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Synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta: Its uptake and enhancement of virus infection in vitro
JOURNAL
OF INVERTEBRATE
Synergistic
Pseudalefia KAORU
54, 15&16’t (1989)
PATHOLOGY
Factor of a Granulosis Virus of the Armyworm, its Uptake and Enhancement of Virus Infection in Vitro
unipuncta: UCHIMA,
DAVID
E. EGERTER,
AND YOSHINORI
TANADA
Department of Entomological Sciences, University ofCalifornia, Berkeley, CalSfornia 94720 Received September 7, 1988; accepted November 18, 1988 A synergistic factor (SF) enhanced the infection of Autograph californica nuclear polyhedrosis virus (AcNPV) in vitro. When the AcNPV was inoculated together with the SF to a Spodopteru frugiperda cell line, the enhancement of AcNPV increased dose-responsively to the SF. Enhancement, however, did not require the coinoculation of AcNPV and SF since it occurred in the pretreatment of the cells with the SF alone and even after a 24-hr interval of SF pretreatment. There was a difference in the activity of the SF on the AcNPV propagated in a cell culture system during various periods of virus propagation. When the SF was treated with phospholipase C, the enhancement of AcNPV infection did not occur in vitro and confirmed our previous study in vivo. The cells appeared to phagocytize the SF and the uptake occurred rapidly (within 1 mitt), but there was no uptake when the SF was treated with phospholipase C. These results indicate that the SF is a binding molecule in which the phospholipid component is essential for its phagocytosis and enhancement of baculovirus infection. The SF appears to have two different binding sites, one for the cell plasma membrane and the other for the enveloped virion. B 1989 Academic press, hc. KEY WORDS: nuclear polyhedrosis virus of Autograph californica; cell line of Spodoptera frueiperda; synergistic factor, uptake; baculovirus infection, enhancement; immunoperoxidase staining.
INTRODUCTION
A synergistic factor (SF) found in the occlusion body (capsule) matrix of the Hawaiian strain of a granulosis virus enhances the infection of a nuclear polyhedrosis virus (NPV) in armyworm ,larvae (Pseudaletia unipuncta) (Tanada and Hukuhara, 1971). The SF is a lipoprotein, MW 93,000 (Hotchkin, 1981) or 126,008 (Yamamoto and Tanada, 1978a), and the phospholipid component is essential for the enhancing activity in vivo (Yamamoto and Tanada, 1978a). The site of action of the SF is the plasma membrane of the microvillus of larval midgut epithelium (Tanada et al., 1975, 1980a, b, 1983; Hukuhara and Zhu, 1988). The SF also enhances in vitro the infection of several NPVs (Ohba and Tanada, 1983,1984a), and its effect is on the cell plasma membrane (Nakagaki et al., 1987; Hukuhara and Zhu, 1988). In our present study, the immunoperoxidase staining for the enveloped virion of NPV was used to study the effect of the SF on virus infection in vitro. The
apparent uptake of the SF into cells was also observed with immunoperoxidase staining. MATERIALS Cells
A cell line of Spodoptera frugiperda (IPLB-SF-21), which was kindly provided by Dr. M. A. Whit1 (University of California, Davis), was maintained in the medium MBL-TC/lO (Gardiner and Stockdale, 1975) containing 10% fetal bovine serum (FBS, Cat. No. 29-101, Flow Laboratories, McLean, Virginia), 100 unit/ml penicillin, 100 p,g/ml streptomycin, and 0.25 l&ml Fungizone (Gibco Lab., Grand Island, New York). The cells were cultured at 27°C and transferred every 48-72 hr. Virus Inoculum Autographa californica nuclear polyhedrosis virus (AcNPV), kindly provided by Dr. M. A. Whitt (University of California, 1.56
0022-201 l/89 $1.50 Copyright All rights
0 1989 by Academic Press, Lnc. of reproduction in any form reserved.
AND METHODS
SYNERGISTIC
FACTOR
Davis), was propagated in IPLB-SF-21. The infected cell culture was centrifuged at 1OOOg and the supematant was used as virus inoculum for the preparation of the different virus inocula. The S. frugiperda cells cultured in 25ml flasks (Falcon 3013, Becton Dikinson and Co., Oxnard, California) were inoculated with AcNPV, incubated for 1 hr, then rinsed twice with medium (4 ml) to remove the excess AcNPV in the cell culture, and replenished with 4 ml of medium. After 48 hr incubation, the medium was collected and centrifuged at lOOOg, and the supematant (virus inoculum) containing mainly enveloped virions was stored at 4°C. The supernatant, when diluted 104-lo5 times, gave 5-30 stained foci/well. The diluted virus inoculum was prepared just prior to its use in the tests, SF and Antibodies The SF was prepared as described by Uchima et al. (1988) and the anti-SF rabbit serum as reported by Yamamoto and Tanada (1978b). The anti-AcNPV-envelope rabbit serum, which was kindly provided by Dr. L. E. Volkman (University of California, Berkeley), had been prepared as described by Volkman et al. (1976). Assay of AcNPV
Infectivity
The assay of AcNPV infectivity was performed with a modification of the method of Volkman and Goldsmith (1982). Microslides containing 12 wells (5 mm diameter, Carlson Scientific Inc., Peotone, Illinois) were first soaked in 1% HCl and 70% ethanol, then rinsed with double-distilled water, and dried and sterilized in an oven. Twenty microliters of cell suspension (lo6 cells/ml) was seeded in each well and cultured overnight at 27°C in a humid chamber. Then the medium was carefully removed from the wells and replaced with 20 ~1 of freshly diluted AcNPV inoculum. After certain periods of AcNPV exposure, the inoculum was removed and replaced with medium containing 0.6% methylcellulose
OF
157
P. unipuncta
(Hink and Vail, 1973), and the cells were incubated for 48 hr at 27°C in a humid chamber. The fixation and immunoperoxidase staining were performed according to Volkman and Goldsmith (1982). The anti-rabbit goat immunoglobulin (Product No. 5001) was purchased from Sigma Chemical Co. (St. Louis, Missouri) and peroxidaseantiperoxidase complex (rabbit serum, Cat. No. 8430-01) from Litton Bionetics (Charleston, South Carolina). Assay of SF Uptake by Cells Cells were seeded in each well of the microslides and cultured overnight, and then the medium in each well was removed and replaced with 20 ~1 of fresh medium containing the SF (100 pg/ml). After the designated period of incubation at 27°C in a humid chamber, the medium was removed from the wells and the attached cells were fixed by dipping the microslides into cold ethanol for 2 min. The fixed cells were rinsed with cold buffered saline (0.15 M NaCl, 50 mM Tris-HCl, pH 7.6) and treated successively with anti-SF rabbit serum, anti-rabbit goat immunoglobulin, and peroxidaseantiperoxidase complex according to the method of microslide preparation described under Assay of AcNPV Infection. Treatment
of SF With Phospholipase
C
One milligram of the SF that had been stored at -20°C in 1.5 ml of 100 mM NaCl and 10 mM sodium phosphate buffer, pH 8.0, were dialyzed in 5 mM CaCl, and 100 mM Tris-HCl, pH 8.0. One milliliter of the SF solution was collected after dialyzation. Phospholipase C (Cat. No. 691950, Boehringer-Mannheim Biochemicals, Indianapolis, Indiana) at 40 units/l0 l~l was added to 1 ml of SF solution and incubated for 2 hr with gentle shaking. The enzyme, undigested SF, and derivatives were removed with a gel filtration column (1.0 x 35 cm) using Fractogel TSK HW-55 (F) (Cat. No. 14981-4, EM Science, Gibbstown, New Jer-
158
UCHIMA,
EGERTER,
sey) as a gel filtration medium and 10 mM Tris-HCI, pH 8.0, as elution buffer. Four hundred micrograms of the SF treated with phospholipase C was recovered and analyzed by immunoprecipitation with the antiSF rabbit serum in a double diffusion test. Statistical Student’s analysis.
