Vol. 66, No. 5
JOURNAL OF VIROLOGY, May 1992, p. 3168-3171
0022-538X/92/053168-04$02.00/0 Copyright © 1992, American Society for Microbiology
Evidence for Zinc Binding by Two Structural Proteins of Plodia interpunctella Granulosis Virust C. JOEL FUNK AND RICHARD A. CONSIGLI*
Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan, Kansas 66506 Received 22 August 1991/Accepted 17 January 1992
Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpuncteUla granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2" specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability. treatment with a chelator (EDTA) and a reducing agent
The occluded virion (capsule) of Plodia interpunctella granulosis virus (PiGV) contains approximately 15 structural proteins which are assembled into four components (25). The occlusion body of this baculovirus surrounds the enveloped nucleocapsid (ENC) or virion. The major occlusion body protein is granulin. A lipid-containing envelope encircles the rod-shaped nucleocapsid (NC), which consists of a capsid shell enclosing a DNA-protein complex at the core. The protein associated with the viral DNA is a very basic, arginine-rich, DNA-binding polypeptide designated VP12 (24, 32). Uncoating of this complex virus in vivo is a multistep process (5). Granulin is dissociated from the virus in the alkaline environment of the insect midgut in conjunction with the alkaline protease associated with the granulin (25). The released ENC then fuses with the midgut cells, allowing the NC to move to the nucleus and align with nuclear pores, delivering the DNA-protein core into the nucleus (20). Workers in our laboratory previously demonstrated that in vitro treatment of NCs with a chelator and a reducing agent caused the NCs to release their DNA-protein complex (24). At that time, we hypothesized that this dissociation of the NC structure was due to a possible virion-associated cation (24, 31). In this report, we provide evidence that zinc is associated with the virus and that two PiGV structural proteins are able to bind zinc, and we propose that this metal ion is important for virus stability. The PiGV capsules were produced in vivo in a laboratory colony of P. interpunctella and purified as described previously (22, 25). Granulin and NCs were then isolated from purified capsules as described previously (23, 25). Briefly, ENCs were isolated by treating capsules with 0.1 M Na2CO3 and 0.1 M NaCl (pH 10.5) for 15 min to solubilize the granulin. Then ENCs were purified by centrifugation on a 30 to 70% glycerol gradient (in 0.01 M Tris [pH 7.5]). The granulin remained as a band at the top of the gradient. NCs were purified by treating ENCs with 1% Nonidet P-40 and centrifugation on a 30 to 70% glycerol gradient (in 0.01 M Tris [pH 8.5]). Tweeten et al. (24) previously demonstrated that a DNAprotein complex was released from the NC in vitro by
*
(dithiothreitol [DTT]). When purified NCs were incubated with 0.01 M EDTA in the absence of DTT, the NCs showed evidence of the initial stage of DNA-protein complex release, partial breakdown at the ends of the capsid (24) (compare Fig. la and b). Optimum breakdown of the capsid and release of the DNA-protein complex was achieved with the addition of DTT (Fig. lc). The DNA released from NCs was found to be associated with protein (Fig. Id). Further analysis of the DNA-protein complex on glycerol gradients allowed the isolation of this complex (unpublished results). By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was determined that the major protein component of the complex was VP12, along with a minor amount of VP31. This confirms earlier results that VP12 is a DNA-binding protein and that it forms a major component of the DNA-protein complex (24, 32). The effect of EDTA on NC stability suggested that a cation is intimately associated with the NC and that the initial stage of in vitro uncoating is mediated by a chelator and not by a reducing agent. However, the release of the DNA-protein complex from the NC and the breakdown of the capsid is facilitated by the reducing agent (Fig. lc). Preliminary studies with X-ray fluorometry and atomic emission indicated that Zn2+, Cu2+, and/or Mg2+ may be associated with the virus. Further analysis for the presence of these elements was made by graphite furnace atomic absorption on purified NC, granulin, and control samples. Control samples were prepared by a mock purification procedure in which the same reagents and procedures were used as for virus purification. The samples were extensively dialyzed against 0.01 M Tris (pH 7.5) before analysis. Virtually no Mg2+ was found in the samples above control levels. Although both Cu2+ and Zn2+ were detected above control levels, the level of Zn2+ was much higher than that of Cu2+, and Zn2+ was found in the highest amount in both NC and granulin preparations (Table 1). In the NC sample, Zn2+ was 116 and 61 times more abundant than Cu2' and Mg2+, respectively, whereas in the granulin sample, Zn2+ was 23 and 14 times more abundant than Cu2+ and Mg2+, respectively. Since Zn2+ was the predominant cation associated with the virus, an additional investigation was performed to determine whether zinc was interacting with specific viral proteins by using a Zn2+ blotting technique. The Zn2+ binding assay was developed under conditions which permit the detection of authentic Zn2+ binding proteins (18). The
Corresponding author.
t Contribution 92-120-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kans.
