SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar

JOURNAL

OF INVERTEBRATE

PATHOLOGY

37, 174- 180 (1981)

SDS-PAGE Comparative Studies on the Polyhedral and Viral Polypeptides of the Nuclear Polyhedrosis Viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar B. MASKOS

CHRISTIAN Institute

of Zoology.

Cell

Biology

AND HERBERTG.MILTENBURGER

Laboratory. Received

D-6100 March

Darmstadt,

Federul

Republic

of Germany

11. 1980

After solubilization of polyhedra of Autographa californica, Lymantriu dispur. and Mamestru brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0-30.0 kdalton). characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides. KEY WORDS: Autographa californicu; Lymantriu dispar: Mamestru brussicae; nuclear polyhedrosis viruses, polyhedral polypeptides. comparison of.

INTRODUCTION

found (McCarthy and Liu, 1976; Cibulsky et al., 1977; Brown et al., 1979; McCarthy and DiCapua, 1979). The present paper demonstrates that NPVs can be distinguished from each other on the basis of their polyhedral protein pattern. This was accomplished by making comparative investigations on the polyhedral polypeptides of Lymantria dispar, Mamestra brassicae, and Autographa californica nuclear polyhedrosis viruses using SDS-PAGE.

A common attribute of nuclear polyhedrosis viruses (NPV) is the proteinous polyhedral inclusion body (PIB) by which enveloped virions are occluded. The PIB protein is designated as granulin for granulosis viruses and as polyhedrin for nuclear polyhedrosis viruses (Summers and Egawa, 1973). Both granulin and polyhedrin isolated from different baculoviruses consist of a polypeptide having a molecular weight of 28,000 ? 2000 dalton. This polypeptide has been shown to be unique for each NPV species by N-terminal analysis and two-dimensional high-voltage electrophoresis of tryptic peptides (Summers and Smith, 1975, 1976). Comparative studies of the polyhedral proteins of the nuclear polyhedrosis viruses have been reported in a number of papers (Kozlov et al., 1975; McCarthy and Liu, 1976; Cibulsky et al., 1977; Merdan et al., 1977; Croizier and Croizier, 1977; Harrap et al., 1977; Summers and Smith, 1978; Brown et al., 1979). In some of these reports only one major polyhedral polypeptide was described (Summers and Smith, 1975, 1976) while in others more than one polypeptide was

MATERIALS

Sources of NPV. We obtained the NPVs of Mamestra brassicae (Mb) from Dr. Groner (Biologische Bundesanstalt fur Land- und Forstwirtschaft, Institut fur biologische Schadlingsbekampfung, Darmstadt, West Germany), the NPVs of Autographa californica (AC) from Dr. Roder (Hoechst AG, Frankfurt/M.) and the NPVs ofLymantria dispar (Ld) from Dr. Knudson (Yale University, New Haven, Conn.). Mb-NPV were replicated in Mb larvae; AC-NPV in larvae of Heliothis virescens (Hv), and Spodoptera littoralis (Sl) cell cultures: and Ld-NPV in Ld larvae. Cell culture. The Mamestra brassicae 174

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AND METHODS

COMPARATIVE

SDS-PAGE

cell line IZD-Mb 0503 used in our experiments was subcultured as described previously (Miltenburger et al., 1977). For the in vitro production of nonoccluded viruses (NOV), infectious hemolymph from Mb larvae infected with AC-NPV was introduced into IZB-Mb 0503 suspension cultures. The infection dose ranged between 0.1 and 10 TCID,,/cell. Only cells in the early log phase of growth were infected. Virus replication was generally accomplished within 72 hr after infection. Polyhedral purification. After centrifugation of cell suspension cultures at 3OOOg, the pellet was homogenized in freshly prepared 4 M urea in 0.1 M Tris buffer, pH 7.4, by four cycles of ultrasonification (Branson-Sonifier B-12; 60 W for 15 set) at 2°C. The sonicate was made 1% (w/v) with respect to sodium dodecyl sulfate (SDS) and was briefly resonicated. The purification procedure of McCarthy and Liu (1976) was adopted, except that after initial purification, the PIB suspension was loaded onto a 50-60% (w/w) sucrose gradient constituted in 0.1% SDS and centrifuged at 91,000g for 120 min at 20°C in a L-2-65 preparative Beckman ultracentrifuge (rotor SW 27). The purification of tissue culture and larvae-derived PIBs (sucrose gradient centrifugation) was repeated several times to yield polyhedra suitable for biochemical analysis. The polyhedral zone was collected, washed, and resuspended with 0.1% SDS and stored at -35°C. Virus purification. Virus particles were released from polyhedra using a method described by Bergold (1947) with moditications reported by Summers and Smith (1978). Purified PIBs, loo-150 mg, were dissolved under continuous stirring for 60 min at 0-4°C or for 30 min at 30°C in 0.1 M Na,CO,, 0.10 M EDTA, 0.17 M NaCl, pH 10.9. The “solubilization” was stopped by fivefold dilution with glass-distilled water and lowering the pH to 8.5-8.9 by addition of 0.1 M Tris buffer, pH 7.8, and 0.01 M EDTA. The undissolved polyhedra were removed by centrifugation at 2000g for 10

