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Virus DNA replication and protein synthesis in Amsacta moorei entomopoxvirus-infected Estigmene acrea cells
JOURNAL
OF INVERTEBRATE
Virus
PATHOLOGY
41, 341-349 (1983)
DNA Replication and Protein Synthesis in Amsacta Entomopoxvirus-Infected Estigmene acrea Cells
moorei
W. H. R. LANGRIDGE Boyce
Thompson
Institute,
Cornell
University,
Tower
Road,
Ithaca,
New
York
14853
Received April 19, 1982; accepted August 23, 1982 Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [3H]thymidine incorporation 12 hr after virus inoculation. Hybridization of 32P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and viruscontaining occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation. KEY WORDS: Entomopoxvirus DNA replication: polyacrylamide gel electrophoresis: entomopoxvirus protein synthesis; DNA hybridization.
formation is available concerning the biosynthesis of entomopoxvirus proteins in Vertebrate poxviruses and entomopoxvirus-infected insect cells. viruses (EPVs) (Matthews, 1979) are simiIn this report, we have infected Estiglar in size and morphology (Granados, rnene acrea cells (BTI-EAA cells) with Am1973). The entomopoxvirus genome is a large (13 1- 200 x 106 daltons), linear, double- sacta EPV and have determined the time stranded DNA molecule equal in size to sequence of DNA replication and biosynvertebrate poxvirus DNA (Langridge and thesis of virus structural proteins and ocRoberts, 1977). The G + C content of EPV clusion body matrix protein. DNA (18-22% G + C) is almost 40% lower MATERIALS AND METHODS than that of vertebrate poxviruses (33% G + C) (Langridge et al., 1977; Langridge and Chemicals Henry, 1981). More than 40 structural pepEagle’s Basal Medium and Vitamin Sotides of an entomopoxvirus from Choris- lution were obtained from Grand Island tune~g biennis have been identified by Biological Company; L-[35S]methionine sodium dodecyl sulfate (SDS)-acrylamide (455 Ci/mM), [a-32P]TTP (20000 Ci/mM), gel electrophoresis (Bilimoria and Arif, and [methyf-3H]thymidine (73 Ci/mM) were 1980). obtained from New England Nuclear; acThe biosynthesis of viral proteins of ver- rylamide and bisacrylamide, bromophenol tebrate poxviruses has been extensively blue, Coomassie blue R-250, and Tris base studied in cell cultures (Holowczak and were obtained from Sigma Chemical ComJoklik, 1967a, b; Katz and Moss, 1969; pany; N’, N’, N’, N’-tetramethylenediamine Moss and Salzman, 1968; Pennington, 1974; (TEMED) was obtained from Bio-Rad; Salzman and Sebring, 1967; Stern and ammonium persulfate was obtained from Dales, 1976). However, at this time no in- Eastman Chemical Company; sodium doINTRODUCTION
341 0022-2011183 $1.50 Copyright AU rights
@ 1983 by Academic Press. Inc. of reproduction in any form reserved.
342
W.
H. R. LANGRIDGE
decyl sulfate (SDS) BDH reagent grade and BDH protein molecular weight markers for SDS-acrylamide gel electrophoresis were obtained from Gallard Schlesinger Chemical Company; dioxane was obtained from Baker Laboratories; 2,5-diphenyloxazole was obtained from Packard Instrument Company; and naphthalene from Matheson, Coleman and Bell.
30% sucrose in 10 mrvr Tris-HCl, pH 8.0, containing 1 mM EDTA (T-E buffer), and centrifuged at 20,000 rpm in a Beckman SW-41 rotor for 1 hr at 4°C. The virus pellet was resuspended overnight at 4°C in T-E buffer overlayered on 40-65% (w/w) sucrose gradients in T-E buffer, and centrifuged at 20,000 rpm in a Beckman SW-27 rotor for 1 hr at 4°C. The single visible virus band was removed from the tube by Cultivation of cells syringe, mixed 3:l with T-E buffer, and Continuous hemocyte cell cultures of the centrifuged at 30,000 rpm in a Beckman lepidopteran Estigmene acrea, designated Ti-50 rotor, for 1 hr at 4°C. The pellet was BTI-EAA cells, were maintained and mulresuspended in 2 ml T-E buffer and a lo-p1 tiplied at 28°C in GTC-100 medium, which aliquot was examined by electron microscopy. The virus was found to be intact and was modified from BML/TClO medium (Knudson and Buckley, 1977) by the addifree from visible contamination. tion of 10% (v/v) fetal calf serum, organic Occluded virus. Five days after virus inacids, and vitamins at those concentrations oculation (m.0.i. = lo), [35S]methioninefound in Grace’s (1962) basal medium. labeled Amsacta EPV-infected cells were lysed by addition of 25 ml of T-E buffer Virion Isolation containing 2% SDS to the cell pellet at 0°C. Nonoccluded virions. BTI-EAA cells (1 Crystalline occlusion bodies, which were x 10Yml medium) were infected by addition not visibly altered in size, shape, or bireof Amsacta EPV nonoccluded virus (NOV) fringence by SDS treatment, were freed at a multiplicity of infection of 5 plaque from cell debris by sonication of the cell forming units (PFU)/cell. After 3 days of lysate with a Lab-line Ultratip sonicator incubation at 28°C cultures were centri(Melrose Park, Ill.) at 20 W for 30 set at fuged in 12-ml conical polystyrene centri0°C. The preparation was centrifuged in a fuge tubes at 1500 rpm in a Sorvall GLC-1 Sorvall SS-34 rotor at 10,000 rpm for 10 min tabletop centrifuge for 10 min at 26°C. The at 4°C and the supernatant was removed. supernatant contained NOV at a concenThe occlusion body pellet was resuspended tration of approximately lo* 50% tissue in 10 ml T-E buffer without SDS, and soniculture infectious dose (TCID,,,) units of cation and centrifugation procedures were virus/ml medium. Radioactively labeled repeated. The occlusion bodies were NOV was produced in BTI-EAA cells by washed three times in distilled water and the same procedure except that [35S]me- checked for purity by phase-contrast mithionine (400 Cifmmol) was added to the croscopy. Virions were released from the culture flask (10 pCi/ml) 6 hr after virus occlusion bodies by incubating 2 ml (1 x 10y inoculation. Approximately 36-40 hr after occlusion bodies/ml) with an equal volume inoculation, the medium containing labeled of 1.6 M sodium carbonate containing 0.1 M NOV and some infected cells containing thioglycolate, pH 10.6, for 3 min at 2°C. virus occlusion bodies was transferred to After release of virions from the occlusion sterile conical polycarbonate centrifuge body matrix protein was complete (3-5 tubes and centrifuged at 1000 rpm for 10 min), the pH of the suspension was admin at 26°C to pellet the cells. Infected cells justed to 8 by the addition of an equal volwere stored at 4°C for extraction of labeled ume of 0.1 M Tris-HCl, pH 7.5. The virion occluded virus. The supernatant containing preparation was overlayered on 40-65% labeled NOV was overlayered on 10 ml of (w/w) sucrose gradients and centrifuged at
EPV
DNA
REPLICATION
AND
PROTEINS
20,000 rpm in a Beckman SW-27 rotor for 1 hr at 4°C. After centrifugation, the gradients were fractionated with an ISCO gradient fractionator equipped with a uv flow cell, and the virus peak was collected, mixed 3: 1 with T-E buffer, and centrifuged at 30,000 rpm for 1 hr at 4°C in a Beckman Ti-50 rotor. The virus pellet was resuspended in 2.0 ml of T-E buffer.