3168
VOL. 66, 1992
.4s a
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3169
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A 2
3
4
2
3
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-VP31 29-
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FIG. 1. Effect of various reagents on virion stability as observed by electron microscopy. (a) Purified NC preparation. (b) NCs incubated in 0.01 M EDTA and 2% Nonidet P-40 in 0.01 M Tris (pH 8.5) for 1 h. The arrows indicate points of NC dissociation. (c) NCs incubated in 2% Nonidet P-40-0.01 M EDTA-1 M NaCI-0.03 M DTT in 0.01 M Tris (pH 8.5) for 1 h. (d) The DNA component of the NCs following release by the same reagents as in panel c except without DTY, following prolonged incubation (12 h). Note the protein attached to the strands of DNA. Samples were stained with uranyl acetate. Bars: a, b, c, 200 nm; d, 2 pum.
gradient-purified NC and PiGV capsule proteins were separated by SDS-PAGE with a 12.5% acrylamide gel and 0.2% bisacrylamide cross-linker. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane sheet, and the membranes were treated overnight at 37°C in phosphate-buffered saline containing 0.1% (wt/vol) sodium azide to promote renaturation of the transferred proteins (3). The nitrocellulose was washed in metal-binding buffer (MBB; 100 mM Tris [pH 6.8], 50 mM NaCl) and then probed with 5 to 10 ,uCi of 65ZnC12 (New England Nuclear Corp., Boston, Mass.) per lane in 10 to 40 ml of MBB for 1 TABLE 1. Atomic absorption analysis of Cu2', in PiGV components
Zn2+, and Mg2+
Concn' (ngip.g of viral protein)
Virus component
Cu22+
Zn22
Mg22
NC NC control" Granulin Granulin control"
0.058 0.0018 0.110 0.00004
6.7 0.084 2.5 0.021
0.110 0.082 0.140 0.160
a Determined with a Varian AA875 atomic absorption spectrophotometer fitted with a Varian GT95 graphite tube analyzer. h Control samples were prepared with the same reagents and gradient conditions used to isolate the viral components. Both virus samples and control samples were dialyzed extensively against 0.01 M Tris (pH 7.5) before analysis.
FIG. 2. Zinc-65 binding analysis of purified PiGV structural proteins following separation by SDS-PAGE and electrophoretic transfer to nitrocellulose. (A) Lanes 1 and 3, molecular weight marker proteins: bovine serum albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,000), alpha-lactalbumin (14,000); lanes 2 and 4, NC proteins (35 p.g of protein). Lanes 1 and 2, amido black stained; lanes 3 and 4, autoradiogram of 65ZnC12-probed nitrocellulose sheet shown in lanes 1 and 2, respectively. (B) Lanes 1 and 3, molecular weight marker proteins (same as in panel A); lanes 2 and 4, occluded PiGV proteins (30 ,ug of protein). Lanes 1 and 2, amido black stained; lanes 3 and 4, autoradiogram of 15ZnCl2-probed nitrocellulose sheet shown in lanes 1 and 2, respectively. G on the right indicates granulin.
h. The nitrocellulose sheet was washed three times with MBB buffer for a total of 15 min, then dried, wrapped in plastic wrap, and exposed to XAR film (Eastman Kodak Co., Rochester, N.Y.), and '5Zn2' binding was determined by autoradiography. Proteins were stained with 0.1% amido black made in 45% methanol-10% acetic acid and destained with 45% methanol-10% acetic acid. The autoradiogram revealed that 15Zn2+ bound to VP12 protein (Fig. 2A, lane 4) and to granulin (Fig. 2B, lane 4), but not to the other viral proteins (compare with the amido black-stained blots, Fig. 2A, lane 2, and Fig. 2B, lane 2). The molecular weight standards (Dalton Mark VII-L; Sigma Chemical Co., St. Louis, Mo.) include a zinc-binding protein, carbonic anhydrase (Mr 29,000), which serves as a positive control on the Zn2+ blot (18) (Fig. 2A, lane 3, and Fig. 2B, lane 3). We also determined that increased amounts of these viral proteins resulted in increased 15Zn2+ binding by these proteins (data not shown). To determine the specificity of cation binding, both NCs and PiGV capsules were subjected to a similar Ca2+ binding assay with 45Ca2+ (12, 14). Neither VP12 nor granulin bound Ca2+ in this assay (data not shown). A molar ratio of Zn2+ to VP12 was calculated from the data obtained with atomic absorption (Table 1) and the percentage of VP12 found in a Coomassie-stained, SDSPAGE-separated NC preparation. Since all the Zn2+ de-
tected in the NC sample was bound to VP12, the molar ratio was
calculated to be 3.24. The molar ratio of Zn2+ to
granulin was calculated to be 1.07, based on a molecular weight of 28,000 for granulin. A number of amino acids could potentially act as a zinc ligand in proteins, but only four amino acid side chains have so far been found to participate in Zn2+ binding (28). In descending order of frequency, they are the ring N atoms of
3170
NOTES
J . VlIROL.
