1887

Abstract

Murine cytomegalovirus (MCMV) immediate-early protein 3 (IE3) is essential for successful viral infection. This study developed MCMVs with an EGFP-fused IE3 gene in order to study IE3 gene expression, subnuclear distribution and biological function, as well as to examine the interaction of IE3 with cellular and viral proteins. The generated viruses included MCMVIE3gfp, in which IE1 was completely removed by the in-frame fusion of exons 3 and 5 and the C terminus of IE3 was tagged with EGFP, and MCMVIE1/3gfp, in which IE1 was kept intact and EGFP was also fused to the C terminus of IE3. Unlike human CMV (HCMV), whose growth was significantly reduced when IE2 (the HCMV homologue of IE3 in MCMV) was tagged with EGFP, MCMVs with IE3–EGFP presented an unchanged replication profile. Using these new constructs, the distribution of IE3 was revealed as well as its interaction with viral and cellular proteins, especially proteins pertaining to DNA replication (M44 and E1) and cellular intrinsic defence [promyelocytic leukemia protein and histone deacetylases (HDACs)]. It was also shown that IE3 domains co-localize with DNA replication domains, and IE3 attracted other required proteins into IE3 domains via protein–protein interactions. In addition, IE3 was shown to interact with HDAC2 and to eliminate the inhibitory effect of HDAC2 on early viral gene production. Together, these results suggest that IE3 acts as a key protein for viral DNA replication by establishing pre-replication domains via recruitment of the required viral and cellular proteins, and by reducing host defences.

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2010-11-01
2019-11-13
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References

  1. Angulo, A., Ghazal, P. & Messerle, M. ( 2000; ). The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth. J Virol 74, 11129–11136.[CrossRef]
    [Google Scholar]
  2. Buhler, B., Keil, G. M., Weiland, F. & Koszinowski, U. H. ( 1990; ). Characterization of the murine cytomegalovirus early transcription unit e1 that is induced by immediate-early proteins. J Virol 64, 1907–1919.
    [Google Scholar]
  3. Caswell, R., Hagemeier, C., Chiou, C. J., Hayward, G., Kouzarides, T. & Sinclair, J. ( 1993; ). The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation. J Gen Virol 74, 2691–2698.[CrossRef]
    [Google Scholar]
  4. Cherrington, J. M. & Mocarski, E. S. ( 1989; ). Human cytomegalovirus ie1 transactivates the α promoter-enhancer via an 18-base-pair repeat element. J Virol 63, 1435–1440.
    [Google Scholar]
  5. Ciocco-Schmitt, G. M., Karabekian, Z., Godfrey, E. W., Stenberg, R. M., Campbell, A. E. & Kerry, J. A. ( 2002; ). Identification and characterization of novel murine cytomegalovirus M112–113 (e1) gene products. Virology 294, 199–208.[CrossRef]
    [Google Scholar]
  6. Eizuru, Y., Inagawa, S. & Minamishima, Y. ( 1984; ). Application of “Hirt supernatant” DNA to the molecular epidemiology of cytomegalovirus infections. J Clin Microbiol 20, 1012–1014.
    [Google Scholar]
  7. Fortunato, E. A., McElroy, A. K., Sanchez, I. & Spector, D. H. ( 2000; ). Exploitation of cellular signaling and regulatory pathways by human cytomegalovirus. Trends Microbiol 8, 111–119.[CrossRef]
    [Google Scholar]
  8. Ghazal, P., Visser, A. E., Gustems, M., García, R., Borst, E. M., Sullivan, K., Messerle, M. & Angulo, A. ( 2005; ). Elimination of ie1 significantly attenuates murine cytomegalovirus virulence but does not alter replicative capacity in cell culture. J Virol 79, 7182–7194.[CrossRef]
    [Google Scholar]
  9. Greaves, R. F. & Mocarski, E. S. ( 1998; ). Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant. J Virol 72, 366–379.
    [Google Scholar]
  10. Hagemeier, C., Walker, S., Caswell, R., Kouzarides, T. & Sinclair, J. ( 1992; ). The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID. J Virol 66, 4452–4456.
    [Google Scholar]
  11. Hengel, H., Lucin, P., Jonjic, S., Ruppert, T. & Koszinowski, U. H. ( 1994; ). Restoration of cytomegalovirus antigen presentation by gamma interferon combats viral escape. J Virol 68, 289–297.
    [Google Scholar]
  12. Jault, F. M., Jault, J. M., Ruchti, F., Fortunato, E. A., Clark, C., Corbeil, J., Richman, D. D. & Spector, D. H. ( 1995; ). Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest. J Virol 69, 6697–6704.
    [Google Scholar]
  13. Keil, G. M., Ebeling-Keil, A. & Koszinowski, U. H. ( 1987; ). Immediate-early genes of murine cytomegalovirus: location, transcripts, and translation products. J Virol 61, 526–533.