SYNTHESIS
IN
INSECT
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343
The pulse-labeled cells were transferred from the plate to a 12-ml conical centrifuge tube. Grace’s medium minus amino acids (2 ml) were added to the tube and the cells were pelleted at 1500 rpm for 10 min at 26°C in a Sorvall GLC centrifuge. The supernatant was removed and the cells were gently resuspended in 5 ml of Grace’s medium minus amino acids and recentrifuged. The cell pellet was resuspended in 0.1 ml T-E buffer and stored at -20°C. To detect the post-translational modification of viral proteins, Estigeme acrea cells were inoculated with Amsacta EPV (m.o.i. = IO) as described above. The medium was replaced with fresh medium minus methionine containing 25 @X/ml of [35S]methionine at 12 and 18 hr after infection. After a I-hr pulse, the radioactive medium was removed and either the cells were immediately harvested in 2 ml of Grace’s medium minus amino acids or incubated for an additional 14 hr in 0.5 ml/well of GTC-100 medium containing 10% fetal calf serum. Infected cells were harvested and stored at -20°C until they were analyzed by SDS - polyacrylamide gel electrophoresis and autoradiography.
Assay of Virus Infectivity BTI-EAA cells in logarithmic growth phase (24 hr cell-doubling time) were adjusted to a cell concentration of 4 x lo5 cells/ml with GTC- 100 culture medium. The cells were inoculated with NOV at an m.o.i. of 10 PFU/cell. Aliquots of the virusinfected cells were distributed into wells (1 ml cells/well) of a 24-well Falcon No. 3008 Multiwell tissue culture plate. The plate was sealed with plastic electrician’s tape and centrifuged at 2400 t-pm (1OOOg) for 1 hr at 26°C in a Sorvall GLC-1 tabletop centrifuge. After centrifugation, the medium was removed, 2 ml of fresh medium was added to each well, and the plate was incubated at 28°C. At various time intervals from 1 to 120 hr after virus infection, cells were removed from four wells of the mi- Electrophoretic Separation of Infected crotiter plate and assayed for the appearance Cell Proteins of nonoccluded virus in the medium by an agarose overlay plaque assay procedure as The frozen cell samples were mixed with described by Wood (1977). 50 ~1 of SDS dissociation buffer and heated in a boiling water bath for 3 min (Laemmli, Isotopic Labeling of Virus-Znfected Cells 1970). To determine the amount of [35S]meTo detect the de novo biosynthesis of thionine incorporation/sample, a LO-p1 aliviral-induced and virus structural protein, quot of each solubilized cell sample was the medium was removed from four mi- transferred to a glass-fiber filter (Whatman crotiter plate wells of infected or uninfected 2.4 cm GF/C). The filters were washed twice cells (4.0 x lo5 cells/well) at 2-hr intervals with 10 ml of 10% trichloroacetic acid (TCA), after virus infection. Grace’s medium once in acetone (Salzman and Sebring, 1967), minus amino acids but containing 10 $X/ml and counted in a Beckman LS-200B scintilla[35S]methionine was added to each well (0.5 tion counter. Aliquots containing 1 X lo5 cpm ml/well) and the cells were incubated at of incorporated y5S Jmethionine were applied 28°C for 1 hr. Following the incubation pe- per lane to an 11% polyacrylamide slab gel riod, the medium containing the labeled with a 4% stacking gel (Laemmli, 1970; methionine was removed by Pasteur Studier, 1973). Electrophoresis was perpipette and replaced with fresh medium (0.5 formed at 300 V for 4 hr at 26°C. After ml/well) containing unlabeled amino acids. electrophoresis, the gel was fixed in 10%
344
W.
H.
R.