STUDIES

ON

NPV

PROTEINS

175

min. The supernatant containing the virions was centrifuged at 87,000g for 1 hr at 4°C (SW 27). The virus pellets were resuspended in glass-distilled water and l- to 2-ml aliquots were layered onto 25-60% (w/w) sucrose gradients constituted in 0.1 M Tris buffer, pH 7.8, and 0.01 M EDTA. The gradient tubes were centrifuged at 91,000g for 2 hr at 4°C (SW 27) and the virus bands were collected. They were washed free from sucrose resuspended in water and stored at -35°C. Polyhedrin purification. Purification of polyhedrin was carried out as described by Summers and Smith (1978). Briefly, for the inactivation of the protease, the PIB suspension was kept in a water bath at 70°C for 2 hr, then resuspended in 0.01 Tris, pH 7.8, and 0.01 M HgCl, and allowed to equilibrate at room temperature overnight. The PIBs were solubilized by adding 0.1 M Na,CO,, 0.17 M NaCl, pH 10.8, for 10 min at a concentration of 5 mg of protein/ml at 4°C. This suspension was centrifuged at 87,000g for 1 hr at 4°C (SW 27) and the upper two-thirds of the supernatant was collected and utilized for PAGE analysis. Protein determination. We followed the procedure described by Lowry et al. (195 I), using crystallized serum albumin as a standard. Samples and standard protein were solubilized in 0.1% (w/v) SDS and 1 N NaOH. Assays of protein by the method of Lowry et al. in samples containing 2mercaptoethanol were carried out as described by Tan (1977). Polyacrylamide gel electrophoreses (PAGE). A slab gel apparatus as described

by Studier (1973) was used. The gels were prepared according to the method of Laemmli (1970) and modified according to Lugtenberg et al. (1975). The separating gel was 1.3 mm thick, 10 cm long, and 16.5 cm wide. Electrophoresis was carried out for approximately 4.5 hr at a constant current of 35 mA (7 W). The gels were stained for 1 hr in a solution of Coomassie Brilliant Blue R-250 and the molecular weights were determined according to the method of Weber

176

MASKOS

AND

and Osborn (1969). The reference proteins used were cytochrome c molecular weight (MW) of 12,400; chymotrypsinogen, MW of 25,000; egg albumin, MW of 45,000: and bovine serum albumine, MW of 67,000 (Serva, Heidelberg, West Germany). Preparation of the samples for SDSPAGE. Highly purified polyhedra were

resuspended in 0.0625 M Tris-HCl pH 6.8, containing 2% sodium dodecyl sulfate (SDS), 5% P-mercaptoethanol, 10% sucrose, and 0.001% bromophenol blue (usually 400-500 pg protein in 40-50 ~1 sample buffer: final protein concentration 10 mgml). The samples were heated at 100°C for 4-5 min and centrifuged for 5 min at 8000g in an Eppendorf centrifuge 5412. Ten to fifteen microliters (30-60 pg protein) of the supernatant were analyzed. Virions. For SDS-PAGE, virus proteins were solubilized by resuspending 250 pg/O.l ml in sample buffer and heating at 100°C for 4- 5 min. Forty to fifty micrograms of protein were analyzed with SDS-PAGE. RESULTS

At most 28% of PIB protein (= 72% insoluble within 10 min of treatment) was recovered from AC inclusion bodies as described above (Summers and Smith, 1978). After 5 min of solubilization of polyhedra in sample buffer, the yield of polyhedral protein was 30%. The SDS-PAGE pattern of polyhedral polypeptides of AC-NPV isolated from Mb and Sl larvae and from Mb and Sl cell cultures are shown in Figure 1. In all cases no significant differences were observed, irrespective of origin and procedure applied: There is only one polypeptide with a molecular weight of 28.0-29.0 kdalton (based on relative mobility from SDS-PAGE). This is in agreement with results published by Summers and Smith (1976) and Croizier and Croizier (1977). When the gels were more heavily loaded (35-50 pg of protein) several additional polypeptide bands were found (Fig. 2). The main aim of our work was to differentiate between polyhedral and viral

MILTENBURGER

67000

**

45000

a.