Analysis t test was used for statistical
RESULTS EfSect of Length of Exposure to AcNPV, With and Without SF, on AcNPV Infection
AND TANADA
Effect of SF Concentration AcNPV Infection
on
The AcNPV was applied to cultured cells together with various concentrations of the SF (O-100 t&ml) and the cells were incubated for 20 hr. The AcNPV infection was enhanced at the SF concentrations higher than 100 &ml and the enhancement increased rapidly, attaining a maximum level at 10 pg/rnl (Fig. 2). The number of stained foci of cells inoculated with the AcNPV together with 10 pg/ml of the SF was about twice as high as that without the SF. Effect of Pretreatment Intervals on AcNPV Infection
Cultured cells were inoculated with AcNPV, with and without the SF (100 ).&ml). Exposure of cells to AcNPV was performed at various intervals (O-24 hr). The number of cells infected in the absence of SF gradually increased during the 24 hr exposure to AcNPV, whereas the number of infected cells in the presence of SF increased rapidly during the first 8 hr of incubation and then increased gradually (Fig. 1). The number of stained foci of cells exposed to AcNPV in the presence of the SF for g-24 hr was more than two times higher than that treated without the SF. The enhancing effect of the SF on AcNPV infection occurred predominantly during the first 8 hr and was near maximum at 24 hr exposure.
With SF
Cells were incubated first with the SF (100 t&ml) for various intervals, then the SF was removed by rinsing with medium, and the cells were exposed to AcNPV for 20 hr. The AcNPV infection increased rapidly and reached a maximal level after 5 min of the SF pretreatment of the cells (Fig. 3). The AcNPV infection was maintained at the maximal level for at least 4 hr SF pretreatment. The number of stained foci of cells pretreated with the SF for more than 5 min was about twice as high as that of cells receiving no SF pretreatment. Uptake of SF by Cells The binding and uptake of the SF by cells 160 r
01 0-‘I Dumtion
of AcNPV
exposure
(hr)
1. Effect of length of exposure to AcNPV, and without the SF, on virus infection. AcNPV exposed to the cells for various periods together 100 pg/ml SF (0) or without SF (0). Each point resents the mean 2 SE from six determinations. FIG.
Concentmtion
with was with rep-
IO’
Id
Id
Id
Id
of SF (rig/ml)
FIG. 2. Effect of SF concentrations on virus infection. AcNPV was inoculated into cells with various concentrations of SF. Cells were incubated in the inoculum at 27°C for 20 hr. Each point represents the mean f SE from six determinations.
SYNERGISTIC
0
Duration
I
2
of
pretreotrnent with
3
4 SF (hr)
FIG. 3. Effect of pretreatment of cells with the SF on virus infection. Cells were pretreated with 100 p.gknl SF for various periods, followed by inoculation of AcNPV. Each point represents the mean 2 SE from six determinations.
were determined by immunoperoxidase staining. Cells were incubated with the SF (100 pg/ml) for various intervals (O-l hr), fixed, and stained as described above. The SF bound rapidly on the cell membrane and appeared to be taken into the cells within I min (Fig. 4). The apparent uptake of the SF by the cells reached a maximal state after 15 min incubation. Effect of Pretreatment of Cells With Various SF Concentrations on AcNPV Infection Cells were incubated with various concentrations of the SF for 1 hr, then rinsed with medium to remove the SF, and incubated with AcNPV for 20 hr. One microgram per milliliter of SF was required for the enhancement of the AcNPV infection (Fig. 5). Virus infection progressively increased with the pretreatment of the cells at higher concentrations of the SF. The number of stained foci of cells pretreated with 100 p&ml SF was 1.7 times higher than that without the SF. Effect of Various Intervals following Pretreatment on AcNPV Infection
159
FACTOR OF P. unipuncra
SF
Cells were treated with the SF for 1 hr, followed by the removal of the SF by rinsing with medium, the incubation for various periods (CL24 hr), and then the exposure to AcNPV for 20 hr. The control group was treated without the SF for 1 hr, followed by
the rinsing procedure and various periods of incubation prior to AcNPV inoculation. The enhancing effect of the SF on AcNPV infection increased gradually with the SF-pretreatment interval of 4 hr and attained a maximal level at about 8 hr (Fig. 6). After 16 hr, the enhancing effect decreased but was still apparent at 24 hr. The number of stained foci in the cells at maximal level of enhancement (8 hr) was about twice that of the control group. Effect of SF on AcNPV Propagated Various Periods of Cell Culture
at
AcNPV was inoculated into cells cultured in flasks (25 ml Falcon) and after 2 hr, the inoculum was removed from each flask by rinsing the cells with three changes of culture medium. Two milliliters of fresh medium was added to each flask and the cells were incubated for 4,8, or 18 hr. After incubation, the medium (supernatant) was collected by centrifugation at 1OOg for 5 min. The supematant was kept at 4°C and used as the inoculum with or without the SF (100 pg/ml). The inoculated cells were incubated for 20 hr. The SF only slightly enhanced the infection of AcNPV propagated for 6 and 10 hr (P < 0.05 and P < 0.1, respectively), and greatly enhanced virus infection in virions produced in 20 hr (P < 0.01) (Fig. 7). Effect of the Treatment Phospholipase C
of SF With
(a) Effect on the infection of AcNPV. Cells were exposed to AcNPV for 20 hr together with the SF or with the SF treated with phospholipase C (100 kg/ml). The control had neither SF nor SF treated with phospholipase C. Two different concentrations of AcNPV were used. The treatment of the SF with phospholipase C resulted in no enhancement of AcNPV infection (Fig. 8), and may have interfered with virus infection since the number of stained foci of cells inoculated with AcNPV and SF treated with phospho-
160
UCHIMA,
EGERTER,
AND
TANADA
FIG. 4. Uptake of SF by cells. Cells were incubs tted with the SF for 0 min (A), 1 min (B), 5 min (C), 15 min (D, E), or 1 hr (F), and stained as described in the text. Photographs of stained cells were taken with bright field microscope (A-D, F) or Nomarsl ti microscope (E). Magnification: bar = 25 pm.