    [Google Scholar]
  14. Kurz, S. K. & Reddehase, M. J. ( 1999; ). Patchwork pattern of transcriptional reactivation in the lungs indicates sequential checkpoints in the transition from murine cytomegalovirus latency to recurrence. J Virol 73, 8612–8622.
    [Google Scholar]
  15. Loh, L. C., Keeler, V. D. & Shanley, J. D. ( 1999; ). Sequence requirements for the nuclear localization of the murine cytomegalovirus M44 gene product pp50. Virology 259, 43–59.[CrossRef]
    [Google Scholar]
  16. Loregian, A. & Coen, D. M. ( 2006; ). Selective anti-cytomegalovirus compounds discovered by screening for inhibitors of subunit interactions of the viral polymerase. Chem Biol 13, 191–200.[CrossRef]
    [Google Scholar]
  17. Lukac, D. M., Harel, N. Y., Tanese, N. & Alwine, J. C. ( 1997; ). TAF-like functions of human cytomegalovirus immediate-early proteins. J Virol 71, 7227–7239.
    [Google Scholar]
  18. Maul, G. G. & Negorev, D. ( 2008; ). Differences between mouse and human cytomegalovirus interactions with their respective hosts at immediate early times of the replication cycle. Med Microbiol Immunol (Berl) 197, 241–249.[CrossRef]
    [Google Scholar]
  19. McElroy, A. K., Dwarakanath, R. S. & Spector, D. H. ( 2000; ). Dysregulation of cyclin E gene expression in human cytomegalovirus-infected cells requires viral early gene expression and is associated with changes in the Rb-related protein p130. J Virol 74, 4192–4206.[CrossRef]
    [Google Scholar]
  20. Mendelson, M., Monard, S., Sissons, P. & Sinclair, J. ( 1996; ). Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors. J Gen Virol 77, 3099–3102.[CrossRef]
    [Google Scholar]
  21. Messerle, M., Buhler, B., Keil, G. M. & Koszinowski, U. H. ( 1992; ). Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3. J Virol 66, 27–36.
    [Google Scholar]
  22. Michaelis, M., Suhan, T., Reinisch, A., Reisenauer, A., Fleckenstein, C., Eikel, D., Gumbel, H., Doerr, H. W., Nau, H. & Cinatl, J., Jr ( 2005; ). Increased replication of human cytomegalovirus in retinal pigment epithelial cells by valproic acid depends on histone deacetylase inhibition. Invest Ophthalmol Vis Sci 46, 3451–3457.[CrossRef]
    [Google Scholar]
  23. Mocarski, E. S., Jr, Shenk, T. & Pass, R. F. ( 2006; ). Cytomegaloviruses. Philadelphia. : Lippincott Williams &Wilkins.
    [Google Scholar]
  24. Nevels, M., Paulus, C. & Shenk, T. ( 2004; ). Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation. Proc Natl Acad Sci U S A 101, 17234–17239.[CrossRef]
    [Google Scholar]
  25. Park, J. J., Kim, Y. E., Pham, H. T., Kim, E. T., Chung, Y. H. & Ahn, J. H. ( 2007; ). Functional interaction of the human cytomegalovirus IE2 protein with histone deacetylase 2 in infected human fibroblasts. J Gen Virol 88, 3214–3223.[CrossRef]
    [Google Scholar]
  26. Reddehase, M. J., Simon, C. O., Seckert, C. K., Lemmermann, N. & Grzimek, N. K. ( 2008; ). Murine model of cytomegalovirus latency and reactivation. Curr Top Microbiol Immunol 325, 315–331.
    [Google Scholar]
  27. Salvant, B. S., Fortunato, E. A. & Spector, D. H. ( 1998; ). Cell cycle dysregulation by human cytomegalovirus: influence of the cell cycle phase at the time of infection and effects on cyclin transcription. J Virol 72, 3729–3741.
    [Google Scholar]
  28. Scully, A. L., Sommer, M. H., Schwartz, R. & Spector, D. H. ( 1995; ). The human cytomegalovirus IE2 86-kilodalton protein interacts with an early gene promoter via site-specific DNA binding and protein–protein associations. J Virol 69, 6533–6540.
    [Google Scholar]
  29. Sourvinos, G., Tavalai, N., Berndt, A., Spandidos, D. A. & Stamminger, T. ( 2007; ). Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells. J Virol 81, 10123–10136.[CrossRef]
    [Google Scholar]
  30. Stenberg, R. M. ( 1996; ). The human cytomegalovirus major immediate-early gene. Intervirology 39, 343–349.
    [Google Scholar]
  31. Stenberg, R. M. & Stinski, M. F. ( 1985; ). Autoregulation of the human cytomegalovirus major immediate-early gene. J Virol 56, 676–682.
