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. 1993 May;67(5):2575–2582. doi: 10.1128/jvi.67.5.2575-2582.1993

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

G S Pari 1, M A Kacica 1, D G Anders 1
PMCID: PMC237578  PMID: 8386266

Abstract

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anders D. G., Irmiere A., Gibson W. Identification and characterization of a major early cytomegalovirus DNA-binding protein. J Virol. 1986 May;58(2):253–262. doi: 10.1128/jvi.58.2.253-262.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anders D. G., Kacica M. A., Pari G., Punturieri S. M. Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. J Virol. 1992 Jun;66(6):3373–3384. doi: 10.1128/jvi.66.6.3373-3384.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anders D. G., Kidd J. R., Gibson W. Immunological characterization of an early cytomegalovirus single-strand DNA-binding protein with similarities to the HSV major DNA-binding protein. Virology. 1987 Dec;161(2):579–588. doi: 10.1016/0042-6822(87)90154-1. [DOI] [PubMed] [Google Scholar]
  4. Anders D. G., Punturieri S. M. Multicomponent origin of cytomegalovirus lytic-phase DNA replication. J Virol. 1991 Feb;65(2):931–937. doi: 10.1128/jvi.65.2.931-937.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  6. Baracchini E., Glezer E., Fish K., Stenberg R. M., Nelson J. A., Ghazal P. An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator. Virology. 1992 Jun;188(2):518–529. doi: 10.1016/0042-6822(92)90506-k. [DOI] [PubMed] [Google Scholar]
  7. Benson J. D., Huang E. S. Two specific topoisomerase II inhibitors prevent replication of human cytomegalovirus DNA: an implied role in replication of the viral genome. J Virol. 1988 Dec;62(12):4797–4800. doi: 10.1128/jvi.62.12.4797-4800.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carmichael E. P., Kosovsky M. J., Weller S. K. Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis. J Virol. 1988 Jan;62(1):91–99. doi: 10.1128/jvi.62.1.91-99.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Challberg M. D. A method for identifying the viral genes required for herpesvirus DNA replication. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9094–9098. doi: 10.1073/pnas.83.23.9094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  11. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  12. Chee M. S., Satchwell S. C., Preddie E., Weston K. M., Barrell B. G. Human cytomegalovirus encodes three G protein-coupled receptor homologues. Nature. 1990 Apr 19;344(6268):774–777. doi: 10.1038/344774a0. [DOI] [PubMed] [Google Scholar]
  13. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cherrington J. M., Khoury E. L., Mocarski E. S. Human cytomegalovirus ie2 negatively regulates alpha gene expression via a short target sequence near the transcription start site. J Virol. 1991 Feb;65(2):887–896. doi: 10.1128/jvi.65.2.887-896.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chiang C. M., Dong G., Broker T. R., Chow L. T. Control of human papillomavirus type 11 origin of replication by the E2 family of transcription regulatory proteins. J Virol. 1992 Sep;66(9):5224–5231. doi: 10.1128/jvi.66.9.5224-5231.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Colberg-Poley A. M., Santomenna L. D., Harlow P. P., Benfield P. A., Tenney D. J. Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression. J Virol. 1992 Jan;66(1):95–105. doi: 10.1128/jvi.66.1.95-105.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ertl P. F., Powell K. L. Physical and functional interaction of human cytomegalovirus DNA polymerase and its accessory protein (ICP36) expressed in insect cells. J Virol. 1992 Jul;66(7):4126–4133. doi: 10.1128/jvi.66.7.4126-4133.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ertl P. F., Thomas M. S., Powell K. L. High level expression of DNA polymerases from herpesviruses. J Gen Virol. 1991 Jul;72(Pt 7):1729–1734. doi: 10.1099/0022-1317-72-7-1729. [DOI] [PubMed] [Google Scholar]
