1887

Abstract

Epstein–Barr virus (EBV) encodes putative helicase–primase proteins BBLF4, BSLF1 and BBLF2/3, which are essential for the lytic phase of viral DNA replication. The BSLF1, BBLF4 and BBLF2/3 proteins were expressed in B95-8 cells after induction of a virus productive cycle, possessing apparent molecular masses of 89 kDa, 90 kDa and 80 kDa, respectively. The anti-BSLF1 or anti-BBLF2/3 protein-specific antibody, which recognizes its target protein in both Western blotting and immunoprecipitation analyses, immunoprecipitated all of the BSLF1, BBLF4 and BBLF2/3 proteins from the extract of the cells with a virus productive cycle, indicating that these viral proteins are assembled together . To characterize their protein–protein interactions in detail, recombinant baculoviruses capable of expressing each of these viral gene products in insect cells were constructed. The assembly of the three virus replication proteins was reproduced in insect cells co- infected with the three recombinant baculoviruses, indicating that complex formation does not require other EBV replication proteins. Furthermore, experiments performed by using the extracts from insect cells co-infected with each pair of the recombinant viruses demonstrated that the BSLF1 protein could interact separately with both the BBLF4 and BBLF2/3 proteins and that the BBLF2/3 protein also interacted with the BBLF4 protein. These observations strongly suggest that within the BBLF4–BSLF1–BBLF2/3 complex each component interacts directly with the other two, resulting in helicase–primase enzyme activity.

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1999-11-01
2019-10-23
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References

