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. 1997 Apr;71(4):2857–2865. doi: 10.1128/jvi.71.4.2857-2865.1997

Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system.

E T Gillock 1, S Rottinghaus 1, D Chang 1, X Cai 1, S A Smiley 1, K An 1, R A Consigli 1
PMCID: PMC191411  PMID: 9060642

Abstract

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.

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Selected References

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  1. Betenbaugh M., Yu M., Kuehl K., White J., Pennock D., Spik K., Schmaljohn C. Nucleocapsid- and virus-like particles assemble in cells infected with recombinant baculoviruses or vaccinia viruses expressing the M and the S segments of Hantaan virus. Virus Res. 1995 Oct;38(2-3):111–124. doi: 10.1016/0168-1702(95)00053-s. [DOI] [PubMed] [Google Scholar]
  2. Brady J. N., Winston V. D., Consigli R. A. Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol. J Virol. 1978 Jul;27(1):193–204. doi: 10.1128/jvi.27.1.193-204.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burton K. S., Consigli R. A. Methylation of the polyomavirus major capsid protein VP1. Virus Res. 1996 Feb;40(2):141–147. doi: 10.1016/0168-1702(95)01266-4. [DOI] [PubMed] [Google Scholar]
  4. Cai X., Chang D., Rottinghaus S., Consigli R. A. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins. J Virol. 1994 Nov;68(11):7609–7613. doi: 10.1128/jvi.68.11.7609-7613.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang D., Cai X., Consigli R. A. Characterization of the DNA binding properties of polyomavirus capsid protein. J Virol. 1993 Oct;67(10):6327–6331. doi: 10.1128/jvi.67.10.6327-6331.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang D., Haynes J. I., 2nd, Brady J. N., Consigli R. A. Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2. Virology. 1992 Dec;191(2):978–983. doi: 10.1016/0042-6822(92)90276-u. [DOI] [PubMed] [Google Scholar]
  7. Chang D., Haynes J. I., 2nd, Brady J. N., Consigli R. A. The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1. Virology. 1992 Aug;189(2):821–827. doi: 10.1016/0042-6822(92)90615-v. [DOI] [PubMed] [Google Scholar]
  8. Delos S. E., Cripe T. P., Leavitt A. D., Greisman H., Garcea R. L. Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres. J Virol. 1995 Dec;69(12):7734–7742. doi: 10.1128/jvi.69.12.7734-7742.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Delos S. E., Montross L., Moreland R. B., Garcea R. L. Expression of the polyomavirus VP2 and VP3 proteins in insect cells: coexpression with the major capsid protein VP1 alters VP2/VP3 subcellular localization. Virology. 1993 May;194(1):393–398. doi: 10.1006/viro.1993.1274. [DOI] [PubMed] [Google Scholar]
  10. Forstová J., Krauzewicz N., Wallace S., Street A. J., Dilworth S. M., Beard S., Griffin B. E. Cooperation of structural proteins during late events in the life cycle of polyomavirus. J Virol. 1993 Mar;67(3):1405–1413. doi: 10.1128/jvi.67.3.1405-1413.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garcea R. L., Benjamin T. L. Isolation and characterization of polyoma nucleoprotein complexes. Virology. 1983 Oct 15;130(1):65–75. doi: 10.1016/0042-6822(83)90118-6. [DOI] [PubMed] [Google Scholar]
  12. Glocker B., Hoopes R. R., Jr, Rohrmann G. F. In vitro transactivation of baculovirus early genes by nuclear extracts from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells. J Virol. 1992 Jun;66(6):3476–3484. doi: 10.1128/jvi.66.6.3476-3484.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Griffith J. P., Griffith D. L., Rayment I., Murakami W. T., Caspar D. L. Inside polyomavirus at 25-A resolution. Nature. 1992 Feb 13;355(6361):652–654. doi: 10.1038/355652a0. [DOI] [PubMed] [Google Scholar]
  14. Haynes J. I., 2nd, Chang D., Consigli R. A. Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly. J Virol. 1993 May;67(5):2486–2495. doi: 10.1128/jvi.67.5.2486-2495.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haynes J. I., 2nd, Consigli R. A. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1. J Virol. 1992 Jul;66(7):4551–4555. doi: 10.1128/jvi.66.7.4551-4555.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  17. Knudson D. L., Tinsley T. W. Replication of a nuclear polyhedrosis virus in a continuous cell line of Spodoptera frugiperda: partial characterization of the viral DNA, comparative DNA-DNA hybridization, and patterns of DNA synthesis. Virology. 1978 Jun 1;87(1):42–57. doi: 10.1016/0042-6822(78)90156-3. [DOI] [PubMed] [Google Scholar]