WW

-0 25 000

rc-

12400

S

A

B

S

C

D

;..=

S

E

FIG. 1. SDSpolyacrylamide slab gel electrophoresis of polyhedral polypeptides. (A) AC-NPV from Mb cell cultures. Polyhedra dissolved in 0.1 M Na,CO,. 0.17 M NaCI. pH 10.8. for 10 min at 4°C according to a procedure described by Summers and Smith (1978). (B) AC-NPV from Mb cell cultures. Polyhedra solubilized in 2% SDS, 5% b-mercaptoethanol in 0.0625 M Tris. pH 6.8, at 100°C for 5 min. (C) AC-NPV from Sl cell cultures. Polyhedra solubilized as described for (B). (D) AC-NPV from Hv larvae. Polyhedra heated (2 hr at 70°C) and treated with HgCI, (0.01 M for 12 hr), then dissolved in 0.1 M Na,CO,, 0.17 M NaCI, pH 10.8, for IO min at 4°C as described by Summers and Smith (1978). (E) AC-NPV from Mb larvae. Polyhedra dissolved as described for (D). (S) Standards: cytochrome c, MW 12.400: chymotrypsinogen, MW 25,000; egg albumin, MW 45,000; and bovine serum albumin, MW 67.000. Amounts of l-2 pg of each protein were electrophoresed in SDS-polyacrylamide gel at a constant current of 35 mA/gel slab.

polypeptides (see Figs. 2-4) and to interpret the bands with molecular weights between 62.0 and 12.4 kdalton. The mobilities of polyhedral and virus polypeptides of AC-NPV and Ld-NPV were compared on the same gel as shown in Figure 2. It can be seen that at least eight polyhedral polypeptides are detectable, whereas under the same conditions 17-20 bands are obtained from purified vu-ions. For example, purified virions of Ld-NPV have at least 20 structural polypeptides ranging from 12.4 to 98.5 kdalton (Fig. 2A). SDS-PAGE analysis of PIB protein obtained by solubilization of polyhedra in sample buffer (2%

COMPARATIVE

VP 98.!5-

3E:SZ 33.029.727 o24 !5-

;gg ,2.4-

SDS-PAGE

STUDIES

ON

NPV

PROTEINS

177

PP “( A

--27.

e

--22.5 -sm.2 -78-l -,a.= mmmdmk --1&z 4 A

0

FIG. 2. SDSPAGE pattern of Ld-NPV and AcNPV virions and polyhedral polypeptides. Releasing of virus particles from polyhedra was induced by alkaline dissolution. Purification was performed by sucrose density gradient centrifugation. Forty to fifty micrograms were electrophoresed in SDS-polyacrylamide gel. Polyhedra were solubilized in 2% SDS, 5% P-mercaptoethanol, 0.0625 M Tris-HCI, pH 6.8, at 100°C for 5 min. For further details see Materials and Methods. Virus polypeptide (VP) of Ld-NPV are shown in (A) and of AC-NPV in (C). Polyhedral polypeptides (PP) of Ld-NPV are shown in (B). and of AC-NPV in (D). The molecular weights of polypeptides are given in kdalton.

SDS, 5% P-mercaptoethanol) in 0.062 M Tri-HCl, pH 6.8) showed that nine polypeptides are present with molecular weights between 12.4 and 59.3 kdalton (Fig. 2B). Four of the polypeptides are predominant, the major one having a molecular weight of 27.8 kdalton. The three others have molecular weights of 18.1, 54.7, and 59.3 kdalton. For AC virions the molecular weights of the structural polypeptides range from 12.4 to 92.5 kdalton (Fig. 2C). Results of these analyses are in agreement with those recently published by Summers and Smith (1978). Eight polypeptides with molecular weights ranging from 12.5 to 62 kdalton were detected by electrophoretic analysis of AC polyhedral polypeptides when polyhedra were solubilized in sample buffer (Fig. 2D) as well as by carbonate

28.2-

20.6-

14.613.712.4-

FIG. 3. Comparison of Mb-NPV from Mb larvae (B); AC-NPV from IZD-Mb Ld-NPV from Ld larvae hedra solubilization and same as described in the

the polyhedral polypeptides. (A); AC-NPV from Hv larvae 0503 cell culture (C): and (D). The conditions for polygel electrophoresis are the legend of Figure 2.

