Polyhedrosis Virus. (LdNPV HA) were obtained from Edward Dougherty, U.S. Department of Agriculture Insect Pathology Laboratory,

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1983, P Vol. 46, No /83/ $02.00/0 Copyright , American Society for Microbiology Characterization of Gypsy Moth (Lymantria dispar) Nuclear Polyhedrosis Virus BRAD STILES,* JOHN P. BURAND, MARTA MEDA, AND H. A. WOOD Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York Received 23 March 1983/Accepted 13 May 1983 Characterization of the proteins and nucleic acid of the gypsy moth nuclear polyhedrosis virus isolated in Ithaca, N.Y. (LdNPV-IT) is presented. A total of 29 viral structural proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis when the virus was isolated in the absence of alkaline protease activity. Fourteen surface envelope viral proteins were identified by lactoperoxidase iodination. Eleven proteins were associated with nucleocapsids prepared by Nonidet P-40 detergent treatment. Distinct alterations of viral proteins were documented when virions were purified in the presence of occlusion bodyassociated alkaline protease(s). Restriction enzyme digests of viral DNA indicated that this isolate was composed of a large number of genetic variants. On the basis of the major molar fragments resulting from EcoRI, BamHI, BglII, and HindIII digests, the molecular weight of the LdNPV genome was approximately 88 x 106. The gypsy moth, Lymantria dispar (a native of Europe), is a serious pest of forest, fruit, and shade trees in the eastern United States. The U.S. Department of Agriculture has been involved in a massive control program which has included the use of the uncloned Hamden isolate of L. dispar nuclear polyhedrosis virus (LdNPV HA) (14). In addition to studies to evaluate LdNPV as a biological control agent, LdNPV has been physically characterized. The viral structural proteins have been studied by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (15, 19, 20, 27), whereas the occlusion body (OB) protein has been dealt with separately by several workers (1, 4, 16). The viral DNA of the Hamden isolate has been partially characterized by restriction endonuclease analysis (17, 21). A clear identification of LdNPV has been complicated by the genetic heterogeneity of uncloned isolates and the presence of an alkaline protease (5) in virus preparations used for structural protein analyses. The present study was initiated to determine whether a local epizootic within the gypsy moth population near Ithaca, N.Y., during 1982 was due to LdNPV. This study includes the first characterization of LdNPV isolated in the absence of alkaline protease activity and characterization of the surface envelope proteins. A detailed analysis of four restriction enzyme digests of the Ithaca isolate (LdNPV IT) DNA is presented. 297 MATERIALS AND METHODS Virus. Samples of the Hamden LdNPV isolate (LdNPV HA) were obtained from Edward Dougherty, U.S. Department of Agriculture Insect Pathology Laboratory, Beltsville, Md. The Ithaca isolate (LdNPV IT) came from infected final instar L. dispar larvae (which exhibited typical gross signs of NPV infection) collected during July 1982 from the Ithaca, N.Y., area. The cadavers were stored at 4 C with 1% (vol/vol) P- mercaptoethanol and 1% (vol/vol) Ivory liquid detergent. Vir purification. After homogenization of diseased larvae in a Waring blender, the homogenate was strained through cheesecloth, and the viral OBs were pelleted by centrifugation (1,000 x g for 15 min). The OBs were then washed three times with water, layered onto linear gradients (40%o [wt/wt] to 63% [wt/wt] sucrose in water), and centrifuged for 1 h at 80,000 x g (5 C). The OBs were collected, diluted, and pelleted by centrifugation at 10,000 x g for 10 min. OBs were treated with 1% SDS (wt/vol) followed by 0.5 M NaCl (including sonication) as described previously by Wood (25). A portion of the purified OBs was heated in a water bath at 800C for 2 h to inactivate the alkaline protease associated with the virus preparation. Protease inactivation was monitored with a modification of the protease assay suggested by Brown et al. (1). Approximately 108 OBs were dissolved in 1 ml of 50 mm Na2CO3 (ph 11) containing 30 mg of azocoll (Sigma Chemical Co.). The mixture was incubated for 30 min at 37 C and then centrifuged for 5 min at 1,000 x g to remove the insoluble azocoll. The amount of dye released from the substrate was quantified by optical density measurements at 520 nm. Virions were released from OBs by treatment with a