lipase C was statistically the control.
less than that of
(6) Uptake by the cells. The cells incubated with the SF or the SF treated with phospholipase C (100 &ml) for 1 hr were fixed and stained immunologically as described under Materials and Methods. Other cells were fixed in cold ethanol and formalin, rinsed twice with buffered saline
pH 7.6), (0.15 M NaCl, 50 mM Tris-HCl, incubated with the SF or the SF treated with the enzyme, and after 1 hr incubation, they were stained with immunoperoxidase. Controls for both fixed and unfixed cells were incubated for 1 hr with neither the SF nor the SF treated with phospholipase C. The SF, not treated with the enzyme, appeared to be taken into both fixed and
SYNERGISTIC
b 20 $ E 1. L,, 10 0
. * IO’ lee
ConcWmtion
. Id
. Id
FACTOR OF P.
4 Id
of SF (rig/ml)
5. Effect of pretreatment of cells with various SF concentrations on virus infection. Cells were pretreated with various concentrations of SF at 27°C for 1 hr and followed by AcNPV inoculation. Each point represents the mean + SE from six determinations. FIG.
unfixed cells (Figs. 8, 9), but the internal staining was more intense in fixed than in unfixed cells. The SF treated with phospholipase C was taken into only fixed cells, and not into unfixed cells. No staining was observed in both of the control groups. DISCUSSION In the replication of AcNPV in S. fiugiperda cells, the virus enters the cells by 1 hr p.i. and uncoats; the virus particles appear in the culture medium after 7 hr and reach a maximum between 24 and 36 hr p.i. (Carstens et al., 1979). The addition of the SF to AcNPV rapidly increases the number of infected cells during the first 8 hr of incubation, suggesting a marked enhancement of virus replication and infection. The synergistic interaction of the SF and baculoviruses results from the binding of
FIG. 7. Effect of SF on AcNPV propagated at various periods of cell culture. AcNPV, propagated for 6 (A), 10 (B), or 20 hr (C) in cell culture, was applied to cells together with 100 &nl SF (a) or without SF (0). Small bar on each column represents the standard error from six determinations. *P < 0.05; **P < 0.1; ***p < 0.01.
the SF to the surfaces of the cell plasma membrane and of the viral envelope. The occurrence of specific binding sites on the plasma membrane is indicated by the SF agglutination of cells susceptible to the virus (Ohba and Tanada, 1984b), by the interference of the SF in NPV infection in Trichoplusia ni cell line (Nakagaki et al., 1987), by the detection of the SF on binding sites with immunofluorescence microscopy (Hukuhara and Zhu, 1988), and with radioreceptor assay using radioactive iodine (Uchima et al., 1988). With the immunoperoxidase staining technique, we have found that the SF enhancement is dependent on the duration of AcNPV exposure to the SF and to the concentration of the SF. The endpoint dilution technique used in an ear40. =u :
30.
J ,” %
20.
$ E
IO.
1 0
6
I2
I8
24
Hours
between
SF ond virus
treatment
6. Effect of various intervals following SF pretreatment on virus infection. Cells were pretreated with 100 t&ml SF (0) or without SF (0). AcNPV was inoculated after various intervals following SF pretreatment. Each point represents the mean f SE from six determinations.
oJIL
nl, A
0
8. Effect of the SF treated with phospholipase C on virus infection. AcNPV was applied to cells together with 100 pg/ml SF (m), with 100 ).&ml SF treated with phospholipase C (D), or virus alone (0). Two different concentrations of AcNPV (A, % x 10m4 of original; B, % x 1O-4 of original) were examined. Small bar on each column represents the standard error from six determinations. FIG.
FIG.
161
unipuncta
162
UCHIMA,
EGERTER,
AND
TANADA
FIG. 9. Cell uptake of SF treated with phospholipase C. Cells were incubated with 100 kg/ml SF (B) or 100 kg/ml SF treated with phospholipase C (C). Previously fixed cells in cold ethanol were also incubated with 100 p&ml SF (E) or 100 &ml SF treated with phospholipase C (F). Control groups for fixed cells (D) and unfixed cells (A) were incubated with neither the SF nor the SF treated with the enzyme. Magnification: bar = 25 pm.