LANGRIDGE
acetic acid:2.0% ethanol and stained for 30 min at 40°C in a solution of 0.27% Coomassie blue R-250 in methanol:acetic acid: water (5:1:5). The gel was dried on Whatman No. 1 filter paper at 60°C under vacuum and labeled proteins were detected by autoradiography on Kodak RP Royal X-Omat R X-ray film. Polypeptide molecular weights were determined in comparison with BDH protein molecular weight markers. Amsacta
EPV DNA
Synthesis
The conditions for virus infection of BTI-EAA cells were as described above with one exception: the control cells were inoculated with Amsacta EPV which had been previously inactivated with uv light by placing the virus suspension in a shallow dish 8.5 in. below a shortwave uv light source (254 nm) for 1 hr. Cultures of cells inoculated with inactivated virus were analyzed in parallel with the virus-infected cells. At selected time intervals after virus inoculation, 1 x lo6 cells were pulse labeled for 15 min in Grace’s medium containing 2 pCi/ml of [3H]thymidine. The cells were collected in 2 ml Grace’s medium minus amino acids and centrifuged for 5 min at 1000 rpm in a Sorvall GLC centrifuge. The cells were resuspended and washed two times in 5 ml of the same medium. The radioactivity incorporated into TCAprecipitable material was determined by liquid scintillation spectrophotometry in a Beckman LS-200B scintillation spectrometer as described by Esteban and Holowczak (1977). To determine when Amsacta EPV DNA replication was initiated in virus-infected cells, BTI-EAA cells were synchronously inoculated with Amsacta EPV (m.o.i. = 10) as described above. At various times after inoculation, the virus-infected cells were harvested (1 x lo6 cells/time interval) and centrifuged at 500 rpm for 10 min in a Sorvall RC-2B centrifuge. The infected cells were resuspended in 4 ml of Tris-HCl buffer, pH 8.0, containing 150 mM NaCl and 10 mM EDTA. Total cellular DNA was
prepared from the infected cells by extracting three times with an equal volume of T-E buffer saturated with freshly distilled phenol (Wigler et al., 1979). The DNA samples were dialyzed for 48 hr at 4°C against 3 X 1 liter of 10 mM Tris-HCl,pH 8.0, buffer containing 1 mM EDTA. Hybridization of 32P-labeled Amsacta EPV DNA to virusinfected cell DNA was performed according to the procedure of Kafatos et al. (1979). Uninfected and virus-infected cellular DNA was denatured in alkali and spotted on nitrocellulose filter paper prewet with 1.0 M ammonium acetate (3 pg DNA/spot) in a hybridot apparatus (Bethesda Research Laboratories, Md.). The DNA spots were washed with 1 M ammonium acetate and the filter dried in vacua at 80°C for 2 hr. The DNA spots were hybridized to Amsacta EPV DNA (Wetmur and Davidson, 1968) previously labeled by nick translation to high specific activity with [a-32P]dTTP by the nick-translation procedure of Rigby et al. (1977). Autoradiography of the nitrocellulose filter (Bonner and Laskey, 1974) permitted detection of the homologous viral DNA hybrids. RESULTS Amsacta EPV occlusion bodies were detected by phase-contrast microscopy as early as 20 hr after virus infection in cells maintained in GTC- 100 medium (Fig. 1). Virus multiplication and occlusion appear to occur almost simultaneously as the percentage of Amsacta EPV infected cells containing occlusion bodies increased at approximately the same time and rate as the increase in the titer of extracellular virus detected by plaque assay (Fig. 1). A large increase in infectious, extracellular, nonoccluded virus was detected in the medium of infected cells by plaque assay between 18 to 20 hr after virus inoculation (Fig. 1). The titer of extracellular virus increased until 72 hr after virus inoculation at which time the cells began to lyse. The percentage of virus-infected cells containing EPV occlusion bodies increased
EPV
DNA
REPLICATION
AND
PROTEINS
HOURS
AFTER
SYNTHESIS
IN
INSECT
CELLS
345
INFECTION
FIG. 1. Extracellular virus growth curve ofAmsacru moorei EPV in BTI-EAA cells. BTI-EAA cells in exponential growth were inoculated with Amsacra EPV nonoccluded virus (m.o.i. = 10). Cells and virus were centrifuged and incubated at 28°C as describedin the text. At selected time intervals, from 1 to 120 hr after inoculation, the medium from the virus-infected cells was assayed for infectious virus by an agarose overlay plaque assay (Wood, 1977). Virus plaques took approximately 5 days to develop at 28°C. Plaque-forming units from 1 to 120 hr = open circles. The percentage of cells containing occlusion bodies detected by phase-contrast light microscopy = closed circles. Two hundred cells were counted/time interval.
from 2.0% at 18 hr to 86% by 94 hr after infection (Fig. 1). A decrease in the rate of formation of occlusion bodies was detected between 30 hr and 40 hr after infection. By 19 hr after infection the incorporation of [3H]thymidine into acid-precipitable radioactivity in intact virus-infected BTI cells (Fig. 2) was approximately 20-fold greater than in cells inoculated with killed virus. The rate of [3H]thymidine incorporation into virus-infected cells increased from 12 to 19 hr after virus inoculation. A significant increase in Amsacta EPV DNA synthesis was detected by dot hybridization from 6 to 12 hr after virus inoculation (Fig. 2, insert). The amount of viral DNA detected in infected cells increased dramatically from 12 to 24 hr after virus inoculation (Fig. 2, insert). In both occluded and nonoccluded forms of Amsacta EPV, 37 virus structural proteins were detected by SDS-polyacrylamide gel electrophoresis and prolonged exposure of the autoradiogram to reveal
labeled proteins present in low concentrations (Fig. 3). The polyacrylamide gel protein patterns of both forms of the virus were identical in number of protein bands. Several proteins were synthesized in larger amounts in either the occluded or the nonoccluded virus. This protein was identical in molecular weight to Amsacta EPV occlusion body matrix protein isolated from sucrose gradient purified occlusion bodies. After the occluded virus was separated from occlusion body matrix protein by two cycles of sucrose density gradient centrifugation (McCarthy et al., 1974), a significant amount of 110,000 M. W. protein was detected after separation of the virus structural proteins by SDS - polyacrylamide gel electro phoresis (Fig. 3A). Antiserum directed against electrophoretically purified Amsacta EPV occlusion body protein precipitated the 110,000 M. W. protein of the occluded virus. Approximately 32 hr after infection, 25 proteins corresponding in molecular
346
W. H. R. LANGRIDGE
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FIG. 2. Kinetics of DNA synthesis in Amsacfa EPV-infected BTI-EAA cells. The conditions for virus infection and uv light inactivation were as described in the text. At selected time intervals after virus infection, 1 x lo6 cells were pulse labeled for 15 min with 2 &i/ml of [3H]thymidine. Cells inoculated with inactivated virus were analyzed in parallel with virus-infected cells. Solid lines are virus-infected cells; dotted lines represent uv-inactivated virus inoculated BTI-EAA cells. Insert: EPV DNA synthesis in BTI-EAA cells by DNA dot hybridization. VirusDetection of Amsacta infected cell DNA from selected time intervals after virus infection was spotted (3.0 pg DNA/spot) on nitrocellulose filter paper and hybridized to “P-labeled Amsacta EPV DNA as described in the text. Time intervals after virus infection are indicated to the left of the corresponding DNA spots.
weights to Amsacta EPV structural proteins were detected in virus-infected cells (Fig. 3, lane 32, diamonds). A significant increase in the rate of [35S]methionine incorporation into virus structural proteins was detected 18 hr after virus inoculation. The majority of the virus structural proteins are synthesized after virus DNA replication, late during virus infection. Early virus protein synthesis was not detected in virus-infected cells maintained in GTC- 100 medium as cellular protein synthesis was not inhibited early in the virus infection process (Fig. 3).