  18. Kosukegawa A., Arisaka F., Takayama M., Yajima H., Kaidow A., Handa H. Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells. Biochim Biophys Acta. 1996 May 21;1290(1):37–45. [PubMed] [Google Scholar]
  19. Lamb J. W., Duncan G. H., Reavy B., Gildow F. E., Mayo M. A., Hay R. T. Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus. J Gen Virol. 1996 Jul;77(Pt 7):1349–1358. doi: 10.1099/0022-1317-77-7-1349. [DOI] [PubMed] [Google Scholar]
  20. Li M., Delos S. E., Montross L., Garcea R. L. Polyomavirus VP1 phosphorylation: coexpression with the VP2 capsid protein modulates VP1 phosphorylation in Sf9 insect cells. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5992–5996. doi: 10.1073/pnas.92.13.5992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mackay R. L., Consigli R. A. Early events in polyoma virus infection: attachment, penetration, and nuclear entry. J Virol. 1976 Aug;19(2):620–636. doi: 10.1128/jvi.19.2.620-636.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Michel M. R., Hirt B., Weil R. Mouse cellular DNA enclosed in polyoma viral capsids (pseudovirions). Proc Natl Acad Sci U S A. 1967 Oct;58(4):1381–1388. doi: 10.1073/pnas.58.4.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Montross L., Watkins S., Moreland R. B., Mamon H., Caspar D. L., Garcea R. L. Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1. J Virol. 1991 Sep;65(9):4991–4998. doi: 10.1128/jvi.65.9.4991-4998.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moreland R. B., Montross L., Garcea R. L. Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1. J Virol. 1991 Mar;65(3):1168–1176. doi: 10.1128/jvi.65.3.1168-1176.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pawlita M., Müller M., Oppenländer M., Zentgraf H., Herrmann M. DNA encapsidation by viruslike particles assembled in insect cells from the major capsid protein VP1 of B-lymphotropic papovavirus. J Virol. 1996 Nov;70(11):7517–7526. doi: 10.1128/jvi.70.11.7517-7526.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rayment I., Baker T. S., Caspar D. L., Murakami W. T. Polyoma virus capsid structure at 22.5 A resolution. Nature. 1982 Jan 14;295(5845):110–115. doi: 10.1038/295110a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salunke D. M., Caspar D. L., Garcea R. L. Self-assembly of purified polyomavirus capsid protein VP1. Cell. 1986 Sep 12;46(6):895–904. doi: 10.1016/0092-8674(86)90071-1. [DOI] [PubMed] [Google Scholar]
  28. Tian T., Medina V., Mayhew D. E., Maeda S., Falk B. W. Beet western yellows luteovirus capsid proteins produced by recombinant baculoviruses assemble into virion-like particles in cells and larvae of Bombyx mori. Virology. 1995 Oct 20;213(1):204–212. doi: 10.1006/viro.1995.1560. [DOI] [PubMed] [Google Scholar]
  29. Trus B. L., Homa F. L., Booy F. P., Newcomb W. W., Thomsen D. R., Cheng N., Brown J. C., Steven A. C. Herpes simplex virus capsids assembled in insect cells infected with recombinant baculoviruses: structural authenticity and localization of VP26. J Virol. 1995 Nov;69(11):7362–7366. doi: 10.1128/jvi.69.11.7362-7366.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tweeten K. A., Bulla L. A., Consigli R. A. Characterization of an extremely basic protein derived from granulosis virus nucleocapsids. J Virol. 1980 Feb;33(2):866–876. doi: 10.1128/jvi.33.2.866-876.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Türler H. Interactions of polyoma and mouse DNA's. II. Polyoma-induced mouse DNA replication and pseudovirion formation. J Virol. 1974 Feb;13(2):285–290. doi: 10.1128/jvi.13.2.285-290.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wilson M. E., Miller L. K. Changes in the nucleoprotein complexes of a baculovirus DNA during infection. Virology. 1986 Jun;151(2):315–328. doi: 10.1016/0042-6822(86)90052-8. [DOI] [PubMed] [Google Scholar]
  33. Yelton D. B., Aposhian H. V. Polyoma pseudovirions. I. Sequence of events in primary mouse embryo cells leading to pseudovirus production. J Virol. 1972 Sep;10(3):340–346. doi: 10.1128/jvi.10.3.340-346.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yuen L. K., Consigli R. A. Generation of capsids from unstable polyoma virions. J Virol. 1983 Sep;47(3):620–625. doi: 10.1128/jvi.47.3.620-625.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yuen L. K., Consigli R. A. Identification and protein analysis of polyomavirus assembly intermediates from infected primary mouse embryo cells. Virology. 1985 Jul 15;144(1):127–138. doi: 10.1016/0042-6822(85)90311-3. [DOI] [PubMed] [Google Scholar]

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