lier study (Ohba and Tanada, 1984a) detected the SF enhancement after 24 hr exposure to the virus, but with immunoperoxidase staining, the activity was detected much earlier, within 4 hr exposure, indicating a rapid virus binding and subsequent enhancement of infection. The enhancement does not require the
coinoculation of SF and AcNPV since it occurs when the SF is applied initially as a pretreatment to the cell culture for up to 24 hr prior to the application of AcNPV. The explanation for the prolonged effect of SF has not been clarified but may be caused by the presence of SF-attached cells, the intracellular effect of the SF (see below), or the
SYNERGISTIC
FACTOR
presence of the SF remaining in the medium after rinsing. The pretreatment requires a higher concentration of the SF as compared to the combined virus-SF treatment. The enhancement during the first 4hr interval increases gradually, followed by a decrease after 16 hr. These results suggest that (1) the bound SF remains on the surface of the cell plasma membrane, (2) the rinse washing does not remove all of the SF from the medium, and (3) the endocytosed SF causes structural changes on the cell plasma membrane to enhance virus binding (see below). We favor the first premise since the agglutination of cells by the SF occurs rapidly and the radioreceptor assay indicates specific binding sites for the SF on the plasma membrane. If the SF is endocytosed by the cells, this may explain the decrease in activity after 16 hr, since such SF may be metabolized within the cells. The enhancing action of the SF occurs rapidly since a maximal level of enhancement occurs within 5 min after the cells have been pretreated with SF prior to the inoculation of the virus. Thus, the SF appears to bind quickly to the cell plasma membrane. This is supported by the observation of Ohba and Tanada (1984b) that the SF agglutinates cells within 10-15 min. The duration of AcNPV propagation in the cell culture system affects the activity of the SF. During this period, various types of uncloned extracellular vu-ions may occur but the activity of the SF appears to result from the presence of two types: (1) virions surrounded by envelopes with peplomers derived from the cell plasma membrane and (2) virions surrounded by envelopes produced in the nucleoplasm. The enveloped virions with peplomers infect cells by endocytosis (see Tanada and Hess, 1984) and those enveloped in the nucleus invade by binding and fusion. Invasion by endocytosis may result in the dissolution of the virions by lysozyme in the endocytic vacuole (Tanada and Hess, 1984) and such dissolution is unlikely to occur in the infection by
OF
P. unipuncta
163
binding and fusion. Hukuhara and Zhu (1988) reported the binding of the SF to the membranes of both types of virions. Thus, the SF may enhance the binding and mode of infection of peplomer-enveloped virions, i.e., infection by fusion as well as by endocytosis. The treatment of the SF with phospholipase C results in the inactivation of the SF in vivo (Yamamoto and Tanada, 1978a), and we have confirmed this inactivation in vitro, indicating that the phospholipid fraction of the SF is essential for enhancement by the SF. According to Tuan and Hou (1988), the phospholipid lecithin (phosphatidyl choline) enhances the NPV infection in the corn earworm, Heliothis armigera. Moreover, the removal of the phospholipid results in a slightly lower level of infection suggesting that such enzymetreated SF blocks the virus receptors on the cell plasma membrane. Although the SF appeared to be endocytosed based on immunoperoxidase staining, there is a possibility that the staining was on the cell surface since the unfixed cells stained much more lightly than fixed cells whose membrane had been altered through fixation. The internalization of the SF offers the possibility that the SF may enhance baculovirus infection through internal activity, e.g., by altering the cell membrane for virus invasion. This premise, however, is unlikely since the level of infection begins to decrease at 8- to 16-hr intervals after SF pretreatment, whereas the coinoculation of SF and virus results in increasing levels of infection even after 8 hr. Moreover, the SF interference of T. ni NPV in the cell line of T. ni but not in that of S. fiugiperdu also suggests an external membrane surface phenomenon (Nakagaki et al., 1987). This aspect of SF internalization and activity needs further study. We conclude that the SF is a binding molecule that enhances baculovirus infections by interacting rapidly with the cell plasma membrane and viral envelope. At least two
164
UCHIMA,
EGERTER,
different binding sites occur on the SF, one for the cell plasma membrane and the other for the virus envelope. ACKNOWLEDGMENTS We thank Ms. Esther M. Omi Olsen and Mr. Shizuo G. Kamita for technical assistance and Mr. Andrew B. Keddie for his assistance in microphotography. This study was partially supported by the National Science Foundation under Grant DCB-8517580.
REFERENCES CARSTENS, E. B., TJIA, S. E., AND DOERFLER, W. 1979. Infection of Spodoptera fiugiperda cells with Autographa cahfornica nuclear polyhedrosis virus. Virology,
99, 386-398.
GARDINER, G. R., AND STOCKDALE, H. 1975. Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J. Znvertebr. Pathol.,
25, 363-370.
HINK, W. F., AND VAIL, P. V. 1973. A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line. .Z. Znvertebr. Pathol., 22, 168-174. HOTCHKIN, P. G. 1981. Comparison of virion proteins and granulin from a granulosis virus produced in two host species. J. Znvertebr. Pathol., 38, 303-304. HUKUHARA, T., AND ZHU, Y. 1988. The effect of the synergistic factor on the in vitro infectivity of nuclear polyhedrosis viruses. In “Invertebrate and Fish Tissue Culture” (Y. Kuroda, E. Kurstak, and K. Maramorosch, Eds.), pp. 159-162. Japan Scientific Societies Press, Tokyo/Springer-Verlag/Berlin. NAKAGAKI, M., OHBA, M., AND TANADA, Y. 1987. Specificity of receptor sites on insect cells for the synergistic factor of an insect baculovitus. J. Znvertebr. Pathol., 50, 169-175. OHBA, M., AND TANADA, Y. 1983. A synergistic factor enhances the in vitro infection of an insect baculovirus. Natutwissenschaften, 70, 613-614. OHBA, M., AND TANADA, Y. 1984a. In vitro enhancement of nuclear polyhedrosis virus infection by the synergistic factor of a granulosis virus of the armyworm, Pseudaletia idae). Ann. Viol.
unipuncta (Lepidoptera: (Inst. Pasteur), 135E,
Noctu-
167-176. OHBA, M., AND TANADA, Y. 1984b. A synergistic factor of an insect granulosis virus agglutinates insect cells. Experientia, 40, 742-744. TANADA, Y., AND HESS, R. T. 1984. The cytopathology of baculovitus infections in insects. In “Insect Ultrastructure” (R. C. King, and H. Akai, Eds.), Vol. 2, pp. 517-556. Plenum, New York. TANADA, Y., HESS, R. T., AND OMI, E. M. 1975. Invasion of a nuclear polyhedrosis virus in midgut of
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TANADA
the armyworm, Pseudaletia unipuncta, and the enhancement of a synergistic enzyme. J. Znvertebr. Pathol., 26, 99-104. TANADA, Y., HESS, R., AND OMI, E. M. 1980a. Localization of esterase activity in the larval midgut of the am~ywora~ (Pseudaletia unipuncta). Insect Eiothem.,
10, 125-128.
TANADA, Y., HESS, R. T., AND OMI, E. M. 1984. The movement and invasion of an insect baculovirus in tracheal cells of the armyworm, Pseudaletia unipuncta. J. Znvertebr. Pathol., 44, 198-208. TANADA, Y., HESS, R. T., OMI, E. M., AND YAMAMOTO, T. 1983. Localization of a Synergistic faCtor of a granulosis virus by its esterase activity in the larval midgut of the armyworm, Pseudaletia unipuncta.
Microbios,
37, 87-93.
TANADA, Y., AND HUKUHARA, T. 1971. Enhanced infection of a nuclear-polyhedrosis virus in larvae of the armyworm, Pseudaletia unipuncta, by a factor in the capsule of a granulosis virus. J. Znvertebr. Pathol.,
17, 116-126.
TANADA, Y., INOUE, H., HESS, R. T., AND OMI, E. M. 1980b. Site of action of a synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta.
J. Znvertebr.
Pathol.,
34, 249-255.
