al., 1992). This finding that the 85-kDa protein is synthesized and released from the insect cells infected with vTREVL, indicates that the T. molitor trehalase undergoes post-translational modifications through the secretion. Consistent with this idea are the following observations: 1) the trehelase cDNA has a putative signal sequence and 5 potential N-glycosylation sites, Asn-XaaSer/Thr (Takiguchi et al., 1992), and 2) the 85-kDa active trehalase was not detected, in the presence of tunicamycin, in the culture medium or the cells (Fig. 3). Since only active trehalase of 62 kDa was detected in the tunicamycin-treated cells (Fig. 3), the active trehalase of 62 kDa appeared to be the non-glycosylated precursor of the trehalase. The results have led us to the idea that the 62-kDa enzyme-precursor with no sugar moieties is Nglycosylated and subsequently secreted as the mature trehalase of 85 kDa into the culture medium, because inhi-
bition of N-glycosylation by tunicamycin prevented the trehalase from being secreted, as such cases described for certain proteins (Gopalakrishnan et al., 1995; Jarvis and Summers, 1989; Jarvis et al., 1990). In the hemolymph of larvae infected with vTREVL, the time in increase of trehalase activity coincided with that in decrease of trehalose [Fig. 4 and Fig. 6(A)]. In general, in insects, glucose in the hemolymph is transferred to the fat body and then converted to trehalose, which is rapidly released from the fat body into the hemolymph to maintain almost constant levels of trehalose (Wyatt, 1967). Therefore, no significant accumulation of glucose has usually been observed in insect hemolymph (Wyatt, 1967), as in the hemolymph of normal larvae shown in Fig. 6(D). The observations in Fig. 6(A), such as a decrease of trehalose and an increase of glucose in the hemolymph of vTREVL-infected larvae, indicate that the total activity of trehalase expressed in the hemolymph was well over the synthetic activity of trehalose from glucose in the fat body, subsequently leading to an accumulation of glucose which was derived from trehalose and finally used by the other tissues until the stage just before larval death. This imbalance in trehalose metabolism may have resulted from the production of the active trehalase in the hemolymph and the lowered synthetic activity due to the damage of the fat-body tissue infected with the virus. In the larvae infected with vERTVL or wt virus, the fat body and the other tissues that can utilize the hemolymph trehalose may have simultaneously been damaged, subsequently resulting in a substantial levels of trehalose in the hemolymph [Fig. 6(B and C)]. As described above, in spite of the essentiality of trehalase in the utilization of hemolymph trehalose by insect tissues, molecular relationship between the enzyme structure and the activity remains unclear. The potential of this baculovirus system for producing a large amount of active trehalase should permit the following studies: (1) structure-function analysis by site-directed mutagenesis as utilized for the investigation of the catalytic mechanism of certain enzymes (Funk et al., 1989; McKegney et al., 1996; Ward et al., 1992), (2) crystallization and subsequent tertiary structure analysis, (3) in vitro elucidation of direct effects of various inhibitors on the trehalase, and (4) tertiary structure analysis of the enzymeinhibitor complex. These studies should pave the way to determine such functional domains as substrate- and inhibitor-binding sites of this enzyme, and to develop trehalase inhibitors effective as insecticides. We first assumed that baculovirus-mediated expression of trehalase in insects would cause the depletion of trehalose in the hemolymph, and then result in the earlier effects with respect to killing these infected insects. Contrary to this assumption, the mortality time of virusinfected 5th instar larvae of M. brassicae increased in the following order: wt virus ⱕ vERTVL ⬍ vTREVL (Fig. 9). Nevertheless, both the recombinant viruses, vTREVL and vERTVL, showed a tendency of slower
BACULOVIRUS-MEDIATED EXPRESSION OF A MEALWORM BEETLE
FIGURE 10. Weights of M. brassicae larvae infected with vTREVL, vERTVL or wt virus. Fifth instar larvae used in Fig. 9 were weighed at 1-day intervals. Each point represents the mean weight of 20 to 24 larvae ± range (n = 2).