2 298 STILES ET AL. solution of 0.1 M Na2CO3, 0.12 M NaCl, and 10 mm EDTA for 30 min at room temperature. Undissolved OBs were pelleted by low-speed centrifugation. The virus suspension was layered over a cushion of 25% (wt/wt) sucrose (1 mm Tris buffer, ph 8.5) and pelleted by centrifugation at 66,000 x g and 5 C for 1 h (this step was omitted for DNA purifications). The virus was next layered onto 25 to 55% (wt/wt) sucrose gradients (1 mm Tris buffer, ph 7.5) and centrifuged at 80,000 x g and 5 C for 1 to 3 h. The virus band was collected, diluted 1:3 with 1 mm Tris buffer (ph 7.5), and pelleted by centrifugation at 80,000 x g and 5 C for 1 h. Purified virions were resuspended in 1 mm Tris buffer (ph 7.5), and the relative particle concentration was determined by optical density measurements at 260 nm. Iodination of viral membrane proteins. Alkaline carbonate-released virions from approximately 109 OBs were banded on sucrose gradients as described above. The virus bands were collected with a minimum of sucrose solution (1 to 2 ml total volume). For iodination, the following was added in order to the virus suspension: 0.2 ml of 0.1 M Tris buffer (ph 7.5), 75,ul of 10%o glucose, 11 mu of glucose oxidase (Sigma), 11 mu of lactoperoxidase (Calbiochem), and 150,uCi of Na'25I (Amersham Corp.) (modification of Hubbard and Cohn [10]). The mixture was incubated at room temperature for 45 min and then diluted with 2 volumes of 1 mm Tris (ph 7.5) to stop the reaction. The labeled virus was immediately layered over a cushion of 35% (wt/wt) sucrose (1 mm Tris, ph 7.5) and centrifuged at 60,000 x g and 5 C for 1 h. The virus pellet was suspended in 1 mm Tris buffer (ph 7.5), and the optical density at 260 nm was measured to determine the relative particle concentration. Trichloroacetic acid-precipitable "25I-incorporated counts were determined with Biofluor cocktail (New England Nuclear Corp.), which had an approximate 80%o counting efficiency. Nucleocapsid purification. Virus (ca. 800,ug) which was purified from unheated OBs was incubated in 5 ml of a 3% (vol/vol) Nonidet P40 (NP-40) solution in 10 mm Tris (ph 7.8)-i mm EDTA (TE buffer) for 18 h at 37 C. The nucleocapsid solution was layered over a two-step sucrose layer composed of 20% (wt/wt) sucrose (TE buffer plus 2% NP-40) and 50% (wt/wt) sucrose (1 mm Tris buffer, ph 7.5) and centrifuged at 80,000 x g and 5 C for 1 h. Pelleted nucleocapsids were suspended in 1 mm Tris buffer (ph 7.5). Nucleocapsids were negatively stained and examined with an electron microscope. PAGE and autoradiography. Virus samples were dissociated and applied to discontinuous SDS-polyacrylamide gels according to the method of Laemmli (12). Gels (15 by 15 by 0.15 cm) with a 4% stacking gel and an 11% running gel were electrophoresed at a constant 6 W per gel (24). Molecular weights (MWs) of nonradioactively labeled proteins were determined with rabbit muscle phosphorylase b, bovine serum albumin, egg white ovalbumin, bovine erythrocyte carbonic anhydrase, soybean trypsin inhibitor, and bovine milk a-lactalbumin (Pharmacia) as standards. For autoradiographic analyses, 14C-methylated phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, and lysozyme (Amersham) were used as MW standards. For visualizing total viral protein patterns, gels were APPL. ENVIRON. MICROBIOL. stained first with Coomassie brilliant blue R (25) and then destained completely and washed in at least five changes (2 h each) of 50% methanol and treated with the alkaline silver stain as described previously by Wray et al. (26). Gels containing "25I-labeled proteins were dried immediately and exposed to Kodak X- Omat X-ray film at -70 C. Stained gels and autoradiograms were scanned with a Helena Quick Scan R&D densitometer. Characterization of viral DNA by restriction endonuclease analysis. Viral DNA was prepared by