    [Google Scholar]
  32. Stenberg, R. M., Fortney, J., Barlow, S. W., Magrane, B. P., Nelson, J. A. & Ghazal, P. ( 1990; ). Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains. J Virol 64, 1556–1565.
    [Google Scholar]
  33. Stinski, M. F. & Isomura, H. ( 2008; ). Role of the cytomegalovirus major immediate early enhancer in acute infection and reactivation from latency. Med Microbiol Immunol (Berl) 197, 223–231.[CrossRef]
    [Google Scholar]
  34. Stinski, M. F. & Petrik, D. T. ( 2008; ). Functional roles of the human cytomegalovirus essential IE86 protein. Curr Top Microbiol Immunol 325, 133–152.
    [Google Scholar]
  35. Tang, Q. & Maul, G. G. ( 2003; ). Mouse cytomegalovirus immediate-early protein 1 binds with host cell repressors to relieve suppressive effects on viral transcription and replication during lytic infection. J Virol 77, 1357–1367.[CrossRef]
    [Google Scholar]
  36. Tang, Q. & Maul, G. ( 2005; ). Immediate early interactions and epigenetic defense mechanisms. In Cytomegaloviruses: Molecular Biology and Immunology. Edited by Reddehase, M. J.. Norwich, UK. : Horizon Scientific Press.
    [Google Scholar]
  37. Tang, Q. & Maul, G. G. ( 2006; ). Mouse cytomegalovirus crosses the species barrier with help from a few human cytomegalovirus proteins. J Virol 80, 7510–7521.[CrossRef]
    [Google Scholar]
  38. Tang, Q., Bell, P., Tegtmeyer, P. & Maul, G. G. ( 2000; ). Replication but not transcription of simian virus 40 DNA is dependent on nuclear domain 10. J Virol 74, 9694–9700.[CrossRef]
    [Google Scholar]
  39. Tang, Q., Li, L., Ishov, A. M., Revol, V., Epstein, A. L. & Maul, G. G. ( 2003; ). Determination of minimum herpes simplex virus type 1 components necessary to localize transcriptionally active DNA to ND10. J Virol 77, 5821–5828.[CrossRef]
    [Google Scholar]
  40. Tang, Q., Li, L. & Maul, G. G. ( 2005; ). Mouse cytomegalovirus early M112/113 proteins control the repressive effect of IE3 on the major immediate-early promoter. J Virol 79, 257–263.[CrossRef]
    [Google Scholar]
  41. Taylor, R. T. & Bresnahan, W. A. ( 2005; ). Human cytomegalovirus immediate-early 2 gene expression blocks virus-induced beta interferon production. J Virol 79, 3873–3877.[CrossRef]
    [Google Scholar]
  42. Wagner, M., Jonjic, S., Koszinowski, U. H. & Messerle, M. ( 1999; ). Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution. J Virol 73, 7056–7060.
    [Google Scholar]
  43. Warming, S., Costantino, N., Court, D. L., Jenkins, N. A. & Copeland, N. G. ( 2005; ). Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33, e36.[CrossRef]
    [Google Scholar]
  44. Wiebusch, L. & Hagemeier, C. ( 1999; ). Human cytomegalovirus 86-kilodalton IE2 protein blocks cell cycle progression in G1. J Virol 73, 9274–9283.
    [Google Scholar]
  45. Wiebusch, L., Neuwirth, A., Grabenhenrich, L., Voigt, S. & Hagemeier, C. ( 2008; ). Cell cycle-independent expression of immediate-early gene 3 results in G1 and G2 arrest in murine cytomegalovirus-infected cells. J Virol 82, 10188–10198.[CrossRef]
    [Google Scholar]
  46. Wilkinson, D. E. & Weller, S. K. ( 2004; ). Recruitment of cellular recombination and repair proteins to sites of herpes simplex virus type 1 DNA replication is dependent on the composition of viral proteins within prereplicative sites and correlates with the induction of the DNA damage response. J Virol 78, 4783–4796.[CrossRef]
    [Google Scholar]
  47. Wright, E., Bain, M., Teague, L., Murphy, J. & Sinclair, J. ( 2005; ). Ets-2 repressor factor recruits histone deacetylase to silence human cytomegalovirus immediate-early gene expression in non-permissive cells. J Gen Virol 86, 535–544.[CrossRef]
    [Google Scholar]
  48. Wu, C. A., Carlson, M. E., Henry, S. C. & Shanley, J. D. ( 1999; ). The murine cytomegalovirus M25 open reading frame encodes a component of the tegument. Virology 262, 265–276.[CrossRef]
    [Google Scholar]
  49. Zhong, L. & Hayward, G. S. ( 1997; ). Assembly of complete, functionally active herpes simplex virus DNA replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays. J Virol 71, 3146–3160.
    [Google Scholar]
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