TABLE 2. Amino acid composition of baculovirus basic proteins Content (mol%)
Amino acid PiGVa
Lys His Arg Asp Thr Ser
Glu Pro Cys Gly Ala Val Met Ile Leu Tyr Phe
1.3 12.5 26.5 1.0
16.3 0.4 4.1 0.3 1.7 1.3 16.9
13.3 0.1 5.1 0.4
AcNPVb
TnNPV'
BmNPVd
OpNPVe
40.6 10.9 17.2
45.1 2.0 7.8 13.7
3.1
3.9
7.8 3.1 1.6
7.8 3.9 2.0 2.0
0.8 40.0 12.6 18.1
4.0 9.0
2.0 2.0
31.0 2.1 11.2 16.6 1.9 4.3 1.9 12.9 2.1 2.8 1.0 0.8
12.6
10.4
1.6 3.1 10.9
11.8
a Data from Tweeten et al. (24). b Data for Autographa califomica nuclear polyhedrosis virus (AcNPV) from Wilson et al. (33). ' Data for Trichoplusia ni nuclear polyhedrosis virus (TnNPV) from Kelly et al. (11). d Data for Bombyx mon nuclear polyhedrosis virus (BmNPV) from Maeda et al. (13). e Data for Orgyia pseudotsugata nuclear polyhedrosis virus (OpNPV) from Russell et al. (16). f-, not detected.
His, the S atoms of Cys, and the carboxylate side chains of Glu and Asp. The amino acid composition of VP12 was previously determined in our laboratory (24) from acid-urea gel-isolated VP12 (Table 2). Under these conditions, the basic VP12 is the only protein that migrates into the gel and is totally free of other PiGV proteins. The amino acid composition of the isolated VP12 was found to contain a high percentage of one of the Zn2+-binding amino acids, namely His. Basic proteins have been identified in a number of other baculoviruses, and the amino acid compositions of several of these are compared in Table 2. It is striking that none of these other proteins have a high percentage of amino acid residues which bind Zn2+. Although there is evidence for cation involvement with other baculoviruses, since EDTA is used to dissociate NCs (21), perhaps other amino acids are used for zinc coordination or a different metal cation stabilizes other baculovirus NCs. It is also interesting that PiGV contains a high percentage of hydrophobic amino acids (16.9% Val, 13.3% Ile). Recently it has been shown that metal ions generally bind in a hydrophilic region which is surrounded by a hydrophobic shell (34). Perhaps these hydrophobic amino acids are structured to facilitate the metal-binding domain. Although the amino acid sequence of PiGV granulin is not known, the granulins of two other granulosis viruses have been sequenced (15). While the proteins do not contain the exact amino acid motif found in other zinc-binding proteins (26, 27), they do have a fairly high percentage of Asp and Glu residues, as well as a smaller percentage of His and Cys residues (15). One region in each granulin has a cluster of His and Cys residues which could potentially bind Zn2+. Starting at amino acid number 12, the consensus sequence is His-X4Cys-X5-His-X12-His, where X equals any amino acid (15). It has been demonstrated for a number of viral systems that divalent cations can play an integral role in maintaining the structural conformation of a viral protein and/or the entire virion. Calcium has been shown to be an important component of several plant viruses and polyomavirus (1, 4, 6, 9). Calcium binding by polyomavirus has been localized to
a specific domain of the major capsid protein VP1 (12). Zinc has been found to interact with reovirus (17), tobacco streak virus (19), and retroviral gag protein products (7, 18). In addition, a number of viral DNA-binding proteins have been demonstrated or predicted to bind zinc (2, 8). The presence of Zn2+ in PiGV and the ability of granulin and VP12 to bind zinc indicate a biological role for this metal cation. We propose that zinc interaction with VP12 is important in maintaining NC stability and that chelation of this cation may be a key event during NC uncoating in vivo. After entry into the host cell, the NC is transported to the nuclear membrane and becomes associated end-on with the nuclear pore. Electron micrographs indicate that the DNAprotein complex is inserted into the nucleus while the capsid remains outside (20). In order for the viral genome to be released into the nucleus, there must be a dissociation of the NC at the terminus. Since VP12 is found in the core of the NC, we suggest that in order for the chelator to initiate NC uncoating, the VP12-associated Zn2+ must be accessible to the chelator at the ends of the capsid. In light of our present findings, we propose that the nuclear membrane may have the ability to remove Zn2+ from the NC, possibly through the action of a zinc-binding protein(s). The initial dissociation is at the termini, as indicated by arrows in Fig. lb, showing subterminal openings in the NC treated with chelator and in the absence of reducing agent. Complete release of the DNA-protein complex is facilitated by a reducing agent. If nuclear microenvironments exist that provide the necessary biological reducing conditions (e.g., glutathione, cysteine), the proposed criteria for uncoating would be fulfilled. Under these conditions, the capsid terminus would be dissociated, allowing the DNA-protein core to be released into the nucleus to initiate the replication cycle. These uncoating events are probably simultaneous with the activation of an NC-associated kinase and phosphorylation of VP12, which is also an important event in NC uncoating (31, 32). It has previously been shown that divalent cations stabilize the oligomeric association of a granulin-related protein, polyhedrin, in the nuclear polyhedrosis virus. The solubili-
VOL. 66, 1992
zation of occlusion bodies is reduced by preincubation of the occluded virions with ZnCl2 (30). Therefore, Zn2+ may play a similar role in the occlusion body of PiGV. It should also be noted that there have been reports in the literature of possible Zn2+ nutritional requirements by baculoviruses. Zinc has been found to enhance baculovirus infections both in vivo and in vitro (10, 29). We report that Zn2+ is present in PiGV and that two structural proteins were found to bind Zn2+. We propose that this cation may have an important biological function for virion stability. It is also possible that Zn2+ plays a significant role in uncoating and assembly events of the PiGV life cycle. This investigation was supported by the Wesley Foundation of Wichita, Kans., and by NASA grants NAGW-1197 and NAGW2328. C.J.F. is a predoctoral fellow of the National Cancer Institute training grant CA09418. We thank Steve Hughes and Frank Padula of the Kansas State University Analytical Laboratory and LaDonna Grenz and Viola
Hill for their technical assistance. We also thank William Mc-
Gaughey and the USDA Grain Marketing Research Laboratory for use
of their facilities.
