1887

Abstract

Evidence suggests that the products of the human cytomegalovirus (HCMV) UL112–113 genes are involved in viral DNA replication during lytic infection. A polyclonal antibody was raised against the UL112 open reading frame (ORF) to characterize its function in detail. Immunoblots utilizing the UL112 antibody identified seven distinct protein bands (p20, p26, p28, p34, p43, p50 and p84) expressed during the HCMV infectious cycle. After screening a cDNA library constructed from cells 72 h after infection with HCMV, only four different cDNA protein-producing constructs were obtained, and their ORFs corresponded to p34, p43, p50 and p84. The proteins p20, p26 and p28 were further shown to be selectively included within mature HCMV particles, virions, non-infectious enveloped particles and dense bodies. Immunoaffinity protein purification was used to prepare the samples for liquid chromatography coupled to tandem mass spectrometry. This analysis revealed that p20, p26 and p28 were derived from the UL112 ORF, most likely through post-translational proteolytic cleavage.

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2009-12-01
2024-04-18
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References

  1. Ahn J. H., Jang W. J., Hayward G. S. 1999; The human cytomegalovirus IE2 and UL112–113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10). J Virol 73:10458–10471
    [Google Scholar]
  2. Belizario J. E., Alves J., Garay-Malpartida M., Occhiucci J. M. 2008; Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif. Curr Protein Pept Sci 9:210–220 [CrossRef]
    [Google Scholar]
  3. Britt W. 2008; Manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease. Curr Top Microbiol Immunol 325:417–470
    [Google Scholar]
  4. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., III, Kouzarides T. other authors 1990; Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154:125–169
    [Google Scholar]
  5. Davison A. J., Dolan A., Akter P., Addison C., Dargan D. J., Alcendor D. J., McGeoch D. J., Hayward G. S. 2003; The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome. J Gen Virol 84:17–28 [CrossRef]
    [Google Scholar]
  6. Dunn W., Chou C., Li H., Hai R., Patterson D., Stolc V., Zhu H., Liu F. 2003; Functional profiling of a human cytomegalovirus genome. Proc Natl Acad Sci U S A 100:14223–14228 [CrossRef]
    [Google Scholar]
  7. Dyson H. J., Wright P. E. 2005; Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6:197–208 [CrossRef]
    [Google Scholar]
  8. Garay-Malpartida H. M., Occhiucci J. M., Alves J., Belizario J. E. 2005; CaSPredictor: a new computer-based tool for caspase substrate prediction. Bioinformatics 21:Suppl. 1i169–i176 [CrossRef]
    [Google Scholar]
  9. Grey F., Antoniewicz A., Allen E., Saugstad J., McShea A., Carrington J. C., Nelson J. 2005; Identification and characterization of human cytomegalovirus-encoded microRNAs. J Virol 79:12095–12099 [CrossRef]
    [Google Scholar]
  10. Grey F., Meyers H., White E. A., Spector D. H., Nelson J. 2007; A human cytomegalovirus-encoded microRNA regulates expression of multiple viral genes involved in replication. PLoS Pathog 3:e163 [CrossRef]
    [Google Scholar]
  11. Irmiere A., Gibson W. 1983; Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology 130:118–133 [CrossRef]
    [Google Scholar]
  12. Iskenderian A. C., Huang L., Reilly A., Stenberg R. M., Anders D. G. 1996; Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes. J Virol 70:383–392
    [Google Scholar]
  13. Iwayama S., Yamamoto T., Furuya T., Kobayashi R., Ikuta K., Hirai K. 1994; Intracellular localization and DNA-binding activity of a class of viral early phosphoproteins in human fibroblasts infected with human cytomegalovirus (Towne strain). J Gen Virol 75:3309–3318 [CrossRef]
    [Google Scholar]
  14. Kerry J. A., Priddy M. A., Jervey T. Y., Kohler C. P., Staley T. L., Vanson C. D., Jones T. R., Iskenderian A. C., Anders D. G., Stenberg R. M. 1996; Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection. J Virol 70:373–382
    [Google Scholar]
  15. Lehner R., Meyer H., Mach M. 1989; Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses. J Virol 63:3792–3800
    [Google Scholar]
  16. Linding R., Jensen L. J., Diella F., Bork P., Gibson T. J., Russell R. B. 2003; Protein disorder prediction: implications for structural proteomics. Structure 11:1453–1459 [CrossRef]
    [Google Scholar]
  17. Mocarski E. S. Jr, Shenk T., Pass R. F. 2007; Cytomegalovirus. In Fields Virology , 5th edn. pp 2701–2772Edited by Knipe M., Howley. Philadelphia, PA: Lippincott Williams & Wilkins;