A viral-induced protein with a molecular weight identical to occlusion body matrix protein (110,000) was detected in increasing amounts from 24 to 32 hr after infection (Fig. 3). No significant change in protein biosynthesis was detected in uninfected BTI-EAA cells from 1 hr through 32 hr incubation. To determine if any of the virus-induced proteins were post-translationally modified, virus-infected cells were pulse labeled at 12 and 18 hr after infection with r5S]methionine for 1 hr. The labeled methionine was chased in medium containing unla-
EPV DNA REPLICATION
A 207,969#
B
2
AND PROTEINS
4
6
6
SYNTHESIS
IN INSECT CELLS
10 12 18 24
32
347
A
; 3
111,886~
; 8 9 :': 12
32
:: 2 15,488)
37
FIG. 3. Autoradiogram of [35S]methionine-labeledAmsacra EPV structural peptides. Lanes A and B are occluded virus and nonoccluded virus, respectively, produced in BTI-EAA cells under continuous labeling conditions of 10 /Xi [9]methionine/2 X lo6 cells/25 ml flask. The labeled amino acid was added 6 hr postinfection as described in the text. Virus was isolated from infected cells as described in Materials and Methods. In lanes A and B, the diamonds indicate quantitative differences detected between virus structural peptides of the nonoccluded and the occluded forms of the virus. Small numbers at the left margin of the gel indicate individual virus structural proteins. Large numbers at far left of gel indicate the position of BDH synthetic peptide molecular weight markers. Lanes 4-32 are Amsacta EPV-infected BTI-EAA cells maintained in GTC-100 medium (m.o.i. = IO), pulse labeled with [W]methionine from 4 to 32 hr after centrifugation. Lane 2 is labeled peptides from uninoculated cells 2 hr after infection. Viral-induced proteins corresponding in molecular weight to viral structural proteins are indicated to the right of lane 32 by diamonds.
beled methionine for 0 hr or 14 hr prior to sacrifice of the cells. Following the chase period, the infected cell proteins were separated by SDS-polyacrylamide gel electrophoresis and the gels autoradiographed as described in the text. No significant changes in the pattern of virus proteins were detected during the two time intervals selected (results not presented). DISCUSSION
In comparison to vaccinia, in which DNA synthesis is detected within 1 hr after virus
infection
(Esteban
and Holowczak, 1977), synthesis is initiated later in the virus infection cycle (6- 12 hr), and requires approximately 7 hr to attain a maximum rate of DNA synthesis in contrast to 2 hr for vaccinia virus (Esteban and Holowczak, 1977). The late initiation of insect poxvirus DNA synthesis may be due, in part, to the lower temperature optimum (28°C) required for virus multiplication in invertebrate cells. Those proteins which are detected in higher concentrations in the occluded virus
Amsacta EPV DNA
348
W.
H.
R.
than in the nonoccluded virus (Fig. 3A, diamonds) may be degradation products of occlusion body protein. This possibility is rather unlikely however, as occlusion body protein isolated from tissue culture derived occlusion bodies yields a single band of 110,000 M. W. tier SDS-polyacrylamide gel electrophoresis (Langridge and Roberts, 1982). Amsacta EPV occlusion body protein is similar in size to the occlusion body matrix protein isolated from Choristoneuru biennis EPV (Bilimoria and Arif, 1979). The biosynthesis of a major viral-induced protein of a molecular weight equivalent to occlusion body protein occurs from 20 to 24 hr after virus infection of both BTI-EAA cells and He&this zea IPLB-1075 cells (unpubl. results). Thus, occlusion body matrix protein may be specified by viral rather than host cell information. The extensive virus protein modification detected in vertebrate poxvirus-infected cells (Katz and Moss, 1970) was not detected in Amsucta EPV-infected BTI-EAA cells. Reasons for the apparent absence of post-translational modification of Amsuctu EPV proteins are not presently understood. ACKNOWLEDGMENTS
LANGRIDGE
GRANADOS, R. R. 1973. Insect poxviruses: Pathology, morphology, and development. Miscellaneous Publications 73-94.
of the Entomological
Society
of America
9,
HOLOWCZAK, J. A., AND JOKLIK, W. K. 1967a. Studies on the structural proteins of vaccinia virus I. Structural proteins of virions and cores. Virology, 33, 717-725.
HOLOWCZAK, J. A., AND JOKLIK, W. K. 1967b. Studies on the structural proteins of vaccinia virus II. Kinetics of the synthesis of individual groups of structural proteins. Virology, 33, 726-739. KAFATOS, F. C., JONES, C. W., AND EFSTRATIADIS, A. 1979. Determination of nucleic acid sequence homologies and relative concentration by a dot hybridization procedure. Nucl. Acids Res., 7, 1541-1552. KATZ, E., AND Moss, B. 1969. Synthesis of vaccinia viral proteins in cytoplasmic extracts I. Incorporation of radioactively labeled amino acids into polypeptides. .I. Virol., 4, 416-422. KATZ, E.. AND Moss, B. 1970. Formation of a vaccinia virus structural polypeptide from a higher molecular weight precursor: Inhibition by vitampicin. Proc.
Nut.
Acad.
Sri.
USA.
66, 677-684.
KNUDSON, D. L., AND BUCKLEY, S. M. 1977. Invertebrate cell culture methods for the study of invertebrate-associated animal viruses. In “Methods of Virology” (K. Maramorosch and H. Koprowski, eds.1, Vol. 6, pp. 323-391. Academic Press, New York. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature
(London),
227, 680-685.
This work was supported in part by Grant USPH AI-0883607 from the National Institute of Allergy and Infectious Diseases to Dr. D. W. Roberts and Dr. R. R. Granados and to Grant NSF-PCM-7816121 from the National Science Foundation. I am grateful to Miss Arpy Barsoumian for technical assistance, and to Dr. A. A. Szalay for helpful discussions.
LANGRIDGE. W. H. R.. BOZARTH, R. F., AND ROBERTS. D. W. 1977. The base composition of entomopoxvirus DNA. Virology, 76, 680-685. LANGRIDGE, W. H. R., AND ROBERTS, D. W. 1977. Molecular weight of DNA from four entomopoxviruses determined by electron microscopy. J.