ADDENDUM IN PROOF Since this article was submitted, Bess et al. (J. W. Bess, Jr., P. J. Powell, H. J. Issaq, L. J. Schumack, M. K. Grimes, L. E. Henderson, and L. 0. Arthur, J. Virol. 66: 840-847, 1992) have described the association of Zn2+ with purified virions of human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses. REFERENCES 1. Abdel-Meguid, S. S., T. Yamane, K. Fukuyama, and M. G. Rossmann. 1981. The location of calcium ions in southern bean mosaic virus. Virology 114:81-85. 2. Berg, J. 1986. Potential metal-binding domains in nucleic acid binding proteins. Science 232:485-487. 3. Birk, H., and H. Koepsell. 1987. Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: renaturation of antigenic sites and reduction of nonspecific antibody binding. Anal. Biochem. 164:12-22. 4. Brady, J. N., V. D. Winston, and R. A. Consigli. 1977. Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J. Virol. 23:717-724. 5. Consigli, R. A., D. L. Russell, and M. E. Wilson. 1986. The biochemistry and molecular biology of the granulosis virus that infects Plodia interpunctella. Curr. Top. Microbiol. Immunol. 131:69-101. 6. Durham, A. C. H., D. A. Hendry, and M. B. Von Wechmar. 1977. Does calcium ion binding control plant virus disassembly? 7.
Virology 77:524-533. Green, L. M., and J. M. Berg. 1990. Retroviral nucleocapsid protein-metal ion interactions: folding and sequence variants.
Proc. Natl. Acad. Sci. USA 87:6403-6407. 8. Gupte, S. S., J. W. Olson, and W. T. Ruyechan. 1991. The major herpes simplex virus type-1 DNA-binding protein is a zinc metalloprotein. J. Biol. Chem. 266:11413-11416. 9. Hogle, J., T. Kirchhausen, and S. C. Harrison. 1983. Divalent cation sites in tomato bushy stunt virus. J. Mol. Biol. 171:95-100. 10. Karabash, Y. A., and A. Y. Karpov. 1973. Aktyvatsiya latentnoho virusu yadernoho polidrozu v guseni kil'chasroho shovkopryada Malacosoma neustna L. [Activation of latent nuclear polyhedrosis virus in Malacosoma neustna L. caterpillars.] Mikrobiol. Zh. (Kiev) 35:781-782. 11. Kelly, D. C., D. A. Brown, M. D. Ayers, C. J. Allen, and I. 0. Walker. 1983. Properties of the major nucleocapsid protein of Heliothis zea singly enveloped nuclear polyhedrosis virus. J. Gen. Virol. 64:399-408. 12. Ludlow, J. W., and R. A. Consigli. 1987. Localization of calcium
NOTES
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on the polyomavirus VP1 capsid protein. J. Virol. 61:2934-2937. 13. Maeda, S., S. G. Kamita, and H. Kataoka. 1991. The basic DNA-binding protein of Bombyx mon nuclear polyhedrosis virus: the existence of an additional arginine repeat. Virology 180:807-810. 14. Maruyama, K., T. Mikawa, and S. Ebaski. 1984. Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biochem. 95:511-519. 15. Rohrmann, G. F. 1986. Polyhedrin structure. J. Gen. Virol. 67:1499-1513. 16. Russell, R. L. Q., and G. F. Rohrmann. 1990. The p6.5 gene region of a nuclear polyhedrosis virus of Orgyia pseudotsugata: DNA sequence and transcriptional analysis of four late genes. J. Gen. Virol. 71:551-560. 17. Schiff, L. A., M. L. Nibert, M. S. Co, E. G. Brown, and B. N. Fields. 1988. Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein a3. Mol. Cell. Biol. 8:273-283. 18. Schiff, L. A., M. L. Nibert, and B. N. Fields. 1988. Characterization of a zinc blotting technique: evidence that a retroviral gag protein binds zinc. Proc. Natl. Acad. Sci. USA 85:4195-4199. 19. Sehnke, P. C., A. M. Mason, S. J. Hood, R. M. Lister, and J. E. Johnson. 1989. A "zinc-finger"-type binding domain in tobacco streak virus coat protein. Virology 168:48-56. 20. Summers, M. D. 1971. Electron microscopic observations on granulosis virus entry, uncoating and replication processes during infection of the midgut cells of Tnchoplusia ni. J. Ultrastruct. Res. 35:606-625. 21. Summers, M. D., and G. E. Smith. 1978. Baculovirus structural polypeptides. Virology 84:390-402. 22. Tweeten, K. A., L. A. Bulla, Jr., and R. A. Consigli. 1977. Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth Plodia interpunctella. Appl. Environ. Microbiol. 34:320-327. 23. Tweeten, K. A., L. A. Bulla, Jr., and R. A. Consigli. 1978. Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella. J. Virol. 26:702-711. 24. Tweeten, K. A., L. A. Bulla, Jr., and R. A. Consigli. 1980. Characterization of an extremely basic protein derived from granulosis virus nucleocapsids. J. Virol. 33:866-876. 25. Tweeten, K. A., L. A. Bulla, Jr., and R. A. Consigli. 1980. Structural polypeptides of the granulosis virus of Plodia interpunctella. J. Virol. 33:877-886. 