  1. Aiyar, A. , Tyree, C. & Sugden, B. ( 1998; ). The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element. EMBO Journal 17, 6394-6403 .[CrossRef]
    [Google Scholar]
  2. Baer, R. , Bankier, A. T. , Biggin, M. D. , Deininger, P. L. , Farrell, P. J. , Gibson, T. J. , Hatfull, G. , Hudson, G. S. , Satchwell, S. C. , Seguin, C. , Tuffnell, P. S. & Barrell, B. G. ( 1984; ). DNA sequence and expression of the B95-8 Epstein–Barr virus genome. Nature 310, 207-211.[CrossRef]
    [Google Scholar]
  3. Calder, J. M. & Stow, N. D. ( 1990; ). Herpes simplex virus helicase–primase: the UL8 protein is not required for DNA- dependent ATPase and DNA helicase activities. Nucleic Acids Research 18, 3573-3578 .[CrossRef]
    [Google Scholar]
  4. Crute, J. J. & Lehman, I. R. ( 1991; ). Herpes simplex virus- 1 helicase–primase. Journal of Biological Chemistry 266, 4484-4488 .
    [Google Scholar]
  5. Crute, J. J. , Tsurumi, T. , Zhu, L. , Weller, S. K. , Olivo, P. D. , Challberg, M. D. , Mocarski, E. S. & Lehman, I. R. ( 1989; ). Herpes simplex virus 1 helicase–primase: a complex of three herpes-encoded gene products. Proceedings of the National Academy of Sciences, USA 86, 2186-2189 .[CrossRef]
    [Google Scholar]
  6. Dodson, M. S. & Lehman, I. R. ( 1991; ). Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase–primase composed of the UL5 and UL52 gene products. Proceedings of the National Academy of Sciences, USA 88, 1105-1109 .[CrossRef]
    [Google Scholar]
  7. Dodson, M. S. , Crute, J. J. , Bruckner, R. C. & Lehman, I. R. ( 1989; ). Overexpression and assembly of the herpes simplex virus type 1 helicase–primase in insect cells. Journal of Biological Chemistry 264, 20835-20838 .
    [Google Scholar]
  8. Dracheva, S. , Koonin, E. V. & Crute, J. J. ( 1995; ). Identification of the primase active site of the herpes simplex virus type 1 helicase–primase. Journal of Biological Chemistry 270, 14148-14153 .[CrossRef]
    [Google Scholar]
  9. Faggioni, A. , Zompetta, C. , Grimaldi, S. , Barile, G. , Frati, L. & Lazdins, J. ( 1986; ). Calcium modulation activates Epstein–Barr virus genome in latently infected cells. Science 232, 1554-1556 .[CrossRef]
    [Google Scholar]
  10. Farrell, P. J. ( 1989; ). Epstein–Barr virus genome. Advances in Viral Oncology 8, 103-132.
    [Google Scholar]
  11. Fixman, E. D. , Hayward, G. S. & Hayward, S. D. ( 1992; ). trans- acting requirements for replication of Epstein–Barr virus ori- Lyt. Journal of Virology 66, 5030-5039 .
    [Google Scholar]
  12. Fixman, E. D. , Hayward, G. S. & Hayward, S. D. ( 1995; ). Replication of Epstein–Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays. Journal of Virology 69, 2998-3006 .
    [Google Scholar]
  13. Fujita, M. , Yamada, C. , Tsurumi, T. , Hanaoka, F. , Matsuzawa, K. & Inagaki, M. ( 1998; ). Cell cycle- and chromatin binding state-dependent phosphorylation of human MCM heterohexameric complexes. Journal of Biological Chemistry 273, 17095-17101 .[CrossRef]
    [Google Scholar]
  14. Gao, Z. , Krithivas, A. , Finan, J. E. , Semmes, O. J. , Zhou, S. , Wang, Y. & Hayward, S. D. ( 1998; ). The Epstein–Barr virus lytic transactivator Zta interacts with the helicase–primase replication proteins. Journal of Virology 72, 8559-8567 .
    [Google Scholar]
  15. Graves-Woodward, K. L. & Weller, S. K. ( 1996; ). Replacement of Gly 815 in helicase motif V alters the single-stranded DNA- dependent ATPase activity of the herpes simplex virus type 1 helicase–primase. Journal of Biological Chemistry 271, 13629-13635 .[CrossRef]
    [Google Scholar]
  16. Graves-Woodward, K. L. , Gottlieb, J. , Challberg, M. D. & Weller, S. K. ( 1997; ). Biochemical analyses of mutations in the HSV-1 helicase–primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding. Journal of Biological Chemistry 272, 4623-4630 .[CrossRef]
    [Google Scholar]
  17. Hammerschmidt, W. & Sugden, B. ( 1988; ). Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein–Barr virus. Cell 55, 427-433.[CrossRef]
    [Google Scholar]
  18. Kiehl, A. & Dorsky, D. I. ( 1995; ). Bipartite DNA-binding region of the Epstein–Barr virus BMRF1 product essential for DNA polymerase accessory function. Journal of Virology 69, 1669-1677 .
    [Google Scholar]
  19. Kitts, P. A. , Ayres, M. D. & Possee, R. D. ( 1990; ). Linearization of baculovirus DNA enhances the recovery of recombinant virus expression. Nucleic Acids Research 18, 5667-5672 .[CrossRef]
    [Google Scholar]
  20. Klinedinst, D. K. & Challberg, M. D. ( 1994; ). Helicase–primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity. Journal of Virology 68, 3693-3701 .
    [Google Scholar]
  21. Luckow, V. A. , Lee, S. C. , Barry, G. F. & Olins, P. O. ( 1993; ). Efficient generation of infectious recombinant baculoviruses by site-specific transposon- mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. Journal of Virology 67, 4566-4579 .
    [Google Scholar]
  22. McLean, G. W. , Abbotts, A. P. , Parry, M. E. , Marsden, H. S. & Stow, N. D. ( 1994; ). The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein. Journal of General Virology 75, 2699-2706 .[CrossRef]
    [Google Scholar]
  23. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  24. Schepers, A. , Pich, D. & Hammerschmidt, W. ( 1996; ). Activation of oriLyt, the lytic origin of DNA replication of Epstein–Barr virus, by BZLF1. Virology 220, 367-376.[CrossRef]
    [Google Scholar]
  25. Spector, F. C. , Giordano, L. H. , Sivaraja, M. & Peterson, M. G. ( 1998; ). Inhibition of herpes simplex virus replication by a 2-amino thiazole via interactions with the helicase component of the UL5–UL8–UL52 complex. Journal of Virology 72, 6979-6987 .
    [Google Scholar]
  26. Takada, K. , Horinouchi, K. , Ono, Y. , Aya, T. , Osato, T. , Takahashi, M. & Hayasaka, S. ( 1991; ). An Epstein–Barr virus- producer line Akata: establishment of the cell line and analysis of viral DNA. Virus Genes 5, 147-156.[CrossRef]
    [Google Scholar]
  27. Tsurumi, T. ( 1993; ). Purification and characterization of the DNA-binding activity of the Epstein–Barr virus DNA polymerase accessory protein BMRF1 gene products, as expressed in insect cells by using the baculovirus system. Journal of Virology 67, 1681-1687 .
    [Google Scholar]
  28. Tsurumi, T. , Kobayashi, A. , Tamai, K. , Daikoku, T. , Kurachi, R. & Nishiyama, Y. ( 1993a; ). Functional expression and characterization of the Epstein–Barr virus DNA polymerase catalytic subunit. Journal of Virology 67, 4651-4658.
    [Google Scholar]
  29. Tsurumi, T. , Daikoku, T. , Kurachi, R. & Nishiyama, Y. ( 1993b; ). Functional interaction between Epstein–Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro. Journal of Virology 67, 7648-7653.
    [Google Scholar]
  30. Tsurumi, T. , Daikoku, T. & Nishiyama, Y. ( 1994; ). Further characterization of the interaction between the Epstein–Barr virus DNA polymerase catalytic subunit and its accessory subunit with regard to the 3′- to-5′ exonuclease activity and stability of initiation complex at primer terminus. Journal of Virology 88, 3354-3363 .
    [Google Scholar]
  31. Tsurumi, T. , Kobayashi, A. , Tamai, K. , Yamada, H. , Daikoku, T. , Yamashita, Y. & Nishiyama, Y. ( 1996; ). Epstein–Barr virus single- stranded DNA-binding protein: purification, characterization, and action on DNA synthesis by the viral DNA polymerase. Virology 222, 352-364.[CrossRef]
    [Google Scholar]
  32. Tsurumi, T. , Kishore, J. , Yokoyama, N. , Fujita, M. , Daikoku, T. , Yamada, H. , Yamashita, Y. & Nishiyama, Y. ( 1998; ). Overexpression, purification and helix-destabilizing properties of Epstein–Barr virus ssDNA- binding protein. Journal of General Virology 79, 1257-1264 .
    [Google Scholar]
  33. Yates, J. L. & Guan, N. ( 1991; ). Epstein–Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells. Journal of Virology 65, 483-488.
    [Google Scholar]
  34. Zhu, L. & Weller, S. K. ( 1992; ). The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase–primase, are essential for its function. Journal of Virology 66, 469-479.
    [Google Scholar]
  35. zur Hausen, H. , O’Neill, F. J. & Freese, U. K. ( 1978; ). Persisting oncogenic herpesvirus induced by the tumor promoter TPA. Nature 272, 373-375.[CrossRef]
    [Google Scholar]
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