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BILIMORIA, S. L., AND ARIF, B. M. 1979. Subunit protein and alkaline protease of entomopoxvirus spheroids. Virology, 96, 596-603. BILIMORIA, S. L., AND ARIF, B. M. 1980. Structural polypeptides of Choristoneuru biennis entomopoxvirus. Virology, 104, 253-2.57. BONNER, W. M., AND LASKEY, R. A. 1974. A film detection method for tritium labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem.,
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W. J., GRANADOS, R. R., AND ROBERTS, D. W. 1974. Isolation and characterization of entomopoxvirions from virus-containing inclusions of
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OF INVERTEBRATE
Virus
PATHOLOGY
41, 341-349 (1983)
DNA Replication and Protein Synthesis in Amsacta Entomopoxvirus-Infected Estigmene acrea Cells
moorei
W. H. R. LANGRIDGE Boyce
Thompson
Institute,
Cornell
University,
Tower
Road,
Ithaca,
New
York
14853
Received April 19, 1982; accepted August 23, 1982 Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [3H]thymidine incorporation 12 hr after virus inoculation. Hybridization of 32P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and viruscontaining occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation. KEY WORDS: Entomopoxvirus DNA replication: polyacrylamide gel electrophoresis: entomopoxvirus protein synthesis; DNA hybridization.
formation is available concerning the biosynthesis of entomopoxvirus proteins in Vertebrate poxviruses and entomopoxvirus-infected insect cells. viruses (EPVs) (Matthews, 1979) are simiIn this report, we have infected Estiglar in size and morphology (Granados, rnene acrea cells (BTI-EAA cells) with Am1973). The entomopoxvirus genome is a large (13 1- 200 x 106 daltons), linear, double- sacta EPV and have determined the time stranded DNA molecule equal in size to sequence of DNA replication and biosynvertebrate poxvirus DNA (Langridge and thesis of virus structural proteins and ocRoberts, 1977). The G + C content of EPV clusion body matrix protein. DNA (18-22% G + C) is almost 40% lower MATERIALS AND METHODS than that of vertebrate poxviruses (33% G + C) (Langridge et al., 1977; Langridge and Chemicals Henry, 1981). More than 40 structural pepEagle’s Basal Medium and Vitamin Sotides of an entomopoxvirus from Choris- lution were obtained from Grand Island tune~g biennis have been identified by Biological Company; L-[35S]methionine sodium dodecyl sulfate (SDS)-acrylamide (455 Ci/mM), [a-32P]TTP (20000 Ci/mM), gel electrophoresis (Bilimoria and Arif, and [methyf-3H]thymidine (73 Ci/mM) were 1980). obtained from New England Nuclear; acThe biosynthesis of viral proteins of ver- rylamide and bisacrylamide, bromophenol tebrate poxviruses has been extensively blue, Coomassie blue R-250, and Tris base studied in cell cultures (Holowczak and were obtained from Sigma Chemical ComJoklik, 1967a, b; Katz and Moss, 1969; pany; N’, N’, N’, N’-tetramethylenediamine Moss and Salzman, 1968; Pennington, 1974; (TEMED) was obtained from Bio-Rad; Salzman and Sebring, 1967; Stern and ammonium persulfate was obtained from Dales, 1976). However, at this time no in- Eastman Chemical Company; sodium doINTRODUCTION
341 0022-2011183 $1.50 Copyright AU rights
@ 1983 by Academic Press. Inc. of reproduction in any form reserved.
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decyl sulfate (SDS) BDH reagent grade and BDH protein molecular weight markers for SDS-acrylamide gel electrophoresis were obtained from Gallard Schlesinger Chemical Company; dioxane was obtained from Baker Laboratories; 2,5-diphenyloxazole was obtained from Packard Instrument Company; and naphthalene from Matheson, Coleman and Bell.
30% sucrose in 10 mrvr Tris-HCl, pH 8.0, containing 1 mM EDTA (T-E buffer), and centrifuged at 20,000 rpm in a Beckman SW-41 rotor for 1 hr at 4°C. The virus pellet was resuspended overnight at 4°C in T-E buffer overlayered on 40-65% (w/w) sucrose gradients in T-E buffer, and centrifuged at 20,000 rpm in a Beckman SW-27 rotor for 1 hr at 4°C. The single visible virus band was removed from the tube by Cultivation of cells syringe, mixed 3:l with T-E buffer, and Continuous hemocyte cell cultures of the centrifuged at 30,000 rpm in a Beckman lepidopteran Estigmene acrea, designated Ti-50 rotor, for 1 hr at 4°C. The pellet was BTI-EAA cells, were maintained and mulresuspended in 2 ml T-E buffer and a lo-p1 tiplied at 28°C in GTC-100 medium, which aliquot was examined by electron microscopy. The virus was found to be intact and was modified from BML/TClO medium (Knudson and Buckley, 1977) by the addifree from visible contamination. tion of 10% (v/v) fetal calf serum, organic Occluded virus. Five days after virus inacids, and vitamins at those concentrations oculation (m.0.i. = lo), [35S]methioninefound in Grace’s (1962) basal medium. labeled Amsacta EPV-infected cells were lysed by addition of 25 ml of T-E buffer Virion Isolation containing 2% SDS to the cell pellet at 0°C. Nonoccluded virions. BTI-EAA cells (1 Crystalline occlusion bodies, which were x 10Yml medium) were infected by addition not visibly altered in size, shape, or bireof Amsacta EPV nonoccluded virus (NOV) fringence by SDS treatment, were freed at a multiplicity of infection of 5 plaque from cell debris by sonication of the cell forming units (PFU)/cell. After 3 days of lysate with a Lab-line Ultratip sonicator incubation at 28°C cultures were centri(Melrose Park, Ill.) at 20 W for 30 set at fuged in 12-ml conical polystyrene centri0°C. The preparation was centrifuged in a fuge tubes at 1500 rpm in a Sorvall GLC-1 Sorvall SS-34 rotor at 10,000 rpm for 10 min tabletop centrifuge for 10 min at 26°C. The at 4°C and the supernatant was removed. supernatant contained NOV at a concenThe occlusion body pellet was resuspended tration of approximately lo* 50% tissue in 10 ml T-E buffer without SDS, and soniculture infectious dose (TCID,,,) units of cation and centrifugation procedures were virus/ml medium. Radioactively labeled repeated. The occlusion bodies were NOV was produced in BTI-EAA cells by washed three times in distilled water and the same procedure except that [35S]me- checked for purity by phase-contrast mithionine (400 Cifmmol) was added to the croscopy. Virions were released from the culture flask (10 pCi/ml) 6 hr after virus occlusion bodies by incubating 2 ml (1 x 10y inoculation. Approximately 36-40 hr after occlusion bodies/ml) with an equal volume inoculation, the medium containing labeled of 1.6 M sodium carbonate containing 0.1 M NOV and some infected cells containing thioglycolate, pH 10.6, for 3 min at 2°C. virus occlusion bodies was transferred to After release of virions from the occlusion sterile conical polycarbonate centrifuge body matrix protein was complete (3-5 tubes and centrifuged at 1000 rpm for 10 min), the pH of the suspension was admin at 26°C to pellet the cells. Infected cells justed to 8 by the addition of an equal volwere stored at 4°C for extraction of labeled ume of 0.1 M Tris-HCl, pH 7.5. The virion occluded virus. The supernatant containing preparation was overlayered on 40-65% labeled NOV was overlayered on 10 ml of (w/w) sucrose gradients and centrifuged at
EPV
DNA
REPLICATION
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20,000 rpm in a Beckman SW-27 rotor for 1 hr at 4°C. After centrifugation, the gradients were fractionated with an ISCO gradient fractionator equipped with a uv flow cell, and the virus peak was collected, mixed 3: 1 with T-E buffer, and centrifuged at 30,000 rpm for 1 hr at 4°C in a Beckman Ti-50 rotor. The virus pellet was resuspended in 2.0 ml of T-E buffer.