26. Vallee, B. L., and D. S. Auld. 1990. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29:5647-5659. 27. Vallee, B. L., J. E. Coleman, and D. S. Auld. 1991. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains. Proc. Natl. Acad. Sci. USA 88:999-1003. 28. Vallee, B. L., and A. Galdes. 1984. The metallobiochemistry of zinc enzymes. Adv. Enzymol. Relat. Areas Mol. Biol. 56:283-430. 29. Weiss, S. A., G. C. Smith, J. L. Vaughn, E. M. Dougherty, and G. J. Tompkins. 1982. Effect of aluminum chloride and zinc sulfate on Autographa califomica nuclear polyhedrosis virus (AcNPV) replication in cell culture. In Vitro 18:937-944. 30. Whitt, M. A., and J. S. Manning. 1988. Stabilization of the Autographa californica nuclear polyhedrosis virus occlusion body matrix by zinc chloride. J. Invertebr. Pathol. 51:278-280. 31. Wilson, M. E., and R. A. Consigli. 1985. Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella. Virology 143: 516-525. 32. Wilson, M. E., and R. A. Consigli. 1985. Functions of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella. Virology 143:526-535. 33. Wilson, M. E., T. H. Mainprize, P. D. Friesen, and L. K. Miller. 1987. Location, transcription, and sequence of a baculovirus gene encoding a small arginine-rich polypeptide. J. Virol. 61: 661-666. 34. Yamashita, M. M., L. Wesson, G. Eisenman, and D. Eisenberg. 1990. Where metal ions bind in proteins. Proc. Natl. Acad. Sci. USA 87:5648-5652.
JOURNAL OF VIROLOGY, May 1992, p. 3168-3171
0022-538X/92/053168-04$02.00/0 Copyright © 1992, American Society for Microbiology
Evidence for Zinc Binding by Two Structural Proteins of Plodia interpunctella Granulosis Virust C. JOEL FUNK AND RICHARD A. CONSIGLI*
Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan, Kansas 66506 Received 22 August 1991/Accepted 17 January 1992
Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpuncteUla granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2" specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability. treatment with a chelator (EDTA) and a reducing agent
The occluded virion (capsule) of Plodia interpunctella granulosis virus (PiGV) contains approximately 15 structural proteins which are assembled into four components (25). The occlusion body of this baculovirus surrounds the enveloped nucleocapsid (ENC) or virion. The major occlusion body protein is granulin. A lipid-containing envelope encircles the rod-shaped nucleocapsid (NC), which consists of a capsid shell enclosing a DNA-protein complex at the core. The protein associated with the viral DNA is a very basic, arginine-rich, DNA-binding polypeptide designated VP12 (24, 32). Uncoating of this complex virus in vivo is a multistep process (5). Granulin is dissociated from the virus in the alkaline environment of the insect midgut in conjunction with the alkaline protease associated with the granulin (25). The released ENC then fuses with the midgut cells, allowing the NC to move to the nucleus and align with nuclear pores, delivering the DNA-protein core into the nucleus (20). Workers in our laboratory previously demonstrated that in vitro treatment of NCs with a chelator and a reducing agent caused the NCs to release their DNA-protein complex (24). At that time, we hypothesized that this dissociation of the NC structure was due to a possible virion-associated cation (24, 31). In this report, we provide evidence that zinc is associated with the virus and that two PiGV structural proteins are able to bind zinc, and we propose that this metal ion is important for virus stability. The PiGV capsules were produced in vivo in a laboratory colony of P. interpunctella and purified as described previously (22, 25). Granulin and NCs were then isolated from purified capsules as described previously (23, 25). Briefly, ENCs were isolated by treating capsules with 0.1 M Na2CO3 and 0.1 M NaCl (pH 10.5) for 15 min to solubilize the granulin. Then ENCs were purified by centrifugation on a 30 to 70% glycerol gradient (in 0.01 M Tris [pH 7.5]). The granulin remained as a band at the top of the gradient. NCs were purified by treating ENCs with 1% Nonidet P-40 and centrifugation on a 30 to 70% glycerol gradient (in 0.01 M Tris [pH 8.5]). Tweeten et al. (24) previously demonstrated that a DNAprotein complex was released from the NC in vitro by
*
