J Korean Med Sci.  2008 Dec;23(6):1046-1052. 10.3346/jkms.2008.23.6.1046.

Human Cytomegalovirus IE1 Protein Enhances Herpes Simplex Virus Type 1-induced Syncytial Formation in U373MG Cells

• Shin KC
• Park CG
• Hwang ES
• Cha CY

• KMID: 1107532
• DOI: http://doi.org/10.3346/jkms.2008.23.6.1046

Abstract

Co-infection of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) is not uncommon in immunocompromised hosts. Importantly, organ transplant recipients concurrently infected with HSV-1 and HCMV have a worse clinical outcome than recipients infected with a single virus. However, factors regulating the pathologic response in HSV-1, HCMV co-infected tissues are unclear. We investigated the potential biologic role of HCMV gene product immediate early 1 (IE1) protein in HSV-1-induced syncytial formation in U373MG cells. We utilized a co-infection model by infecting HSV-1 to U373MG cells constitutively expressing HCMV IE1 protein, UMG1-2. Syncytial formation was assessed by enumerating nuclei number per syncytium and number of syncytia. HSV-1-induced syncytial formation was enhanced after 24 hr in UMG1-2 cells compared with U373MG controls. The amplified phenotype in UMG1-2 cells was effectively suppressed by roscovitine in addition to inhibitors of viral replication. This is the first study to provide histological evidence of the contribution of HCMV IE1 protein to enhanced cytopathogenic responses in active HSV-1 infection.

Keyword

Herpesvirus 1, Human; Human Cytomegalovirus; IE1 Protein 1, Cytomegalovirus; Syncytial Formation; Roscovitine

MeSH Terms

Cell Line, Tumor
Giant Cells/*virology
Herpesvirus 1, Human/*growth & development
Humans
Immediate-Early Proteins/biosynthesis/*metabolism
Protein Kinase Inhibitors/pharmacology
Purines/pharmacology
Transfection
Virus Replication/drug effects

Figure

• 

  Fig. 1 Enhanced syncytial formation in HSV-1-infected UMG1-2 cells. (A) UMG1-2 and U373MG control cells were infected with HSV-1 for 24 hr on a chamber slide. Note the increased syncytial formation in UMG1-2 cells compared with U373MG controls (100× magnification). (B) Enumeration of nuclei per syncytium and number of syncytia after 24 hr. Note the increased syncytial formation, especially nuclei number of syncytium in UMG1-2 cells (*p<0.05, †p<0.01, ‡p<0.001). Values are expressed as mean±standard error and represent data from 3 independent experiments.

• 

  Fig. 2 Roscovitine ameliorates augmented syncytial formation in HSV-1-infected UMG1-2 cells. (A) Cells were plated on a 96-well plate and treated with described reagents simultaneously at the time of HSV-1 infection for 24 hr. MHSVI-116, ganciclovir, mitomycin C, and nocodazole robustly suppressed syncytial formation of both groups of cells. Roscovitine (20 µM) also effectively inhibited syncytial formation in UMG1-2 cells as well as U373MG control cells (*p<0.05, †p<0.01). TPCK, N-tosyl-1-phenylalanine-chloromethyl ketone. Values are expressed as mean±standard error and represent data from 3 independent experiments. (B) Comparison of immunohistochemistry results of roscovitine vs. media-only treated wells 24 hr after HSV-1 infection.

• 

  Fig. 3 Dose-dependent inhibition of syncytial formation by roscovitine. Syncytial formation in both groups of cells are suppressed with an increasing dose of roscovitine. Note the enhanced syncytial formation in UMG1-2 cells is suppressed to the level of U373MG controls at concentrations 20 µM or more (*p<0.05). Values are expressed as mean±standard error and represent data from 3 independent experiments.

Reference

1. Corey L, Spear PG. Infections with herpes simplex viruses (1). N Engl J Med. 1986. 314:686–691.

2. Najioullah F, Bosshard S, Thouvenot D, Boibieux A, Menager B, Biron F, Aymard M, Lina B. Diagnosis and surveillance of herpes simplex virus infection of the central nervous system. J Med Virol. 2000. 61:468–473.

3. Ecob-Johnston MS, Whetsell WO Jr. Host-cell response to herpes virus infection in central and peripheral nervous tissue in vitro. J Gen Virol. 1979. 44:747–757.

4. Yoffe B, Lewis DE, Petrie BL, Noonan CA, Melnick JL, Hollinger FB. Fusion as a mediator of cytolysis in mixtures of uninfected CD4+ lymphocytes and cells infected by human immunodeficiency virus. Proc Natl Acad Sci USA. 1987. 84:1429–1433.

5. Stegmann T, Doms RW, Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989. 18:187–211.

6. White JM. Membrane fusion. Science. 1992. 258:917–924.

7. Browne H, Bruun B, Minson T. Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL. J Gen Virol. 2001. 82:1419–1422.

8. Turner A, Bruun B, Minson T, Browne H. Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J Virol. 1998. 72:873–875.

9. Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, Miedema F, Schellekens PT, Tersmette M. Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. Ann Intern Med. 1993. 118:681–688.