SYNTHESIS
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The pulse-labeled cells were transferred from the plate to a 12-ml conical centrifuge tube. Grace’s medium minus amino acids (2 ml) were added to the tube and the cells were pelleted at 1500 rpm for 10 min at 26°C in a Sorvall GLC centrifuge. The supernatant was removed and the cells were gently resuspended in 5 ml of Grace’s medium minus amino acids and recentrifuged. The cell pellet was resuspended in 0.1 ml T-E buffer and stored at -20°C. To detect the post-translational modification of viral proteins, Estigeme acrea cells were inoculated with Amsacta EPV (m.o.i. = IO) as described above. The medium was replaced with fresh medium minus methionine containing 25 @X/ml of [35S]methionine at 12 and 18 hr after infection. After a I-hr pulse, the radioactive medium was removed and either the cells were immediately harvested in 2 ml of Grace’s medium minus amino acids or incubated for an additional 14 hr in 0.5 ml/well of GTC-100 medium containing 10% fetal calf serum. Infected cells were harvested and stored at -20°C until they were analyzed by SDS - polyacrylamide gel electrophoresis and autoradiography.
Assay of Virus Infectivity BTI-EAA cells in logarithmic growth phase (24 hr cell-doubling time) were adjusted to a cell concentration of 4 x lo5 cells/ml with GTC- 100 culture medium. The cells were inoculated with NOV at an m.o.i. of 10 PFU/cell. Aliquots of the virusinfected cells were distributed into wells (1 ml cells/well) of a 24-well Falcon No. 3008 Multiwell tissue culture plate. The plate was sealed with plastic electrician’s tape and centrifuged at 2400 t-pm (1OOOg) for 1 hr at 26°C in a Sorvall GLC-1 tabletop centrifuge. After centrifugation, the medium was removed, 2 ml of fresh medium was added to each well, and the plate was incubated at 28°C. At various time intervals from 1 to 120 hr after virus infection, cells were removed from four wells of the mi- Electrophoretic Separation of Infected crotiter plate and assayed for the appearance Cell Proteins of nonoccluded virus in the medium by an agarose overlay plaque assay procedure as The frozen cell samples were mixed with described by Wood (1977). 50 ~1 of SDS dissociation buffer and heated in a boiling water bath for 3 min (Laemmli, Isotopic Labeling of Virus-Znfected Cells 1970). To determine the amount of [35S]meTo detect the de novo biosynthesis of thionine incorporation/sample, a LO-p1 aliviral-induced and virus structural protein, quot of each solubilized cell sample was the medium was removed from four mi- transferred to a glass-fiber filter (Whatman crotiter plate wells of infected or uninfected 2.4 cm GF/C). The filters were washed twice cells (4.0 x lo5 cells/well) at 2-hr intervals with 10 ml of 10% trichloroacetic acid (TCA), after virus infection. Grace’s medium once in acetone (Salzman and Sebring, 1967), minus amino acids but containing 10 $X/ml and counted in a Beckman LS-200B scintilla[35S]methionine was added to each well (0.5 tion counter. Aliquots containing 1 X lo5 cpm ml/well) and the cells were incubated at of incorporated y5S Jmethionine were applied 28°C for 1 hr. Following the incubation pe- per lane to an 11% polyacrylamide slab gel riod, the medium containing the labeled with a 4% stacking gel (Laemmli, 1970; methionine was removed by Pasteur Studier, 1973). Electrophoresis was perpipette and replaced with fresh medium (0.5 formed at 300 V for 4 hr at 26°C. After ml/well) containing unlabeled amino acids. electrophoresis, the gel was fixed in 10%
344
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LANGRIDGE
acetic acid:2.0% ethanol and stained for 30 min at 40°C in a solution of 0.27% Coomassie blue R-250 in methanol:acetic acid: water (5:1:5). The gel was dried on Whatman No. 1 filter paper at 60°C under vacuum and labeled proteins were detected by autoradiography on Kodak RP Royal X-Omat R X-ray film. Polypeptide molecular weights were determined in comparison with BDH protein molecular weight markers. Amsacta
EPV DNA
Synthesis
The conditions for virus infection of BTI-EAA cells were as described above with one exception: the control cells were inoculated with Amsacta EPV which had been previously inactivated with uv light by placing the virus suspension in a shallow dish 8.5 in. below a shortwave uv light source (254 nm) for 1 hr. Cultures of cells inoculated with inactivated virus were analyzed in parallel with the virus-infected cells. At selected time intervals after virus inoculation, 1 x lo6 cells were pulse labeled for 15 min in Grace’s medium containing 2 pCi/ml of [3H]thymidine. The cells were collected in 2 ml Grace’s medium minus amino acids and centrifuged for 5 min at 1000 rpm in a Sorvall GLC centrifuge. The cells were resuspended and washed two times in 5 ml of the same medium. The radioactivity incorporated into TCAprecipitable material was determined by liquid scintillation spectrophotometry in a Beckman LS-200B scintillation spectrometer as described by Esteban and Holowczak (1977). To determine when Amsacta EPV DNA replication was initiated in virus-infected cells, BTI-EAA cells were synchronously inoculated with Amsacta EPV (m.o.i. = 10) as described above. At various times after inoculation, the virus-infected cells were harvested (1 x lo6 cells/time interval) and centrifuged at 500 rpm for 10 min in a Sorvall RC-2B centrifuge. The infected cells were resuspended in 4 ml of Tris-HCl buffer, pH 8.0, containing 150 mM NaCl and 10 mM EDTA. Total cellular DNA was