(dithiothreitol [DTT]). When purified NCs were incubated with 0.01 M EDTA in the absence of DTT, the NCs showed evidence of the initial stage of DNA-protein complex release, partial breakdown at the ends of the capsid (24) (compare Fig. la and b). Optimum breakdown of the capsid and release of the DNA-protein complex was achieved with the addition of DTT (Fig. lc). The DNA released from NCs was found to be associated with protein (Fig. Id). Further analysis of the DNA-protein complex on glycerol gradients allowed the isolation of this complex (unpublished results). By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was determined that the major protein component of the complex was VP12, along with a minor amount of VP31. This confirms earlier results that VP12 is a DNA-binding protein and that it forms a major component of the DNA-protein complex (24, 32). The effect of EDTA on NC stability suggested that a cation is intimately associated with the NC and that the initial stage of in vitro uncoating is mediated by a chelator and not by a reducing agent. However, the release of the DNA-protein complex from the NC and the breakdown of the capsid is facilitated by the reducing agent (Fig. lc). Preliminary studies with X-ray fluorometry and atomic emission indicated that Zn2+, Cu2+, and/or Mg2+ may be associated with the virus. Further analysis for the presence of these elements was made by graphite furnace atomic absorption on purified NC, granulin, and control samples. Control samples were prepared by a mock purification procedure in which the same reagents and procedures were used as for virus purification. The samples were extensively dialyzed against 0.01 M Tris (pH 7.5) before analysis. Virtually no Mg2+ was found in the samples above control levels. Although both Cu2+ and Zn2+ were detected above control levels, the level of Zn2+ was much higher than that of Cu2+, and Zn2+ was found in the highest amount in both NC and granulin preparations (Table 1). In the NC sample, Zn2+ was 116 and 61 times more abundant than Cu2' and Mg2+, respectively, whereas in the granulin sample, Zn2+ was 23 and 14 times more abundant than Cu2+ and Mg2+, respectively. Since Zn2+ was the predominant cation associated with the virus, an additional investigation was performed to determine whether zinc was interacting with specific viral proteins by using a Zn2+ blotting technique. The Zn2+ binding assay was developed under conditions which permit the detection of authentic Zn2+ binding proteins (18). The
Corresponding author.
t Contribution 92-120-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kans.
3168
VOL. 66, 1992
.4s a
.~' ~. ~ -
IO0
NOTES
3169
B
A 2
3
4
2
3
4
t
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-VP 31
-VP31 29-
-G
..
24 -
.,0'
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b
20 -
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-VP12
4-
14-
La %~~~~~~~~~~~~~~~~~~~-
_ii;
_ _~
.44 :,'
*..
..e
I
FIG. 1. Effect of various reagents on virion stability as observed by electron microscopy. (a) Purified NC preparation. (b) NCs incubated in 0.01 M EDTA and 2% Nonidet P-40 in 0.01 M Tris (pH 8.5) for 1 h. The arrows indicate points of NC dissociation. (c) NCs incubated in 2% Nonidet P-40-0.01 M EDTA-1 M NaCI-0.03 M DTT in 0.01 M Tris (pH 8.5) for 1 h. (d) The DNA component of the NCs following release by the same reagents as in panel c except without DTY, following prolonged incubation (12 h). Note the protein attached to the strands of DNA. Samples were stained with uranyl acetate. Bars: a, b, c, 200 nm; d, 2 pum.
gradient-purified NC and PiGV capsule proteins were separated by SDS-PAGE with a 12.5% acrylamide gel and 0.2% bisacrylamide cross-linker. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane sheet, and the membranes were treated overnight at 37°C in phosphate-buffered saline containing 0.1% (wt/vol) sodium azide to promote renaturation of the transferred proteins (3). The nitrocellulose was washed in metal-binding buffer (MBB; 100 mM Tris [pH 6.8], 50 mM NaCl) and then probed with 5 to 10 ,uCi of 65ZnC12 (New England Nuclear Corp., Boston, Mass.) per lane in 10 to 40 ml of MBB for 1 TABLE 1. Atomic absorption analysis of Cu2', in PiGV components
Zn2+, and Mg2+
Concn' (ngip.g of viral protein)
Virus component
Cu22+
Zn22
Mg22
NC NC control" Granulin Granulin control"
0.058 0.0018 0.110 0.00004
6.7 0.084 2.5 0.021
0.110 0.082 0.140 0.160
a Determined with a Varian AA875 atomic absorption spectrophotometer fitted with a Varian GT95 graphite tube analyzer. h Control samples were prepared with the same reagents and gradient conditions used to isolate the viral components. Both virus samples and control samples were dialyzed extensively against 0.01 M Tris (pH 7.5) before analysis.