incubating gradient-purified virus for 6 h at 37 C in TE buffer plus 1.0% (wt/vol) SDS and proteinase K (100,ug/ml) (3). This was followed by four extractions with TE buffersaturated phenol and dialysis against 0.1 x SSC (1x SSC is 1.5 M NaCl, 0.15 M sodium citrate). All restriction enzyme digests except for HindIII were carried out in a solution containing 33 mm Tris-acetate (ph 7.9), 66 mm potassium acetate, 10 mm magnesium acetate, and 0.5 mm dithiothreitol. The HindIII digest was conducted in Core Buffer (Bethesda Research Laboratories) composed of 50 mm Tris-hydrochloride (ph 8.0), 10 mm MgCl2, and 50 mm NaCI. All digestions were incubated at 37 C for 10 to 18 h in the presence of 100,ug of bovine serum albumin per ml. Restricted DNAs were electrophoresed in 0.65% agarose gel, visualized by ethidium bromide staining, and scanned as previously outlined for PAGE gels. Determination of MWs and relative concentration of DNA restriction enzyme fragments. MW estimates of restriction fragments were determined by the procedure of Southern (22), using the corrected value for the migration of DNA bands in the gel. Lambda DNA restricted with KpnI or Hindlll was used as MW standards. The concentration of each fragment relative to its presence in major or minor molar amounts was determined from the relationship of the area of the peak from the gel scan to the distance migrated in the gel (2). RESULTS Viral structural proteins. A total of 29 viral structural proteins (Fig. 1, lane A, indicated by arrows) were consistently identified in six determinations by silver or Coomassie blue staining of the LdNPV IT isolate after heat inactivation of the alkaline protease associated with the OB preparation. As shown, additional bands were detected by silver staining; however, owing to the inconsistency of their detection, they were not included as structural proteins. Under these conditions the OB matrix protein was determined to have an MW of 30,000. Since the distribution pattern of viral proteins did not agree with reports for the LdNPV HA isolate, the analysis was repeated on virus purified in the presence of active alkaline protease. Under these conditions a total of 28 viral proteins were consistently observed (Fig. 1, lane B), and the protein pattern was similar to those previously published (15, 19, 20, 27). When protease activity was present, four structural viral proteins (vp 95, vp 22, vp 19, and vp 16) were not detected, and four additional protein bands (vp 69, vp

3 VOL. 46, _.* a t t; 6 a 14' 13- * 1i3 FIG. 1. SDS-PAGE analysis of LdNPV proteins. (A) LdNPV structural proteins from protease-inactive OB preparation, silver stained; (B) LdNPV structural proteins from protease-active OB preparation, silver stained; (C) LdNPV 125I-labeled surface envelope proteins from protease-inactive OBs; (D) LdNPV 125- labeled surface envelope proteins from protease-active OBs. Approximate MWs (x103) for proteins are indicated in the margin. The asterisks (*) indicate bands missing or reduced in protease-active preparations, whereas the dots (0) indicate protein bands not found in protease-inactive preparations. 29.5, vp 29, and vp 13) were distinctly reduced in concentration. Three viral proteins (vp 74, vp 25, and vp 18) were only detected in proteaseactive samples. Surface envelope proteins. To identify the viral surface envelope proteins and further define the action of the protease upon the virus, 1251 lactoperoxidase labeling was performed (Fig. 1, lanes C and D). Only 9 surface envelope proteins were consistently observed when the protease was active (lane D), whereas 14 proteins were consistently present when the protease was inactivated (lane C). In the protease-active sample, vp 109, vp 95, vp 59, vp 29, vp 22, vp 16, and vp 13 were never detected, and vp 69 was always greatly reduced in label intensity. Two proteins, GYPSY MOTH NUCLEAR POLYHEDROSIS VIRUS 299 vp 18 and vp 15, were only observed in proteaseactive samples. Nucleocapsid proteins. Viral nucleocapsids were prepared from virions purified from protease-active OBs by treatment with the detergent NP-40 (7, 8, 23, 25). A total of 11 nucleocapsidassociated proteins were consistently identified by silver staining (Fig. 2, lane A). As previously reported by Wood (25), it was not possible to A ie.7s 54 _- 42 m.g*i I _ l1 31 P- l _o 16P-_0 17.B C D E '109 a95 '88 '81 '69 '59 '42 '39 '33 '30 '29 '22 '16 FIG. 2. LdNPV nucleocapsid preparations. (A) Nucleocapsids produced by NP-40 treatment of LdNPV purified from protease-active OBs, silver stained; (B) intact virus (from protease active OBs) after 1" I surface iodination followed by NP-40 treatment; (C) intact virus (from protease-active OBs) after "25I surface iodination without NP-40 treatment; (D) intact virus (protease-active OBs) after 1251 surface iodination followed by NP-40 treatment; the asterisk (*) indicates a single missing band after NP-40 treatment; (E) 125I-labeled intact virus (protease inactive) without NP-40 treatment. Approximate MWs (x 103) for proteins are indicated in the margins. 13

4 300 STILES ET AL. remove the virus envelope by NP-40 treatment if virions from protease-inactivated OBs were used. Detergent treatment of these virions removed only vp 16, as demonstrated by 125i labeling (Fig. 2, lanes D and E). Removal of the viral envelope from proteaseactive preparations was verified by electron microscopic observation (data not shown) and by the fact that NP-40 treatment of 125I-labeled virions eliminated all but six labeled proteins (Fig. 2, lanes B and C). These six remaining proteins may be nucleocapsid proteins which were nonspecifically labeled (9) or may represent a low percentage of naked nucleocapsids present in purified virus preparations. Restriction enzyme analysis of LdNPV IT DNA. The results of the digestion of LdNPV IT DNA with the restriction enzymes EcoRI, BamHI, BglII, and HindIII are presented in Fig. 3 and summarized in Table 1. The presence of a large number of minor molar fragments evident in the restriction enzyme digests shown in Fig. 3 indicated the presence of a wide range of genetic variation within LdNPV IT. These minor bands made it impossible to accurately determine the size of the virus genome. However, the genome size of the major component of this virus population estimated from the major molar restriction enzyme bands was ± 8.9 kildbases (kb), corresponding to an approximate DNA MW of 88 x 106. In Fig. 4 we have identified several of the more dramatic of these differences. A comparison of LdNPV IT and HA viral DNAs restricted with HindIII, BglII, and EcoRI is presented in Fig. 4. These data indicate that although there are some minor differences, these two isolates are quite similar. DISCUSSION The pattern of viral structural proteins for the Ithaca isolate (from protease-active preparations) matched well those of earlier studies (15, 19, 20, 27) that were also conducted with an active protease. Our experiments did demonstrate a greater number of viral proteins due to the higher sensitivity of the silver stain (26) used on the gels. However, when the protease activity was inactivated before OB dissolution, the viral structural protein pattern was distinctly different (Fig. 1). In the absence of protease activity there was an increase in staining intensity of four bands, the appearance of four new protein bands, and the disappearance of three proteins (Fig. 1, lanes A and B). These changes were further illustrated in the 125I labeling experiment (Fig. 1, lanes C and D) in which the surface envelope proteins of an NPV have been identified for the first time. Except for vp 109 and vp 59 (Fig. 1, lane C), all of the iodinated proteins of protease-active virus observed to be APPL. ENVIRON. MICROBIOL. affected were also shown to undergo protease degradation in silver-stained gels (Fig. 1, lane B). Based on the fact that vp 109 and vp 59 were not observed in the nucleocapsid preparations (Fig. 2, lane A), it appears that these two structural proteins undergo a minor degradation in the presence of protease activity. We conclude, therefore, that under these conditions the primary site of action of the protease is on the A B C D E F FIG. 3. Ethidium bromide-stained 0.65% agarose gel electrophoresis of LdNPV IT DNA after digestion with EcoRI (B), BamHI (C), BgIII (D), or HindIII (E). The numbers indicate kb of lambda DNA digested with KpnI (A) and HindlIl (F).