chloride treatment although the alkaline protease was inactivated by heat and HgCl, treatment. In this latter case the polyhedral polypeptide pattern was also highly specific. In particular three polypeptides with molecular weights of 16.1, 17.9, and 20.0 kdalton were reproducibly detected. Unexpectedly, SDS-PAGE analysis of solubilized protein from AC polyhedra isolated from Hv larvae yielded a qualitative difference in the polypeptide composition when compared to NPVs replicated in cell culture (Fig. 3). A polypeptide with a molecular weight of 33.8 kdalton could be detected both in carbonate chloride-dissolved samples of AC polyhedra replicated in Hv larvae after inactivation of the associated protease by heat and HgCl, treatment and in directly solubilized AC poly-

178

MASKOS

AND

hedra when heated in sample buffer. This polypeptide could not be found in carbonate chloride-solubilized polyhedra replicated in Mb and Sl cell cultures. The reason for this is not yet known. Alkaliliberated virions of Mb-NPV showed at least 17 polypeptide bands with molecular weights ranging from 12.4 to 85.0 kdalton (Fig. 4C). The polypeptide pattern of Mb polyhedra is shown in Figures 4A and B. The major polyhedral polypeptide with a molecular weight of 28.2 kdalton is evident as the most pronounced band. The four smallest polypeptides with molecular weights of 13.7, 14.8, 18.2, and 20.6 kdalton can be regularly distinguished. In some cases a small amount of a polypeptide with a molecular weight of 16.0 kdalton is found when the gels were more heavily loaded (60 pg protein). In addition, in gels loaded with an excessive amount of polyhedral protein, several additional bands are present. They most probably represent viral polypeptides (Figs. 4A, B). In this case, the polyhedra solubiiized in sample buffer showed a protein pattern similar to that from alkaliliberated virions. It is of interest that the relatively heavy load of the gels with the proteins from whole polyhedra did not disturb the appropriate resolution of the viral polypeptides. When samples of polyhedral polypeptides (30 pg protein) were coelectrophoresed with viral polypeptides (30 pg) the relative mobilities of both protein preparations were not changed. DISCUSSION

The paracrystalline matrix of nuclear polyhedrosis virus PIBs contains a major polypeptide with a molecular weight between 25.0 and 31.0 kdalton as determined by SDS-PAGE (Summers and Smith, 1976, 1978; Croizier and Croizier, 1977). It was also reported (McCarthy and DiCapua, 1979) that the major component of the undegraded solubilized proteins of larval (the alkaline protease was inactivated)- and tissue culture-derived polyhedra of Ld- and AC-NPVs has a molecular weight of 30.0-

MILTENBURGER

-0.

A

B

C

--

_I

61.556.4-

-65.0 73.0 167.0 63.6

_ _ _ -

28.2-

_ -

20.618.2-

14.8 13.7-

-

12.4

-

_ _

57.6 49.6 46.6 44.0 42.3 39.3 34.2 29.5

26.7 22.7 18.3

14.6 12.2

FIG. 4. SDS-PAGE of polyhedral and viral polypeptides of Mb-NPVs. Polyhedra were solubilized in 2% SDS, 5% P-mercatoethanol. and 0.0625 M Tris-HCI, pH 6.8, at 100°C for 5 min. Fifty micrograms (A) and thirty micrograms (BJ were electrophoresed in SDS-PAG. Mb viral structural polypeptides are shown in (C). The virions were liberated from polyhedra by carbonate chloride treatment and purified as described under Materials and Methods. Forty micrograms of viral proteins were electrophoresed. The molecular weights are given in kdalton.

32.0 kdalton. A minor component has been shown to have a molecular weight of 59.063.0 kdalton. There is no detectable difference in this protein obtained from the alkaline dissolution of either Ld- or’ AcNPVs (McCarthy and DiCapua, 1979), using the SDS-PAGE of Maize1 (1971). In an investigation on polyhedral polypeptides from five nuclear polyhedrosis viruses, 8- 11 polyhedral polypeptides were found with molecular weights ranging from 9.0-88.0 kdalton (Cibulsky et al., 1977). It should be recalled that the presence of an endogenous protease complicated the