TUAN, S.-J., AND Hou, R. F. 1988. Enhancement of nuclear polyhedrosis virus infection by lecithin in the corn eanvorm, Heliothis armigera. J. Znvertebr. Pathol., 52, 180-182. UCHIMA, K., HARVEY, J. P., OMI, E. M., AND TANADA, Y. 1988. Binding sites on the midgut cell membrane for the synergistic factor of G granulosis virus of the armyworm (Pseudaletia unipuncta). Znsect Biochem.,
645-650.
VOLKMAN, L. E., AND GOLDSMITH, P. A. 1982. Generalized immunoassay for Autographa californica nuclear polyhedrosis virus infectivity in vitro. Appl. Environ.
Microbial.,
44, 227-233.
VOLKMAN, L. E., SUMMERS, M. D., AND HSIEH, C.-H. 1976. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: Comparative neutralization, comparative infectivity, and in vitro growth studies. J. Viral., 19, 820-832. YAMAMOTO, T., AND TANADA, Y. 1978a. Phospholipid, an enhancing component in the synergistic factor of a granulosis virus of the armyworm, Pseudaletia
unipuncta.
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31, 48-56.
YAMAMOTO, T., AND TANADA, Y. 1978b. Protein components of two strains of granulosis virus of the armyworm, Pseudaletia unipuncta (Lepidoptera, Noctuidae). J. Znvertebr. Pathoi., 32, 158-170. YAMAMOTO, T., AND TANADA, Y. 1980. Physicochemical properties and location of capsule components, in particular the synergistic factor, in the occlusion body of a granulosis virus of the &myworm, Pseudaletia
unipuncta.
Virology,
107, 4344lO.
OF INVERTEBRATE
Synergistic
Pseudalefia KAORU
54, 15&16’t (1989)
PATHOLOGY
Factor of a Granulosis Virus of the Armyworm, its Uptake and Enhancement of Virus Infection in Vitro
unipuncta: UCHIMA,
DAVID
E. EGERTER,
AND YOSHINORI
TANADA
Department of Entomological Sciences, University ofCalifornia, Berkeley, CalSfornia 94720 Received September 7, 1988; accepted November 18, 1988 A synergistic factor (SF) enhanced the infection of Autograph californica nuclear polyhedrosis virus (AcNPV) in vitro. When the AcNPV was inoculated together with the SF to a Spodopteru frugiperda cell line, the enhancement of AcNPV increased dose-responsively to the SF. Enhancement, however, did not require the coinoculation of AcNPV and SF since it occurred in the pretreatment of the cells with the SF alone and even after a 24-hr interval of SF pretreatment. There was a difference in the activity of the SF on the AcNPV propagated in a cell culture system during various periods of virus propagation. When the SF was treated with phospholipase C, the enhancement of AcNPV infection did not occur in vitro and confirmed our previous study in vivo. The cells appeared to phagocytize the SF and the uptake occurred rapidly (within 1 mitt), but there was no uptake when the SF was treated with phospholipase C. These results indicate that the SF is a binding molecule in which the phospholipid component is essential for its phagocytosis and enhancement of baculovirus infection. The SF appears to have two different binding sites, one for the cell plasma membrane and the other for the enveloped virion. B 1989 Academic press, hc. KEY WORDS: nuclear polyhedrosis virus of Autograph californica; cell line of Spodoptera frueiperda; synergistic factor, uptake; baculovirus infection, enhancement; immunoperoxidase staining.
INTRODUCTION
A synergistic factor (SF) found in the occlusion body (capsule) matrix of the Hawaiian strain of a granulosis virus enhances the infection of a nuclear polyhedrosis virus (NPV) in armyworm ,larvae (Pseudaletia unipuncta) (Tanada and Hukuhara, 1971). The SF is a lipoprotein, MW 93,000 (Hotchkin, 1981) or 126,008 (Yamamoto and Tanada, 1978a), and the phospholipid component is essential for the enhancing activity in vivo (Yamamoto and Tanada, 1978a). The site of action of the SF is the plasma membrane of the microvillus of larval midgut epithelium (Tanada et al., 1975, 1980a, b, 1983; Hukuhara and Zhu, 1988). The SF also enhances in vitro the infection of several NPVs (Ohba and Tanada, 1983,1984a), and its effect is on the cell plasma membrane (Nakagaki et al., 1987; Hukuhara and Zhu, 1988). In our present study, the immunoperoxidase staining for the enveloped virion of NPV was used to study the effect of the SF on virus infection in vitro. The
apparent uptake of the SF into cells was also observed with immunoperoxidase staining. MATERIALS Cells
A cell line of Spodoptera frugiperda (IPLB-SF-21), which was kindly provided by Dr. M. A. Whit1 (University of California, Davis), was maintained in the medium MBL-TC/lO (Gardiner and Stockdale, 1975) containing 10% fetal bovine serum (FBS, Cat. No. 29-101, Flow Laboratories, McLean, Virginia), 100 unit/ml penicillin, 100 p,g/ml streptomycin, and 0.25 l&ml Fungizone (Gibco Lab., Grand Island, New York). The cells were cultured at 27°C and transferred every 48-72 hr. Virus Inoculum Autographa californica nuclear polyhedrosis virus (AcNPV), kindly provided by Dr. M. A. Whitt (University of California, 1.56
0022-201 l/89 $1.50 Copyright All rights
0 1989 by Academic Press, Lnc. of reproduction in any form reserved.