    [Google Scholar]
  18. Murphy E., Yu D., Grimwood J., Schmutz J., Dickson M., Jarvis M. A., Hahn G., Nelson J. A., Myers R. M., Shenk T. E. 2003; Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc Natl Acad Sci U S A 100:14976–14981 [CrossRef]
    [Google Scholar]
  19. Murphy E., Vanicek J., Robins H., Shenk T., Levine A. J. 2008; Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs: implications for latency. Proc Natl Acad Sci U S A 105:5453–5458 [CrossRef]
    [Google Scholar]
  20. Pari G. S., Kacica M. A., Anders D. G. 1993; Open reading frames UL44, IRS1/TRS1, and UL36–38 are required for transient complementation of human cytomegalovirus ori Lyt-dependent DNA synthesis. J Virol 67:2575–2582
    [Google Scholar]
  21. Park M. Y., Kim Y. E., Seo M. R., Lee J. R., Lee C. H., Ahn J. H. 2006; Interactions among four proteins encoded by the human cytomegalovirus UL112–113 region regulate their intranuclear targeting and the recruitment of UL44 to pre-replication foci. J Virol 80:2718–2727 [CrossRef]
    [Google Scholar]
  22. Penfold M. E., Mocarski E. S. Jr 1997; Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis. Virology 239:46–61 [CrossRef]
    [Google Scholar]
  23. Piwko W., Jentsch S. 2006; Proteasome-mediated protein processing by bidirectional degradation initiated from an internal site. Nat Struct Mol Biol 13:691–697 [CrossRef]
    [Google Scholar]
  24. Rechsteiner M. 1990; PEST sequences are signals for rapid intracellular proteolysis. Semin Cell Biol 1:433–440
    [Google Scholar]
  25. Rechsteiner M., Rogers S. W. 1996; PEST sequences and regulation by proteolysis. Trends Biochem Sci 21:267–271 [CrossRef]
    [Google Scholar]
  26. Rogers S., Wells R., Rechsteiner M. 1986; Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234:364–368 [CrossRef]
    [Google Scholar]
  27. Staprans S. I., Spector D. H. 1986; 2.2-kilobase class of early transcripts encoded by cell-related sequences in human cytomegalovirus strain AD169. J Virol 57:591–602
    [Google Scholar]
  28. Talbot P., Almeida J. D. 1977; Human cytomegalovrius: purification of enveloped virions and dense bodies. J Gen Virol 36:345–349 [CrossRef]
    [Google Scholar]
  29. Tompa P., Buzder-Lantos P., Tantos A., Farkas A., Szilágyi A., Bánóczi Z., Hudecz F., Friedrich P. 2004; On the sequential determinants of calpain cleavage. J Biol Chem 279:20775–20785 [CrossRef]
    [Google Scholar]
  30. Tyan Y. C., Wu H. Y., Su W. C., Chen P. W., Liao P. C. 2005; Proteomic analysis of human pleural effusion. Proteomics 5:1062–1074 [CrossRef]
    [Google Scholar]
  31. Varnum S. M., Streblow D. N., Monroe M. E., Smith P., Auberry K. J., Pasa-Tolic L., Wang D., Camp D. G., II, Rodland K. other authors 2004; Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J Virol 78:10960–10966 [CrossRef]
    [Google Scholar]
  32. Wang S.-K., Duh C.-Y., Chang T.-T. 2000; Cloning and identification of regulatory gene UL76 of human cytomegalovirus. J Gen Virol 81:2407–2416
    [Google Scholar]
  33. Wang S. K., Duh C. Y., Wu C. W. 2004; Human cytomegalovirus UL76 encodes a novel virion-associated protein that is able to inhibit viral replication. J Virol 78:9750–9762 [CrossRef]
    [Google Scholar]
  34. Wells R., Stensland L., Vieira J. 2009; The HCMV UL112–113 locus can activate the full KSHV lytic replication cycle. J Virol 83:4695–4699 [CrossRef]
    [Google Scholar]
  35. Winkler M., Stamminger T. 1996; A specific subform of the human cytomegalovirus transactivator protein pUL69 is contained within the tegument of virus particles. J Virol 70:8984–8987
    [Google Scholar]
  36. Wright D. A., Spector D. H. 1989; Posttranscriptional regulation of a class of human cytomegalovirus phosphoproteins encoded by an early transcription unit. J Virol 63:3117–3127
    [Google Scholar]
  37. Wright D. A., Staprans S. I., Spector D. H. 1988; Four phosphoproteins with common amino termini are encoded by human cytomegalovirus AD169. J Virol 62:331–340
    [Google Scholar]
  38. Yamamoto T., Suzuki S., Radsak K., Hirai K. 1998; The UL112/113 gene products of human cytomegalovirus which colocalize with viral DNA in infected cell nuclei are related to efficient viral DNA replication. Virus Res 56:107–114 [CrossRef]
    [Google Scholar]
  39. Yao F., Courtney R. J. 1992; Association of ICP0 but not ICP27 with purified virions of herpes simplex virus type 1. J Virol 66:2709–2716
    [Google Scholar]
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