COMPARATIVE

SDS-PAGE

characterization of the alkali-solubilized polyhedral polypeptides (Summers and Smith, 1975, 1976). Proteolytic degradation of major polypeptides may lead to the formation of polypeptides with lower molecular weights after alkaline dissolution (McCarthy and Liu, 1976; Eppstein et al., 1975). This is in contrast to our results which demonstrate multiple polypeptide bands of the polyhedral protein. These multiple polypeptide bands of the polyhedral proteins did not disappear after inactivation of the alkaline protease. Furthermore when the inactivated polyhedra of AC-, Ld-, and Mb-NPVs were solubilized in sample buffer at pH 6.8, we could demonstrate distinct differences in the polypeptides of the three baculovirus species, each of which showed characteristic bands. They could be identified as polyhedral polypeptides by comparisons with the structural polypeptides from purified virions. This means that NPVs from both larvae and cell cultures as well as from different species can be identified by SDSPAGE without releasing the virions from the PIBs by alkaline dissolution and without gradient purification of the viral components. Using this fast, reliable, and quite simple method, the identification of baculoviruses can be performed reproducibly by establishing characteristic polypeptide patterns of PIBs. ACKNOWLEDGMENTS We thank Mrs. W. Zipp and Mrs. E. Magel for skillful and dedicated technical assistance. This research was supported by grants from the Federal Ministry of Research and Technology (PTB 8051 and P’l-B 8041).

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R. T. 1977. Biochemical comparison of polyhedral protein from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae). J. Invertebr. Pathol., 29, 182-191. CROIZIER, G., AND CROIZIER, L., 1977. Evaluation du poids moltculaire de la protCine des corps d’inclusion de divers baculovirus d’insectes. Arch. Viral., 55, 247-250. EPPSTEIN, D. A., THOMA, J. A., SCOTT, H. A., AND YOUNG, S. Y. 1975. Degradation of matrix protein from a nuclear-polyhedrosis virus of Trichoplusia ni by an endogenous protease. Virology, 67, 591-594. HARRAP, K. A., PAYNE, CH.C., AND ROBERTSON, J. S. 1977. The properties of three bacdoviruses from closely related hosts. Virology, 79, 14-31. KOZLOV, E. A., LEVITINA, T. L., SIDOROVA, N. M., RADAVSKI, YU.L., AND SEREBRYANI, S. B. 1975. Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella. J. Invertebr. Pathol., 25, 103-107. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 26.5-275. LUGTENBERG, B., MEIJERS, J., PETERS, R., VAN DER HOFK, P., AND VAN ALPHEN, L. 1975. Electrophoretic resolution of the major outer membrane protein of Escherichia coli K 12 into four bands. FEBS Lett., 58(l), 254-258. MAIZEL, J. V. 1971. Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5, pp. 179-263. Academic Press, New York. MCCARTHY, W. J., AND LIU, S. Y. 1976. Electrophoretic and serological characterization of Porthetria dispar polyhedron protein. J. Invertebr. Pathol., 28, 57-65. MCCARTHY, W. J., AND DICAPUA, R. A. 1979. Characterization of solubilized proteins from tissue cultureand host-derived nuclear polyhedra of Lymantria dispar and Autographa californica. Intervirology, 11, 174-181. MERDAN, A., CROIZIER, L., VEYRUNES, J.-C., AND CROIZIER, G. 1977. etude cornparke des prottines des polyCdres et des virions de trois isolats de baculovirus de Spodoptera littoralis. Entomophaga, 22, 413-420. MILTENBURGER, H. G., DAVID, P., MAHR, U., UND ZIPP, W. 1977. ijber die erstellung von Lepidopteren-Dauerzellinien und die in vitro-replikation von insektenpathogenen viren. 1. Mamestra brassicae L. (Kohleule)-Zellinien und NPV-replikation. Z. Angeu. Entomol., 82, 306-323. STUDIER, W. F. 1973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol.. 79, 237-248.

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SUMMERS, M. D., AND EGAWA. K. 1973. Physical and chemical properties of 7’richoplusiu ni granulosis virus granulin. J. Viral.. 1092L 1103. SUMMERS, M.D., AND SMITH. G. E. 1975. Trich~phsiu ni granulosis virus granulin: A phenol-soluble, phosphorylated protein. J. Viral.. I1081116. SUMMERS, M. D., AND SMITH, G. E. 1976. Comparative studies of baculovirus granulins and polyhedrins. Intervirology. 6, 1688 180.

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SUMMERS, M. D., AND SMITH, G. E. 1978. Baculovirus structural polypeptides. Virolog?.. 84, 39&402. TAN, K. K. 1978. Assay of proteins by TAowry‘s method in samples containing 2-mercaptoethanol. And. Biochem., 86(l). 327-331. WERER. K., AND OSBORN, M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem..

244,

4406~4412.