AND METHODS
SYNERGISTIC
FACTOR
Davis), was propagated in IPLB-SF-21. The infected cell culture was centrifuged at 1OOOg and the supematant was used as virus inoculum for the preparation of the different virus inocula. The S. frugiperda cells cultured in 25ml flasks (Falcon 3013, Becton Dikinson and Co., Oxnard, California) were inoculated with AcNPV, incubated for 1 hr, then rinsed twice with medium (4 ml) to remove the excess AcNPV in the cell culture, and replenished with 4 ml of medium. After 48 hr incubation, the medium was collected and centrifuged at lOOOg, and the supematant (virus inoculum) containing mainly enveloped virions was stored at 4°C. The supernatant, when diluted 104-lo5 times, gave 5-30 stained foci/well. The diluted virus inoculum was prepared just prior to its use in the tests, SF and Antibodies The SF was prepared as described by Uchima et al. (1988) and the anti-SF rabbit serum as reported by Yamamoto and Tanada (1978b). The anti-AcNPV-envelope rabbit serum, which was kindly provided by Dr. L. E. Volkman (University of California, Berkeley), had been prepared as described by Volkman et al. (1976). Assay of AcNPV
Infectivity
The assay of AcNPV infectivity was performed with a modification of the method of Volkman and Goldsmith (1982). Microslides containing 12 wells (5 mm diameter, Carlson Scientific Inc., Peotone, Illinois) were first soaked in 1% HCl and 70% ethanol, then rinsed with double-distilled water, and dried and sterilized in an oven. Twenty microliters of cell suspension (lo6 cells/ml) was seeded in each well and cultured overnight at 27°C in a humid chamber. Then the medium was carefully removed from the wells and replaced with 20 ~1 of freshly diluted AcNPV inoculum. After certain periods of AcNPV exposure, the inoculum was removed and replaced with medium containing 0.6% methylcellulose
OF
157
P. unipuncta
(Hink and Vail, 1973), and the cells were incubated for 48 hr at 27°C in a humid chamber. The fixation and immunoperoxidase staining were performed according to Volkman and Goldsmith (1982). The anti-rabbit goat immunoglobulin (Product No. 5001) was purchased from Sigma Chemical Co. (St. Louis, Missouri) and peroxidaseantiperoxidase complex (rabbit serum, Cat. No. 8430-01) from Litton Bionetics (Charleston, South Carolina). Assay of SF Uptake by Cells Cells were seeded in each well of the microslides and cultured overnight, and then the medium in each well was removed and replaced with 20 ~1 of fresh medium containing the SF (100 pg/ml). After the designated period of incubation at 27°C in a humid chamber, the medium was removed from the wells and the attached cells were fixed by dipping the microslides into cold ethanol for 2 min. The fixed cells were rinsed with cold buffered saline (0.15 M NaCl, 50 mM Tris-HCl, pH 7.6) and treated successively with anti-SF rabbit serum, anti-rabbit goat immunoglobulin, and peroxidaseantiperoxidase complex according to the method of microslide preparation described under Assay of AcNPV Infection. Treatment
of SF With Phospholipase
C
One milligram of the SF that had been stored at -20°C in 1.5 ml of 100 mM NaCl and 10 mM sodium phosphate buffer, pH 8.0, were dialyzed in 5 mM CaCl, and 100 mM Tris-HCl, pH 8.0. One milliliter of the SF solution was collected after dialyzation. Phospholipase C (Cat. No. 691950, Boehringer-Mannheim Biochemicals, Indianapolis, Indiana) at 40 units/l0 l~l was added to 1 ml of SF solution and incubated for 2 hr with gentle shaking. The enzyme, undigested SF, and derivatives were removed with a gel filtration column (1.0 x 35 cm) using Fractogel TSK HW-55 (F) (Cat. No. 14981-4, EM Science, Gibbstown, New Jer-
158
UCHIMA,
EGERTER,
sey) as a gel filtration medium and 10 mM Tris-HCI, pH 8.0, as elution buffer. Four hundred micrograms of the SF treated with phospholipase C was recovered and analyzed by immunoprecipitation with the antiSF rabbit serum in a double diffusion test. Statistical Student’s analysis.
Analysis t test was used for statistical
RESULTS EfSect of Length of Exposure to AcNPV, With and Without SF, on AcNPV Infection
AND TANADA
Effect of SF Concentration AcNPV Infection
on
The AcNPV was applied to cultured cells together with various concentrations of the SF (O-100 t&ml) and the cells were incubated for 20 hr. The AcNPV infection was enhanced at the SF concentrations higher than 100 &ml and the enhancement increased rapidly, attaining a maximum level at 10 pg/rnl (Fig. 2). The number of stained foci of cells inoculated with the AcNPV together with 10 pg/ml of the SF was about twice as high as that without the SF. Effect of Pretreatment Intervals on AcNPV Infection
Cultured cells were inoculated with AcNPV, with and without the SF (100 ).&ml). Exposure of cells to AcNPV was performed at various intervals (O-24 hr). The number of cells infected in the absence of SF gradually increased during the 24 hr exposure to AcNPV, whereas the number of infected cells in the presence of SF increased rapidly during the first 8 hr of incubation and then increased gradually (Fig. 1). The number of stained foci of cells exposed to AcNPV in the presence of the SF for g-24 hr was more than two times higher than that treated without the SF. The enhancing effect of the SF on AcNPV infection occurred predominantly during the first 8 hr and was near maximum at 24 hr exposure.
With SF
Cells were incubated first with the SF (100 t&ml) for various intervals, then the SF was removed by rinsing with medium, and the cells were exposed to AcNPV for 20 hr. The AcNPV infection increased rapidly and reached a maximal level after 5 min of the SF pretreatment of the cells (Fig. 3). The AcNPV infection was maintained at the maximal level for at least 4 hr SF pretreatment. The number of stained foci of cells pretreated with the SF for more than 5 min was about twice as high as that of cells receiving no SF pretreatment. Uptake of SF by Cells The binding and uptake of the SF by cells 160 r
01 0-‘I Dumtion
of AcNPV
exposure
(hr)
1. Effect of length of exposure to AcNPV, and without the SF, on virus infection. AcNPV exposed to the cells for various periods together 100 pg/ml SF (0) or without SF (0). Each point resents the mean 2 SE from six determinations. FIG.
Concentmtion
with was with rep-
IO’
Id
Id
Id
Id
of SF (rig/ml)
FIG. 2. Effect of SF concentrations on virus infection. AcNPV was inoculated into cells with various concentrations of SF. Cells were incubated in the inoculum at 27°C for 20 hr. Each point represents the mean f SE from six determinations.
SYNERGISTIC
0
Duration
I
2
of
pretreotrnent with
3
4 SF (hr)
FIG. 3. Effect of pretreatment of cells with the SF on virus infection. Cells were pretreated with 100 p.gknl SF for various periods, followed by inoculation of AcNPV. Each point represents the mean 2 SE from six determinations.