5 VOL. 46, 1983 GYPSY MOTH NUCLEAR POLYHEDROSIS VIRUS 301 TABLE 1. Summary of restriction endonuclease analysis of LdNPV IT DNA' EcoRI BamHI BglII HindIII Fragmentb KbC Fragment Kb Fragment Kb Fragment Kb A 36.6 A 25.0 A 29.6 A 25.6 B C B 22.8 B C 21.9 C B D 14.9 DE D F 6.6 C 12.3 E 13.2 E 11.6 G 3.2 D 11.5 F H 1.9 E F 10.2 I 1.0 F 9.9 G G H I 7.4 H J 6.3 I K G,H J 2.0 I,J 3.3 L 4.9 KL 1.8 K,L M 1.5 M 3.8 N 1.2 N a The genome size was obtained by the summation of restriction enzyme fragments determined to be major components of the virus genome. For EcoRI, the genome size (in Kb) was 121.5; for BamHI, 138.6; for BglII, 138.3; and for HindIII, b Restriction enzyme fragments determined to be major components of the LdNPV viral genome are identified by a letter whereas minor molar components are identified numerically. c The size of each restriction fragment was determined from scans of agarose gels following the procedure of Southern (22). Lambda DNA restricted with KpnI, HindIII, or SstI served as standards. virion envelope, specifically affecting proteins which are exposed to some degree on the surface of the virion envelope. These data corroborate the conclusion of Wood (25) that if protease activity is present in an NPV preparation it must be inactivated before conducting studies of the viral structural proteins. Although a total of 14 bands was consistently observed in all surface iodination experiments (Fig. 1, lane C), this number probably represents a minimum, as 3 additional bands were occasionally resolved, though not frequently enough to warrant inclusion in our findings. The large amount of vp 30, the OB matrix protein, still present on the surface of virions (Fig. 1, protease-inactive preparation) probably interfered with the labeling of some envelope proteins, causing their erratic appearance. When viral nucleocapsids were prepared by NP-40 detergent treatment (7, 8, 23, 25), the majority of the structural proteins were undetected (Fig. 2, lane B). A total of 11 nucleocapsid proteins were consistently detected with silver staining (Fig. 2, lane A). The vp 31 nucleocapsid protein was not identified in whole virus preparations, probably because it was masked on the gels by the abundant vp 30 polyhedral matrix protein. We noted that four nucleocapsid proteins (vp 42, vp 39, vp 33, and vp 16) were also labeled in surface iodination experiments (Fig. 2, lane E, protease-inactive virus). These may represent pairs of comigrating nucleocapsid and envelope proteins or contaminating envelope proteins in the nucleocapsid preparation. In addition, the 18,000-MW nucleocapsid protein was observed in protease-active but not in proteaseinactive preparations (Fig. 1, lanes A and B) which indicated that it would be preferable to isolate nucleocapsids from protease-inactive OB preparations. As reported by Wood (25), NP-40 treatment does not remove the viral envelope if the virions have been isolated in the absence of an active alkaline protease, i.e., tissue culturederived or heat-treated insect-derived OBs. Without protease degradation, NP-40 was capable of removing only the vp 16 envelope protein (Fig. 2, lane D). We used restriction enzyme analysis to identify and characterize the NPV responsible for the epizootic observed near Ithaca, N.Y. On the basis of the previously reported restriction enzyme digests of LdNPV DNA with EcoRI (21) and HindIII (17), we tentatively identified the Ithaca isolate as LdNPV. To verify this, digests