FIG. 2. Zinc-65 binding analysis of purified PiGV structural proteins following separation by SDS-PAGE and electrophoretic transfer to nitrocellulose. (A) Lanes 1 and 3, molecular weight marker proteins: bovine serum albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,000), alpha-lactalbumin (14,000); lanes 2 and 4, NC proteins (35 p.g of protein). Lanes 1 and 2, amido black stained; lanes 3 and 4, autoradiogram of 65ZnC12-probed nitrocellulose sheet shown in lanes 1 and 2, respectively. (B) Lanes 1 and 3, molecular weight marker proteins (same as in panel A); lanes 2 and 4, occluded PiGV proteins (30 ,ug of protein). Lanes 1 and 2, amido black stained; lanes 3 and 4, autoradiogram of 15ZnCl2-probed nitrocellulose sheet shown in lanes 1 and 2, respectively. G on the right indicates granulin.
h. The nitrocellulose sheet was washed three times with MBB buffer for a total of 15 min, then dried, wrapped in plastic wrap, and exposed to XAR film (Eastman Kodak Co., Rochester, N.Y.), and '5Zn2' binding was determined by autoradiography. Proteins were stained with 0.1% amido black made in 45% methanol-10% acetic acid and destained with 45% methanol-10% acetic acid. The autoradiogram revealed that 15Zn2+ bound to VP12 protein (Fig. 2A, lane 4) and to granulin (Fig. 2B, lane 4), but not to the other viral proteins (compare with the amido black-stained blots, Fig. 2A, lane 2, and Fig. 2B, lane 2). The molecular weight standards (Dalton Mark VII-L; Sigma Chemical Co., St. Louis, Mo.) include a zinc-binding protein, carbonic anhydrase (Mr 29,000), which serves as a positive control on the Zn2+ blot (18) (Fig. 2A, lane 3, and Fig. 2B, lane 3). We also determined that increased amounts of these viral proteins resulted in increased 15Zn2+ binding by these proteins (data not shown). To determine the specificity of cation binding, both NCs and PiGV capsules were subjected to a similar Ca2+ binding assay with 45Ca2+ (12, 14). Neither VP12 nor granulin bound Ca2+ in this assay (data not shown). A molar ratio of Zn2+ to VP12 was calculated from the data obtained with atomic absorption (Table 1) and the percentage of VP12 found in a Coomassie-stained, SDSPAGE-separated NC preparation. Since all the Zn2+ de-
tected in the NC sample was bound to VP12, the molar ratio was
calculated to be 3.24. The molar ratio of Zn2+ to
granulin was calculated to be 1.07, based on a molecular weight of 28,000 for granulin. A number of amino acids could potentially act as a zinc ligand in proteins, but only four amino acid side chains have so far been found to participate in Zn2+ binding (28). In descending order of frequency, they are the ring N atoms of
3170
NOTES
J . VlIROL.
TABLE 2. Amino acid composition of baculovirus basic proteins Content (mol%)
Amino acid PiGVa
Lys His Arg Asp Thr Ser
Glu Pro Cys Gly Ala Val Met Ile Leu Tyr Phe
1.3 12.5 26.5 1.0
16.3 0.4 4.1 0.3 1.7 1.3 16.9
13.3 0.1 5.1 0.4
AcNPVb
TnNPV'
BmNPVd
OpNPVe
40.6 10.9 17.2
45.1 2.0 7.8 13.7
3.1
3.9
7.8 3.1 1.6
7.8 3.9 2.0 2.0
0.8 40.0 12.6 18.1
4.0 9.0
2.0 2.0
31.0 2.1 11.2 16.6 1.9 4.3 1.9 12.9 2.1 2.8 1.0 0.8
12.6
10.4
1.6 3.1 10.9
11.8
a Data from Tweeten et al. (24). b Data for Autographa califomica nuclear polyhedrosis virus (AcNPV) from Wilson et al. (33). ' Data for Trichoplusia ni nuclear polyhedrosis virus (TnNPV) from Kelly et al. (11). d Data for Bombyx mon nuclear polyhedrosis virus (BmNPV) from Maeda et al. (13). e Data for Orgyia pseudotsugata nuclear polyhedrosis virus (OpNPV) from Russell et al. (16). f-, not detected.