10. Katzenstein TL. Molecular biological assessment methods and understanding the course of the HIV infection. APMIS Suppl. 2003. 1–37.

11. Pass RF. Epidemiology and transmission of cytomegalovirus. J Infect Dis. 1985. 152:243–248.

12. Laskin OL, Stahl-Bayliss CM, Morgello S. Concomitant herpes simplex virus type 1 and cytomegalovirus ventriculoencephalitis in acquired immunodeficiency syndrome. Arch Neurol. 1987. 44:843–847.

13. Vital C, Monlun E, Vital A, Martin-Negrier ML, Cales V, Leger F, Longy-Boursier M, Le Bras M, Bloch B. Concurrent herpes simplex type 1 necrotizing encephalitis, cytomegalovirus ventriculoencephalitis and cerebral lymphoma in an AIDS patient. Acta Neuropathol (Berl). 1995. 89:105–108.

14. Dunn DL, Matas AJ, Fryd DS, Simmons RL, Najarian JS. Association of concurrent herpes simplex virus and cytomegalovirus with detrimental effects after renal transplantation. Arch Surg. 1984. 119:812–817.

15. Kim J, Kwon YJ, Park ES, Sung B, Kim JH, Park CG, Hwang ES, Cha CY. Human cytomegalovirus (HCMV) IE1 plays role in resistance to apoptosis with etoposide in cancer cell line by Cdk2 accumulation. Microbiol Immunol. 2003. 47:959–967.

16. Cha CY, Hwang ES, Kook YH. Production and characterization of monoclonal antibodies specific to herpes simplex viruses. J Korean Soc Microbiol. 1988. 23:505–515.

17. Mahboobi S, Pongratz H, Hufsky H, Hockemeyer J, Frieser M, Lyssenko A, Paper DH, Burgermeister J, Bohmer FD, Fiebig HH, Burger AM, Baasner S, Beckers T. Synthetic 2-aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents. J Med Chem. 2001. 44:4535–4553.

18. Wing BA, Johnson RA, Huang ES. Identification of positive and negative regulatory regions involved in regulating expression of the human cytomegalovirus UL94 late promoter: role of IE2-86 and cellular p53 in mediating negative regulatory function. J Virol. 1998. 72:1814–1825.

19. Sydiskis RJ, Owen DG, Lohr JL, Rosler KH, Blomster RN. Inactivation of enveloped viruses by anthraquinones extracted from plants. Antimicrob Agents Chemother. 1991. 35:2463–2466.

20. Sodeik B, Ebersold MW, Helenius A. Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol. 1997. 136:1007–1021.

21. Colberg-Poley AM, Isom HC, Rapp F. Reactivation of herpes simplex virus type 2 from a quiescent state by human cytomegalovirus. Proc Natl Acad Sci USA. 1979. 76:5948–5951.

22. Colberg-Poley AM, Isom HC, Rapp F. Involvement of an early human cytomegalovirus function in reactivation of quiescent herpes simplex virus type 2. J Virol. 1981. 37:1051–1059.

23. Wathen MW, Stinski MF. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. J Virol. 1982. 41:462–477.

24. Wilkinson GW, Kelly C, Sinclair JH, Rickards C. Disruption of PML-associated nuclear bodies mediated by the human cytomegalovirus major immediate early gene product. J Gen Virol. 1998. 79(Pt 5):1233–1245.

25. Stenberg RM, Depto AS, Fortney J, Nelson JA. Regulated expression of early and late RNAs and proteins from the human cytomegalovirus immediate-early gene region. J Virol. 1989. 63:2699–2708.

26. Malone CL, Vesole DH, Stinski MF. Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins. J Virol. 1990. 64:1498–1506.

27. Luu P, Flores O. Binding of SP1 to the immediate-early protein-responsive element of the human cytomegalovirus DNA polymerase promoter. J Virol. 1997. 71:6683–6691.

28. Hayhurst GP, Bryant LA, Caswell RC, Walker SM, Sinclair JH. CCAAT box-dependent activation of the TATA-less human DNA polymerase alpha promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein. J Virol. 1995. 69:182–188.

29. Lukac DM, Harel NY, Tanese N, Alwine JC. TAF-like functions of human cytomegalovirus immediate-early proteins. J Virol. 1997. 71:7227–7239.

30. Lafemina RL, Pizzorno MC, Mosca JD, Hayward GS. Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes. Virology. 1989. 172:584–600.

31. Zhu H, Shen Y, Shenk T. Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol. 1995. 69:7960–7970.

32. Pajovic S, Wong EL, Black AR, Azizkhan JC. Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins. Mol Cell Biol. 1997. 17:6459–6464.

33. Poma EE, Kowalik TF, Zhu L, Sinclair JH, Huang ES. The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter. J Virol. 1996. 70:7867–7877.

34. Johnson RA, Yurochko AD, Poma EE, Zhu L, Huang ES. Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 protein-protein interaction and the possible functional consequences. J Gen Virol. 1999. 80:1293–1303.