were determined by immunoperoxidase staining. Cells were incubated with the SF (100 pg/ml) for various intervals (O-l hr), fixed, and stained as described above. The SF bound rapidly on the cell membrane and appeared to be taken into the cells within I min (Fig. 4). The apparent uptake of the SF by the cells reached a maximal state after 15 min incubation. Effect of Pretreatment of Cells With Various SF Concentrations on AcNPV Infection Cells were incubated with various concentrations of the SF for 1 hr, then rinsed with medium to remove the SF, and incubated with AcNPV for 20 hr. One microgram per milliliter of SF was required for the enhancement of the AcNPV infection (Fig. 5). Virus infection progressively increased with the pretreatment of the cells at higher concentrations of the SF. The number of stained foci of cells pretreated with 100 p&ml SF was 1.7 times higher than that without the SF. Effect of Various Intervals following Pretreatment on AcNPV Infection
159
FACTOR OF P. unipuncra
SF
Cells were treated with the SF for 1 hr, followed by the removal of the SF by rinsing with medium, the incubation for various periods (CL24 hr), and then the exposure to AcNPV for 20 hr. The control group was treated without the SF for 1 hr, followed by
the rinsing procedure and various periods of incubation prior to AcNPV inoculation. The enhancing effect of the SF on AcNPV infection increased gradually with the SF-pretreatment interval of 4 hr and attained a maximal level at about 8 hr (Fig. 6). After 16 hr, the enhancing effect decreased but was still apparent at 24 hr. The number of stained foci in the cells at maximal level of enhancement (8 hr) was about twice that of the control group. Effect of SF on AcNPV Propagated Various Periods of Cell Culture
at
AcNPV was inoculated into cells cultured in flasks (25 ml Falcon) and after 2 hr, the inoculum was removed from each flask by rinsing the cells with three changes of culture medium. Two milliliters of fresh medium was added to each flask and the cells were incubated for 4,8, or 18 hr. After incubation, the medium (supernatant) was collected by centrifugation at 1OOg for 5 min. The supematant was kept at 4°C and used as the inoculum with or without the SF (100 pg/ml). The inoculated cells were incubated for 20 hr. The SF only slightly enhanced the infection of AcNPV propagated for 6 and 10 hr (P < 0.05 and P < 0.1, respectively), and greatly enhanced virus infection in virions produced in 20 hr (P < 0.01) (Fig. 7). Effect of the Treatment Phospholipase C
of SF With
(a) Effect on the infection of AcNPV. Cells were exposed to AcNPV for 20 hr together with the SF or with the SF treated with phospholipase C (100 kg/ml). The control had neither SF nor SF treated with phospholipase C. Two different concentrations of AcNPV were used. The treatment of the SF with phospholipase C resulted in no enhancement of AcNPV infection (Fig. 8), and may have interfered with virus infection since the number of stained foci of cells inoculated with AcNPV and SF treated with phospho-
160
UCHIMA,
EGERTER,
AND
TANADA
FIG. 4. Uptake of SF by cells. Cells were incubs tted with the SF for 0 min (A), 1 min (B), 5 min (C), 15 min (D, E), or 1 hr (F), and stained as described in the text. Photographs of stained cells were taken with bright field microscope (A-D, F) or Nomarsl ti microscope (E). Magnification: bar = 25 pm.
lipase C was statistically the control.
less than that of
(6) Uptake by the cells. The cells incubated with the SF or the SF treated with phospholipase C (100 &ml) for 1 hr were fixed and stained immunologically as described under Materials and Methods. Other cells were fixed in cold ethanol and formalin, rinsed twice with buffered saline
pH 7.6), (0.15 M NaCl, 50 mM Tris-HCl, incubated with the SF or the SF treated with the enzyme, and after 1 hr incubation, they were stained with immunoperoxidase. Controls for both fixed and unfixed cells were incubated for 1 hr with neither the SF nor the SF treated with phospholipase C. The SF, not treated with the enzyme, appeared to be taken into both fixed and
SYNERGISTIC
b 20 $ E 1. L,, 10 0
. * IO’ lee
ConcWmtion
. Id
. Id
FACTOR OF P.
4 Id
of SF (rig/ml)
5. Effect of pretreatment of cells with various SF concentrations on virus infection. Cells were pretreated with various concentrations of SF at 27°C for 1 hr and followed by AcNPV inoculation. Each point represents the mean + SE from six determinations. FIG.
unfixed cells (Figs. 8, 9), but the internal staining was more intense in fixed than in unfixed cells. The SF treated with phospholipase C was taken into only fixed cells, and not into unfixed cells. No staining was observed in both of the control groups. DISCUSSION In the replication of AcNPV in S. fiugiperda cells, the virus enters the cells by 1 hr p.i. and uncoats; the virus particles appear in the culture medium after 7 hr and reach a maximum between 24 and 36 hr p.i. (Carstens et al., 1979). The addition of the SF to AcNPV rapidly increases the number of infected cells during the first 8 hr of incubation, suggesting a marked enhancement of virus replication and infection. The synergistic interaction of the SF and baculoviruses results from the binding of
FIG. 7. Effect of SF on AcNPV propagated at various periods of cell culture. AcNPV, propagated for 6 (A), 10 (B), or 20 hr (C) in cell culture, was applied to cells together with 100 &nl SF (a) or without SF (0). Small bar on each column represents the standard error from six determinations. *P < 0.05; **P < 0.1; ***p < 0.01.
the SF to the surfaces of the cell plasma membrane and of the viral envelope. The occurrence of specific binding sites on the plasma membrane is indicated by the SF agglutination of cells susceptible to the virus (Ohba and Tanada, 1984b), by the interference of the SF in NPV infection in Trichoplusia ni cell line (Nakagaki et al., 1987), by the detection of the SF on binding sites with immunofluorescence microscopy (Hukuhara and Zhu, 1988), and with radioreceptor assay using radioactive iodine (Uchima et al., 1988). With the immunoperoxidase staining technique, we have found that the SF enhancement is dependent on the duration of AcNPV exposure to the SF and to the concentration of the SF. The endpoint dilution technique used in an ear40. =u :
30.
J ,” %
20.
$ E
IO.
1 0
6
I2
I8
24
Hours
between
SF ond virus
treatment
6. Effect of various intervals following SF pretreatment on virus infection. Cells were pretreated with 100 t&ml SF (0) or without SF (0). AcNPV was inoculated after various intervals following SF pretreatment. Each point represents the mean f SE from six determinations.
oJIL
nl, A
0
8. Effect of the SF treated with phospholipase C on virus infection. AcNPV was applied to cells together with 100 pg/ml SF (m), with 100 ).&ml SF treated with phospholipase C (D), or virus alone (0). Two different concentrations of AcNPV (A, % x 10m4 of original; B, % x 1O-4 of original) were examined. Small bar on each column represents the standard error from six determinations. FIG.
FIG.