prepared from the infected cells by extracting three times with an equal volume of T-E buffer saturated with freshly distilled phenol (Wigler et al., 1979). The DNA samples were dialyzed for 48 hr at 4°C against 3 X 1 liter of 10 mM Tris-HCl,pH 8.0, buffer containing 1 mM EDTA. Hybridization of 32P-labeled Amsacta EPV DNA to virusinfected cell DNA was performed according to the procedure of Kafatos et al. (1979). Uninfected and virus-infected cellular DNA was denatured in alkali and spotted on nitrocellulose filter paper prewet with 1.0 M ammonium acetate (3 pg DNA/spot) in a hybridot apparatus (Bethesda Research Laboratories, Md.). The DNA spots were washed with 1 M ammonium acetate and the filter dried in vacua at 80°C for 2 hr. The DNA spots were hybridized to Amsacta EPV DNA (Wetmur and Davidson, 1968) previously labeled by nick translation to high specific activity with [a-32P]dTTP by the nick-translation procedure of Rigby et al. (1977). Autoradiography of the nitrocellulose filter (Bonner and Laskey, 1974) permitted detection of the homologous viral DNA hybrids. RESULTS Amsacta EPV occlusion bodies were detected by phase-contrast microscopy as early as 20 hr after virus infection in cells maintained in GTC- 100 medium (Fig. 1). Virus multiplication and occlusion appear to occur almost simultaneously as the percentage of Amsacta EPV infected cells containing occlusion bodies increased at approximately the same time and rate as the increase in the titer of extracellular virus detected by plaque assay (Fig. 1). A large increase in infectious, extracellular, nonoccluded virus was detected in the medium of infected cells by plaque assay between 18 to 20 hr after virus inoculation (Fig. 1). The titer of extracellular virus increased until 72 hr after virus inoculation at which time the cells began to lyse. The percentage of virus-infected cells containing EPV occlusion bodies increased
EPV
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AFTER
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345
INFECTION
FIG. 1. Extracellular virus growth curve ofAmsacru moorei EPV in BTI-EAA cells. BTI-EAA cells in exponential growth were inoculated with Amsacra EPV nonoccluded virus (m.o.i. = 10). Cells and virus were centrifuged and incubated at 28°C as describedin the text. At selected time intervals, from 1 to 120 hr after inoculation, the medium from the virus-infected cells was assayed for infectious virus by an agarose overlay plaque assay (Wood, 1977). Virus plaques took approximately 5 days to develop at 28°C. Plaque-forming units from 1 to 120 hr = open circles. The percentage of cells containing occlusion bodies detected by phase-contrast light microscopy = closed circles. Two hundred cells were counted/time interval.
from 2.0% at 18 hr to 86% by 94 hr after infection (Fig. 1). A decrease in the rate of formation of occlusion bodies was detected between 30 hr and 40 hr after infection. By 19 hr after infection the incorporation of [3H]thymidine into acid-precipitable radioactivity in intact virus-infected BTI cells (Fig. 2) was approximately 20-fold greater than in cells inoculated with killed virus. The rate of [3H]thymidine incorporation into virus-infected cells increased from 12 to 19 hr after virus inoculation. A significant increase in Amsacta EPV DNA synthesis was detected by dot hybridization from 6 to 12 hr after virus inoculation (Fig. 2, insert). The amount of viral DNA detected in infected cells increased dramatically from 12 to 24 hr after virus inoculation (Fig. 2, insert). In both occluded and nonoccluded forms of Amsacta EPV, 37 virus structural proteins were detected by SDS-polyacrylamide gel electrophoresis and prolonged exposure of the autoradiogram to reveal
labeled proteins present in low concentrations (Fig. 3). The polyacrylamide gel protein patterns of both forms of the virus were identical in number of protein bands. Several proteins were synthesized in larger amounts in either the occluded or the nonoccluded virus. This protein was identical in molecular weight to Amsacta EPV occlusion body matrix protein isolated from sucrose gradient purified occlusion bodies. After the occluded virus was separated from occlusion body matrix protein by two cycles of sucrose density gradient centrifugation (McCarthy et al., 1974), a significant amount of 110,000 M. W. protein was detected after separation of the virus structural proteins by SDS - polyacrylamide gel electro phoresis (Fig. 3A). Antiserum directed against electrophoretically purified Amsacta EPV occlusion body protein precipitated the 110,000 M. W. protein of the occluded virus. Approximately 32 hr after infection, 25 proteins corresponding in molecular
346
W. H. R. LANGRIDGE
15 -
F-l ‘0x > a 0
0
*
6
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24
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f ‘*0
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TIME
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FIG. 2. Kinetics of DNA synthesis in Amsacfa EPV-infected BTI-EAA cells. The conditions for virus infection and uv light inactivation were as described in the text. At selected time intervals after virus infection, 1 x lo6 cells were pulse labeled for 15 min with 2 &i/ml of [3H]thymidine. Cells inoculated with inactivated virus were analyzed in parallel with virus-infected cells. Solid lines are virus-infected cells; dotted lines represent uv-inactivated virus inoculated BTI-EAA cells. Insert: EPV DNA synthesis in BTI-EAA cells by DNA dot hybridization. VirusDetection of Amsacta infected cell DNA from selected time intervals after virus infection was spotted (3.0 pg DNA/spot) on nitrocellulose filter paper and hybridized to “P-labeled Amsacta EPV DNA as described in the text. Time intervals after virus infection are indicated to the left of the corresponding DNA spots.
weights to Amsacta EPV structural proteins were detected in virus-infected cells (Fig. 3, lane 32, diamonds). A significant increase in the rate of [35S]methionine incorporation into virus structural proteins was detected 18 hr after virus inoculation. The majority of the virus structural proteins are synthesized after virus DNA replication, late during virus infection. Early virus protein synthesis was not detected in virus-infected cells maintained in GTC- 100 medium as cellular protein synthesis was not inhibited early in the virus infection process (Fig. 3).