35. Zhang Z, Huong SM, Wang X, Huang DY, Huang ES. Interactions between human cytomegalovirus IE1-72 and cellular p107: functional domains and mechanisms of up-regulation of cyclin E/cdk2 kinase activity. J Virol. 2003. 77:12660–12670.

36. Sanchez V, McElroy AK, Yen J, Tamrakar S, Clark CL, Schwartz RA, Spector DH. Cyclin-dependent kinase activity is required at early times for accurate processing and accumulation of the human cytomegalovirus UL122-123 and UL37 immediate-early transcripts and at later times for virus production. J Virol. 2004. 78:11219–11232.

37. Sanchez V, Spector DH. Cyclin-dependent kinase activity is required for efficient expression and posttranslational modification of human cytomegalovirus proteins and for production of extracellular particles. J Virol. 2006. 80:5886–5896.

38. Schang LM, Phillips J, Schaffer PA. Requirement for cellular cyclin-dependent kinases in herpes simplex virus replication and transcription. J Virol. 1998. 72:5626–5637.

39. Hossain A, Holt T, Ciacci-Zanella J, Jones C. Analysis of cyclin-dependent kinase activity after herpes simplex virus type 2 infection. J Gen Virol. 1997. 78:3341–3348.

40. Habran L, Bontems S, Di Valentin E, Sadzot-Delvaux C, Piette J. Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity. J Biol Chem. 2005. 280:29135–29143.

41. Benson C, White J, De Bono J, O'Donnell A, Raynaud F, Cruickshank C, McGrath H, Walton M, Workman P, Kaye S, Cassidy J, Gianella-Borradori A, Judson I, Twelves C. A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days. Br J Cancer. 2007. 96:29–37.

42. De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strnad M, Kim SH. Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. Eur J Biochem. 1997. 243:518–526.

43. Schang LM. Cyclin-dependent kinases as cellular targets for antiviral drugs. J Antimicrob Chemother. 2002. 50:779–792.

44. Schang LM, Rosenberg A, Schaffer PA. Transcription of herpes simplex virus immediate-early and early genes is inhibited by roscovitine, an inhibitor specific for cellular cyclin-dependent kinases. J Virol. 1999. 73:2161–2172.

45. Schang LM, Rosenberg A, Schaffer PA. Roscovitine, a specific inhibitor of cellular cyclin-dependent kinases, inhibits herpes simplex virus DNA synthesis in the presence of viral early proteins. J Virol. 2000. 74:2107–2120.

46. Davido DJ, Leib DA, Schaffer PA. The cyclin-dependent kinase inhibitor roscovitine inhibits the transactivating activity and alters the posttranslational modification of herpes simplex virus type 1 ICP0. J Virol. 2002. 76:1077–1088.

47. Diwan P, Lacasse JJ, Schang LM. Roscovitine inhibits activation of promoters in herpes simplex virus type 1 genomes independently of promoter-specific factors. J Virol. 2004. 78:9352–9365.

48. Sambucetti LC, Cherrington JM, Wilkinson GW, Mocarski ES. NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. Embo J. 1989. 8:4251–4258.

49. Jiang HY, Petrovas C, Sonenshein GE. RelB-p50 NF-kappa B complexes are selectively induced by cytomegalovirus immediate-early protein 1: differential regulation of Bcl-x(L) promoter activity by NF-kappa B family members. J Virol. 2002. 76:5737–5747.

50. Kim S, Yu SS, Kim VN. Essential role of NF-kappa B in transactivation of the human immunodeficiency virus long terminal repeat by the human cytomegalovirus 1E1 protein. J Gen Virol. 1996. 77:83–91.

51. Patel A, Hanson J, McLean TI, Olgiate J, Hilton M, Miller WE, Bachenheimer SL. Herpes simplex type 1 induction of persistent NF-kappa B nuclear translocation increases the efficiency of virus replication. Virology. 1998. 247:212–222.

52. Andersen DO, Weber ND, Wood SG, Hughes BG, Murray BK, North JA. In vitro virucidal activity of selected anthraquinones and anthraquinone derivatives. Antiviral Res. 1991. 16:185–196.

53. Gao M, Robertson BJ, McCann PJ, O'Boyle DR, Weller SK, Newcomb WW, Brown JC, Weinheimer SP. Functional conservations of the alkaline nuclease of herpes simplex type 1 and human cytomegalovirus. Virology. 1998. 249:460–470.

54. Reid GG, Ellsmore V, Stow ND. An analysis of the requirements for human cytomegalovirus oriLyt-dependent DNA synthesis in the presence of the herpes simplex virus type 1 replication fork proteins. Virology. 2003. 308:303–316.

55. Ng TI, Talarico C, Burnette TC, Biron K, Roizman B. Partial substitution of the functions of the herpes simplex virus 1 UL13 gene by the human cytomegalovirus UL97 gene. Virology. 1996. 225:347–358.

56. Preston CM, Nicholl MJ. Human cytomegalovirus tegument protein pp71 directs long-term gene expression from quiescent herpes simplex virus genomes. J Virol. 2005. 79:525–535.