6 302 STILES ET AL. A B C Hi Hi Hi FIG. 4. Comparison of LdNPV IT (I) and HA (H) DNAs digested with HindIII (A), BgIll, (B), or EcoRI (C). The white dots indicate differences in the restriction enzyme fragment patterns of these two isolates. of viral DNA from a known gypsy moth virus isolate (LdNPV HA) were performed. As expected, both virus isolates were composed of a large number of genetic variants as evidenced by the presence of submolar restriction enzyme fragments (Fig. 4). Comparable genetic variation has been reported for wild isolates of several baculoviruses (6, 11, 13, 18, 20). Although the restriction enzyme patterns of HA and IT viral DNA were quite similar, some minor differences were apparent. In Fig. 4 we have identified several of the more dramatic of these differences. For example, DNA from the IT isolate has three fragments, EcoRI-E (11.6 kb), BglII-B (17.6 kb), and HindIII-I (2.7 kb), which are major components of this isolate but minor molar components of the HA isolate. Also, DNA from the HA isolate has a major molar BglII fragment of 13.1 kb which is a minor molar APPL. ENVIRON. MICROBIOL. component of IT viral DNA (BglII-4). These differences reflect different levels of particular genetic variants within each wild population and can be used to distinguish between the HA and the IT isolate. It appears that restriction enzyme analysis would be an interesting and useful tool for following and identifying changes in the virus gene pool after introduction of a well-characterized virus isolate into an insect population. ACKNOWLEDGMENTS We would like to thank M. Verbanic and K. Carrol for their help in field-collecting diseased larvae and their technical assistance in the laboratory. Major support for this project was provided by the Jessie Smith Noyes Foundation and by the Rockefeller Foundation grant no LITERATURE CITED 1. Brown, S. E., F. S. Kaczmarek, N. R. Dubois, R. T. Zerlo, J. Holleman, J. P. Breillat, and H. M. Mazone Comparative properties of the inclusion body proteins of the nucleopolyhedrosis viruses of Neodiprion sertifer and Lymantria dispar. Arch. Virol. 59: Burand, J. P., and M. D. Summers Alteration of Autographa californica nuclear polyhedrosis virus DNA upon serial passage in cell culture. Virology 119: Burand, J. P., M. D. Summers, and G. E. Snith Transfection with baculovirus DNA. Virology 101: Crozier, G., and L. Crozier Evaluation du poids moleculaire de la proteine des corps d'inclusion de divers baculovirus d'insectes. Arch. Virol. 55: Faulkner, P Baculovirus, p In E. W. Davidson (ed.), Pathogenesis of invertebrate microbial diseases. Allanheld, Osmun, New York. 6. Gettig, R. R., and W. J. McCarthy Genotypic variation among wild isolates of Heliothis spp. nuclear polyhedrosis viruses from different geographical regions. Virology 117: Harrap, K. A., and J. F. Longworth An evaluation of purification methods for baculoviruses. J. Invertebr. Pathol. 24: Harrap, K. A., C. C. Payne, and J. S. Robertson The properties of three baculoviruses from closely related hosts. Virology 79: Hubbard, A. L., and Z. A. Cohn The enzymatic iodination of the red cell membrane. J. Cell Biol. 55: Hubbard. A. L., and Z. A. Cohn Specific labels for cell surfaces, p In A. H. Maddy (ed.), Biochemical analysis of membranes. John Wiley & Sons, Inc., New York. 11. Knell, J. D., and M. D. Summers Investigation of genetic heterogeneity in wild isolates of Spodopterafrugiperda nuclear polyhedrosis virus by restriction endonuclease analysis of plaque-purified variants. Virology 112: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Lee, H. H., and L. K. Miller Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. J. Virol. 27: Lewis, F. B., W. D. Rollinson, and W. G. Yendol Gypsy moth nuclear polyhedrosis virus, p In C. C. Doane and M. L. McManus (ed.), The gypsy moth: research towards integrated pest management. U.S. Department of Agriculture Technical Bulletin Maskos, C. B., and H. G. Miltenburger SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra

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