A viral-induced protein with a molecular weight identical to occlusion body matrix protein (110,000) was detected in increasing amounts from 24 to 32 hr after infection (Fig. 3). No significant change in protein biosynthesis was detected in uninfected BTI-EAA cells from 1 hr through 32 hr incubation. To determine if any of the virus-induced proteins were post-translationally modified, virus-infected cells were pulse labeled at 12 and 18 hr after infection with r5S]methionine for 1 hr. The labeled methionine was chased in medium containing unla-
EPV DNA REPLICATION
A 207,969#
B
2
AND PROTEINS
4
6
6
SYNTHESIS
IN INSECT CELLS
10 12 18 24
32
347
A
; 3
111,886~
; 8 9 :': 12
32
:: 2 15,488)
37
FIG. 3. Autoradiogram of [35S]methionine-labeledAmsacra EPV structural peptides. Lanes A and B are occluded virus and nonoccluded virus, respectively, produced in BTI-EAA cells under continuous labeling conditions of 10 /Xi [9]methionine/2 X lo6 cells/25 ml flask. The labeled amino acid was added 6 hr postinfection as described in the text. Virus was isolated from infected cells as described in Materials and Methods. In lanes A and B, the diamonds indicate quantitative differences detected between virus structural peptides of the nonoccluded and the occluded forms of the virus. Small numbers at the left margin of the gel indicate individual virus structural proteins. Large numbers at far left of gel indicate the position of BDH synthetic peptide molecular weight markers. Lanes 4-32 are Amsacta EPV-infected BTI-EAA cells maintained in GTC-100 medium (m.o.i. = IO), pulse labeled with [W]methionine from 4 to 32 hr after centrifugation. Lane 2 is labeled peptides from uninoculated cells 2 hr after infection. Viral-induced proteins corresponding in molecular weight to viral structural proteins are indicated to the right of lane 32 by diamonds.
beled methionine for 0 hr or 14 hr prior to sacrifice of the cells. Following the chase period, the infected cell proteins were separated by SDS-polyacrylamide gel electrophoresis and the gels autoradiographed as described in the text. No significant changes in the pattern of virus proteins were detected during the two time intervals selected (results not presented). DISCUSSION
In comparison to vaccinia, in which DNA synthesis is detected within 1 hr after virus
infection
(Esteban
and Holowczak, 1977), synthesis is initiated later in the virus infection cycle (6- 12 hr), and requires approximately 7 hr to attain a maximum rate of DNA synthesis in contrast to 2 hr for vaccinia virus (Esteban and Holowczak, 1977). The late initiation of insect poxvirus DNA synthesis may be due, in part, to the lower temperature optimum (28°C) required for virus multiplication in invertebrate cells. Those proteins which are detected in higher concentrations in the occluded virus
Amsacta EPV DNA
348
W.
H.
R.
than in the nonoccluded virus (Fig. 3A, diamonds) may be degradation products of occlusion body protein. This possibility is rather unlikely however, as occlusion body protein isolated from tissue culture derived occlusion bodies yields a single band of 110,000 M. W. tier SDS-polyacrylamide gel electrophoresis (Langridge and Roberts, 1982). Amsacta EPV occlusion body protein is similar in size to the occlusion body matrix protein isolated from Choristoneuru biennis EPV (Bilimoria and Arif, 1979). The biosynthesis of a major viral-induced protein of a molecular weight equivalent to occlusion body protein occurs from 20 to 24 hr after virus infection of both BTI-EAA cells and He&this zea IPLB-1075 cells (unpubl. results). Thus, occlusion body matrix protein may be specified by viral rather than host cell information. The extensive virus protein modification detected in vertebrate poxvirus-infected cells (Katz and Moss, 1970) was not detected in Amsucta EPV-infected BTI-EAA cells. Reasons for the apparent absence of post-translational modification of Amsuctu EPV proteins are not presently understood. ACKNOWLEDGMENTS
LANGRIDGE
GRANADOS, R. R. 1973. Insect poxviruses: Pathology, morphology, and development. Miscellaneous Publications 73-94.
of the Entomological
Society
of America
9,
HOLOWCZAK, J. A., AND JOKLIK, W. K. 1967a. Studies on the structural proteins of vaccinia virus I. Structural proteins of virions and cores. Virology, 33, 717-725.
HOLOWCZAK, J. A., AND JOKLIK, W. K. 1967b. Studies on the structural proteins of vaccinia virus II. Kinetics of the synthesis of individual groups of structural proteins. Virology, 33, 726-739. KAFATOS, F. C., JONES, C. W., AND EFSTRATIADIS, A. 1979. Determination of nucleic acid sequence homologies and relative concentration by a dot hybridization procedure. Nucl. Acids Res., 7, 1541-1552. KATZ, E., AND Moss, B. 1969. Synthesis of vaccinia viral proteins in cytoplasmic extracts I. Incorporation of radioactively labeled amino acids into polypeptides. .I. Virol., 4, 416-422. KATZ, E.. AND Moss, B. 1970. Formation of a vaccinia virus structural polypeptide from a higher molecular weight precursor: Inhibition by vitampicin. Proc.
Nut.
Acad.
Sri.
USA.
66, 677-684.
KNUDSON, D. L., AND BUCKLEY, S. M. 1977. Invertebrate cell culture methods for the study of invertebrate-associated animal viruses. In “Methods of Virology” (K. Maramorosch and H. Koprowski, eds.1, Vol. 6, pp. 323-391. Academic Press, New York. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature
(London),
227, 680-685.
This work was supported in part by Grant USPH AI-0883607 from the National Institute of Allergy and Infectious Diseases to Dr. D. W. Roberts and Dr. R. R. Granados and to Grant NSF-PCM-7816121 from the National Science Foundation. I am grateful to Miss Arpy Barsoumian for technical assistance, and to Dr. A. A. Szalay for helpful discussions.
LANGRIDGE. W. H. R.. BOZARTH, R. F., AND ROBERTS. D. W. 1977. The base composition of entomopoxvirus DNA. Virology, 76, 680-685. LANGRIDGE, W. H. R., AND ROBERTS, D. W. 1977. Molecular weight of DNA from four entomopoxviruses determined by electron microscopy. J.
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