speed of viral growth in the larvae than wt virus, because there was about a 4-fold difference in the titer (TCID50) of extracellular virus in the hemolymph of virus-infected larvae between the recombinant viruses and wt virus at 4 days p.i. However, the weight gains of virus-infected larvae were scarcely affected by such differences in virus-type as vTREVL, vERTVL and wt virus (Fig. 10). Further, significant differences in the symptoms of virusinfected larvae of M. brassicae were observed among vTREVL, vERTVL and wt virus (Fig. 11). The typical symptoms of NPV infection such as the browning and liquefying of the host body were observed in wt virusinfected larvae. The symptoms of NPV infection were significantly moderated in vTREVL-infected larvae. The symptoms of vERTVL-infected larvae were intermediate between those of vTREVL-infected and wt virus-infected
FIGURE 11. vTREVL-, vERTVL- or wt virus-infected M. brassicae larva shortly after death. Fifth instar larvae used in Fig. 9 were monitored until death. (A): vTREVL-infected larva; (B) vERTVL-infected larva; and (C): wt virus-infected larva.
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larvae. These results appear to be consistent with those of recent reports (O’Reilly et al., 1995; Wood et al., 1993) have shown that the lack of polyhedrin production and the further expression of another gene reduce the insecticidal activity of the virus. The expression of the trehalase gene may be involved in the increased mortality time and moderated symptom of infected larvae (Fig. 9 and Fig. 11), in addition to the lack of polyhedrin protein. However, a recombinant virus expressing both polyhedrin gene and inserted foreign gene has been shown to increase the mortality time and reduce the weight gain of infected larvae, compared to the wt virus as a control (Bonning et al., 1992). Therefore, to understand the relationship between gene expressions of trehalase and polyhedrin, and the increased mortality time and moderated symptom of infected larvae, it is further required to examine whether a recombinant virus that can express both the polyhedrin and secretory-trehalase genes, expresses much higher or lower insecticidal activity than wt virus. REFERENCES Ando O., Nakajima M., Kifune M., Fang H. and Tanzawa K. (1995) Trehazolin, a slow, tight-binding inhibitor of silkworm trehalase. Biochim. Biophys. Acta 1244, 295–302. Asano N., Takeuchi M., Kameda Y., Matsui K. and Kono Y. (1990) Trehalase inhibitors, validoxylamine A and related compounds as insecticides. . J. Antibiot. 43, 722–726. Benz, G.A. (1986) Introduction: historical perspectives. In The Biology of Baculoviruses (Edited by Granados R. R. and Federici B. A.), Vol. I, pp. 1–35. CRC Press, Boca Raton, Fla. Bonning B.C., Hirst M., Posse R.D. and Hammock B.D. (1992) Further development of a recombinant baculovirus insecticide expressing the enzyme juvenile hormone esterase from Heliothis virescens. Insect Biochem. Molec. Biol. 22, 453–458. Bonning B.C., Hoover K., Booth T.F., Duffey S. and Hammock B.D. (1995) Development of a recombinant baculovirus expressing a modified juvenile hormone esterase with potential for insect control. Arch. Insect. Biochem. Physiol. 30, 177–194. Bonning B.C. and Hammock B.D. (1996) Development of recombinant baculoviruses for insect control. Annu. Rev. Entomol. 41, 191–210. Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248–254. Chejanovsky N., Zilberberg N., Rivkin H., Zlotkin E. and Gurevitz M. (1995) Functional expression of an alpha anti-insect scorpion neurotoxin in insect cells and lepidopterous larvae. FEBS Lett. 376, 181–184. Funk C.D., Gunne H., Steiner H., Izumi T. and Samuelssen B. (1989) Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system. Proc. Natl. Acad. Sci U.S.A. 86, 2592–2596. Gopalakrishnan B., Muthukrishnan S. and Kramer K.J. (1995) Baculovirus-mediated expression of a Manduca sexta chitinase gene: properties of the recombinant protein. Insect Biochem. Molec. Biol. 25, 255–265. Hammock B.D., Bonning B.C., Posse R.D., Hanzlik T.N. and Maeda S. (1990) Expression and effects of the juvenile hormone esterase in a baculovirus vector. Nature 344, 458–461. Hink W.F. (1970) Established insect cell line from the cabbage looper Trichoplusia ni.. Nature 226, 466–467. Hugget A.S.G. and Nixon D.A. (1957) Enzymatic determination of blood glucose. Biochem J. 66, 12. Jarvis D.L. and Summers M.D. (1989) Glycosylation and secretion of
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Acknowledgements—The authors thank T. Uchiyama and Y. Sato (Hokko Chemical Industry Co., Atsugi, Kanagawa, Japan) for their kind supplies of Mamestra brassicae eggs.