161
unipuncta
162
UCHIMA,
EGERTER,
AND
TANADA
FIG. 9. Cell uptake of SF treated with phospholipase C. Cells were incubated with 100 kg/ml SF (B) or 100 kg/ml SF treated with phospholipase C (C). Previously fixed cells in cold ethanol were also incubated with 100 p&ml SF (E) or 100 &ml SF treated with phospholipase C (F). Control groups for fixed cells (D) and unfixed cells (A) were incubated with neither the SF nor the SF treated with the enzyme. Magnification: bar = 25 pm.
lier study (Ohba and Tanada, 1984a) detected the SF enhancement after 24 hr exposure to the virus, but with immunoperoxidase staining, the activity was detected much earlier, within 4 hr exposure, indicating a rapid virus binding and subsequent enhancement of infection. The enhancement does not require the
coinoculation of SF and AcNPV since it occurs when the SF is applied initially as a pretreatment to the cell culture for up to 24 hr prior to the application of AcNPV. The explanation for the prolonged effect of SF has not been clarified but may be caused by the presence of SF-attached cells, the intracellular effect of the SF (see below), or the
SYNERGISTIC
FACTOR
presence of the SF remaining in the medium after rinsing. The pretreatment requires a higher concentration of the SF as compared to the combined virus-SF treatment. The enhancement during the first 4hr interval increases gradually, followed by a decrease after 16 hr. These results suggest that (1) the bound SF remains on the surface of the cell plasma membrane, (2) the rinse washing does not remove all of the SF from the medium, and (3) the endocytosed SF causes structural changes on the cell plasma membrane to enhance virus binding (see below). We favor the first premise since the agglutination of cells by the SF occurs rapidly and the radioreceptor assay indicates specific binding sites for the SF on the plasma membrane. If the SF is endocytosed by the cells, this may explain the decrease in activity after 16 hr, since such SF may be metabolized within the cells. The enhancing action of the SF occurs rapidly since a maximal level of enhancement occurs within 5 min after the cells have been pretreated with SF prior to the inoculation of the virus. Thus, the SF appears to bind quickly to the cell plasma membrane. This is supported by the observation of Ohba and Tanada (1984b) that the SF agglutinates cells within 10-15 min. The duration of AcNPV propagation in the cell culture system affects the activity of the SF. During this period, various types of uncloned extracellular vu-ions may occur but the activity of the SF appears to result from the presence of two types: (1) virions surrounded by envelopes with peplomers derived from the cell plasma membrane and (2) virions surrounded by envelopes produced in the nucleoplasm. The enveloped virions with peplomers infect cells by endocytosis (see Tanada and Hess, 1984) and those enveloped in the nucleus invade by binding and fusion. Invasion by endocytosis may result in the dissolution of the virions by lysozyme in the endocytic vacuole (Tanada and Hess, 1984) and such dissolution is unlikely to occur in the infection by
OF
P. unipuncta
163
binding and fusion. Hukuhara and Zhu (1988) reported the binding of the SF to the membranes of both types of virions. Thus, the SF may enhance the binding and mode of infection of peplomer-enveloped virions, i.e., infection by fusion as well as by endocytosis. The treatment of the SF with phospholipase C results in the inactivation of the SF in vivo (Yamamoto and Tanada, 1978a), and we have confirmed this inactivation in vitro, indicating that the phospholipid fraction of the SF is essential for enhancement by the SF. According to Tuan and Hou (1988), the phospholipid lecithin (phosphatidyl choline) enhances the NPV infection in the corn earworm, Heliothis armigera. Moreover, the removal of the phospholipid results in a slightly lower level of infection suggesting that such enzymetreated SF blocks the virus receptors on the cell plasma membrane. Although the SF appeared to be endocytosed based on immunoperoxidase staining, there is a possibility that the staining was on the cell surface since the unfixed cells stained much more lightly than fixed cells whose membrane had been altered through fixation. The internalization of the SF offers the possibility that the SF may enhance baculovirus infection through internal activity, e.g., by altering the cell membrane for virus invasion. This premise, however, is unlikely since the level of infection begins to decrease at 8- to 16-hr intervals after SF pretreatment, whereas the coinoculation of SF and virus results in increasing levels of infection even after 8 hr. Moreover, the SF interference of T. ni NPV in the cell line of T. ni but not in that of S. fiugiperdu also suggests an external membrane surface phenomenon (Nakagaki et al., 1987). This aspect of SF internalization and activity needs further study. We conclude that the SF is a binding molecule that enhances baculovirus infections by interacting rapidly with the cell plasma membrane and viral envelope. At least two
164
UCHIMA,
EGERTER,
different binding sites occur on the SF, one for the cell plasma membrane and the other for the virus envelope. ACKNOWLEDGMENTS We thank Ms. Esther M. Omi Olsen and Mr. Shizuo G. Kamita for technical assistance and Mr. Andrew B. Keddie for his assistance in microphotography. This study was partially supported by the National Science Foundation under Grant DCB-8517580.
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GARDINER, G. R., AND STOCKDALE, H. 1975. Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J. Znvertebr. Pathol.,
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HINK, W. F., AND VAIL, P. V. 1973. A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line. .Z. Znvertebr. Pathol., 22, 168-174. HOTCHKIN, P. G. 1981. Comparison of virion proteins and granulin from a granulosis virus produced in two host species. J. Znvertebr. Pathol., 38, 303-304. HUKUHARA, T., AND ZHU, Y. 1988. The effect of the synergistic factor on the in vitro infectivity of nuclear polyhedrosis viruses. In “Invertebrate and Fish Tissue Culture” (Y. Kuroda, E. Kurstak, and K. Maramorosch, Eds.), pp. 159-162. Japan Scientific Societies Press, Tokyo/Springer-Verlag/Berlin. NAKAGAKI, M., OHBA, M., AND TANADA, Y. 1987. Specificity of receptor sites on insect cells for the synergistic factor of an insect baculovitus. J. Znvertebr. Pathol., 50, 169-175. OHBA, M., AND TANADA, Y. 1983. A synergistic factor enhances the in vitro infection of an insect baculovirus. Natutwissenschaften, 70, 613-614. OHBA, M., AND TANADA, Y. 1984a. In vitro enhancement of nuclear polyhedrosis virus infection by the synergistic factor of a granulosis virus of the armyworm, Pseudaletia idae). Ann. Viol.
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TUAN, S.-J., AND Hou, R. F. 1988. Enhancement of nuclear polyhedrosis virus infection by lecithin in the corn eanvorm, Heliothis armigera. J. Znvertebr. Pathol., 52, 180-182. UCHIMA, K., HARVEY, J. P., OMI, E. M., AND TANADA, Y. 1988. Binding sites on the midgut cell membrane for the synergistic factor of G granulosis virus of the armyworm (Pseudaletia unipuncta). Znsect Biochem.,
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VOLKMAN, L. E., SUMMERS, M. D., AND HSIEH, C.-H. 1976. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: Comparative neutralization, comparative infectivity, and in vitro growth studies. J. Viral., 19, 820-832. YAMAMOTO, T., AND TANADA, Y. 1978a. Phospholipid, an enhancing component in the synergistic factor of a granulosis virus of the armyworm, Pseudaletia
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