His, the S atoms of Cys, and the carboxylate side chains of Glu and Asp. The amino acid composition of VP12 was previously determined in our laboratory (24) from acid-urea gel-isolated VP12 (Table 2). Under these conditions, the basic VP12 is the only protein that migrates into the gel and is totally free of other PiGV proteins. The amino acid composition of the isolated VP12 was found to contain a high percentage of one of the Zn2+-binding amino acids, namely His. Basic proteins have been identified in a number of other baculoviruses, and the amino acid compositions of several of these are compared in Table 2. It is striking that none of these other proteins have a high percentage of amino acid residues which bind Zn2+. Although there is evidence for cation involvement with other baculoviruses, since EDTA is used to dissociate NCs (21), perhaps other amino acids are used for zinc coordination or a different metal cation stabilizes other baculovirus NCs. It is also interesting that PiGV contains a high percentage of hydrophobic amino acids (16.9% Val, 13.3% Ile). Recently it has been shown that metal ions generally bind in a hydrophilic region which is surrounded by a hydrophobic shell (34). Perhaps these hydrophobic amino acids are structured to facilitate the metal-binding domain. Although the amino acid sequence of PiGV granulin is not known, the granulins of two other granulosis viruses have been sequenced (15). While the proteins do not contain the exact amino acid motif found in other zinc-binding proteins (26, 27), they do have a fairly high percentage of Asp and Glu residues, as well as a smaller percentage of His and Cys residues (15). One region in each granulin has a cluster of His and Cys residues which could potentially bind Zn2+. Starting at amino acid number 12, the consensus sequence is His-X4Cys-X5-His-X12-His, where X equals any amino acid (15). It has been demonstrated for a number of viral systems that divalent cations can play an integral role in maintaining the structural conformation of a viral protein and/or the entire virion. Calcium has been shown to be an important component of several plant viruses and polyomavirus (1, 4, 6, 9). Calcium binding by polyomavirus has been localized to
a specific domain of the major capsid protein VP1 (12). Zinc has been found to interact with reovirus (17), tobacco streak virus (19), and retroviral gag protein products (7, 18). In addition, a number of viral DNA-binding proteins have been demonstrated or predicted to bind zinc (2, 8). The presence of Zn2+ in PiGV and the ability of granulin and VP12 to bind zinc indicate a biological role for this metal cation. We propose that zinc interaction with VP12 is important in maintaining NC stability and that chelation of this cation may be a key event during NC uncoating in vivo. After entry into the host cell, the NC is transported to the nuclear membrane and becomes associated end-on with the nuclear pore. Electron micrographs indicate that the DNAprotein complex is inserted into the nucleus while the capsid remains outside (20). In order for the viral genome to be released into the nucleus, there must be a dissociation of the NC at the terminus. Since VP12 is found in the core of the NC, we suggest that in order for the chelator to initiate NC uncoating, the VP12-associated Zn2+ must be accessible to the chelator at the ends of the capsid. In light of our present findings, we propose that the nuclear membrane may have the ability to remove Zn2+ from the NC, possibly through the action of a zinc-binding protein(s). The initial dissociation is at the termini, as indicated by arrows in Fig. lb, showing subterminal openings in the NC treated with chelator and in the absence of reducing agent. Complete release of the DNA-protein complex is facilitated by a reducing agent. If nuclear microenvironments exist that provide the necessary biological reducing conditions (e.g., glutathione, cysteine), the proposed criteria for uncoating would be fulfilled. Under these conditions, the capsid terminus would be dissociated, allowing the DNA-protein core to be released into the nucleus to initiate the replication cycle. These uncoating events are probably simultaneous with the activation of an NC-associated kinase and phosphorylation of VP12, which is also an important event in NC uncoating (31, 32). It has previously been shown that divalent cations stabilize the oligomeric association of a granulin-related protein, polyhedrin, in the nuclear polyhedrosis virus. The solubili-
VOL. 66, 1992
zation of occlusion bodies is reduced by preincubation of the occluded virions with ZnCl2 (30). Therefore, Zn2+ may play a similar role in the occlusion body of PiGV. It should also be noted that there have been reports in the literature of possible Zn2+ nutritional requirements by baculoviruses. Zinc has been found to enhance baculovirus infections both in vivo and in vitro (10, 29). We report that Zn2+ is present in PiGV and that two structural proteins were found to bind Zn2+. We propose that this cation may have an important biological function for virion stability. It is also possible that Zn2+ plays a significant role in uncoating and assembly events of the PiGV life cycle. This investigation was supported by the Wesley Foundation of Wichita, Kans., and by NASA grants NAGW-1197 and NAGW2328. C.J.F. is a predoctoral fellow of the National Cancer Institute training grant CA09418. We thank Steve Hughes and Frank Padula of the Kansas State University Analytical Laboratory and LaDonna Grenz and Viola
Hill for their technical assistance. We also thank William Mc-
Gaughey and the USDA Grain Marketing Research Laboratory for use
of their facilities.
ADDENDUM IN PROOF Since this article was submitted, Bess et al. (J. W. Bess, Jr., P. J. Powell, H. J. Issaq, L. J. Schumack, M. K. Grimes, L. E. Henderson, and L. 0. Arthur, J. Virol. 66: 840-847, 1992) have described the association of Zn2+ with purified virions of human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses. REFERENCES 1. Abdel-Meguid, S. S., T. Yamane, K. Fukuyama, and M. G. Rossmann. 1981. The location of calcium ions in southern bean mosaic virus. Virology 114:81-85. 2. Berg, J. 1986. Potential metal-binding domains in nucleic acid binding proteins. Science 232:485-487. 3. Birk, H., and H. Koepsell. 1987. Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: renaturation of antigenic sites and reduction of nonspecific antibody binding. Anal. Biochem. 164:12-22. 4. Brady, J. N., V. D. Winston, and R. A. Consigli. 1977. Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J. Virol. 23:717-724. 5. Consigli, R. A., D. L. Russell, and M. E. Wilson. 1986. The biochemistry and molecular biology of the granulosis virus that infects Plodia interpunctella. Curr. Top. Microbiol. Immunol. 131:69-101. 6. Durham, A. C. H., D. A. Hendry, and M. B. Von Wechmar. 1977. Does calcium ion binding control plant virus disassembly? 7.
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