  19. Fixman E. D., Hayward G. S., Hayward S. D. trans-acting requirements for replication of Epstein-Barr virus ori-Lyt. J Virol. 1992 Aug;66(8):5030–5039. doi: 10.1128/jvi.66.8.5030-5039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fleckenstein B., Müller I., Collins J. Cloning of the complete human cytomegalovirus genome in cosmids. Gene. 1982 Apr;18(1):39–46. doi: 10.1016/0378-1119(82)90054-3. [DOI] [PubMed] [Google Scholar]
  21. Ghazal P., Young J., Giulietti E., DeMattei C., Garcia J., Gaynor R., Stenberg R. M., Nelson J. A. A discrete cis element in the human immunodeficiency virus long terminal repeat mediates synergistic trans activation by cytomegalovirus immediate-early proteins. J Virol. 1991 Dec;65(12):6735–6742. doi: 10.1128/jvi.65.12.6735-6742.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goldstein D. J., Weller S. K. An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis. J Virol. 1988 Aug;62(8):2970–2977. doi: 10.1128/jvi.62.8.2970-2977.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gottlieb J., Marcy A. I., Coen D. M., Challberg M. D. The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase processivity. J Virol. 1990 Dec;64(12):5976–5987. doi: 10.1128/jvi.64.12.5976-5987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Guo Z. S., DePamphilis M. L. Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication. Mol Cell Biol. 1992 Jun;12(6):2514–2524. doi: 10.1128/mcb.12.6.2514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hagemeier C., Walker S. M., Sissons P. J., Sinclair J. H. The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements. J Gen Virol. 1992 Sep;73(Pt 9):2385–2393. doi: 10.1099/0022-1317-73-9-2385. [DOI] [PubMed] [Google Scholar]
  26. Hammerschmidt W., Sugden B. Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus. Cell. 1988 Nov 4;55(3):427–433. doi: 10.1016/0092-8674(88)90028-1. [DOI] [PubMed] [Google Scholar]
  27. Hamzeh F. M., Lietman P. S., Gibson W., Hayward G. S. Identification of the lytic origin of DNA replication in human cytomegalovirus by a novel approach utilizing ganciclovir-induced chain termination. J Virol. 1990 Dec;64(12):6184–6195. doi: 10.1128/jvi.64.12.6184-6195.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hardwick J. M., Lieberman P. M., Hayward S. D. A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen. J Virol. 1988 Jul;62(7):2274–2284. doi: 10.1128/jvi.62.7.2274-2284.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Heilbronn R., Jahn G., Bürkle A., Freese U. K., Fleckenstein B., zur Hausen H. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene. J Virol. 1987 Jan;61(1):119–124. doi: 10.1128/jvi.61.1.119-124.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hermiston T. W., Malone C. L., Stinski M. F. Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter. J Virol. 1990 Jul;64(7):3532–3536. doi: 10.1128/jvi.64.7.3532-3536.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hernandez T. R., Lehman I. R. Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein. J Biol Chem. 1990 Jul 5;265(19):11227–11232. [PubMed] [Google Scholar]
  32. Huang E. S. Human cytomegalovirus. III. Virus-induced DNA polymerase. J Virol. 1975 Aug;16(2):298–310. doi: 10.1128/jvi.16.2.298-310.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Huang E. S. Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid. J Virol. 1975 Dec;16(6):1560–1565. doi: 10.1128/jvi.16.6.1560-1565.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Jones T. R., Muzithras V. P. A cluster of dispensable genes within the human cytomegalovirus genome short component: IRS1, US1 through US5, and the US6 family. J Virol. 1992 Apr;66(4):2541–2546. doi: 10.1128/jvi.66.4.2541-2546.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kiehl A., Dorsky D. I. Cooperation of EBV DNA polymerase and EA-D(BMRF1) in vitro and colocalization in nuclei of infected cells. Virology. 1991 Sep;184(1):330–340. doi: 10.1016/0042-6822(91)90849-7. [DOI] [PubMed] [Google Scholar]
  36. Li J. S., Zhou B. S., Dutschman G. E., Grill S. P., Tan R. S., Cheng Y. C. Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity. J Virol. 1987 Sep;61(9):2947–2949. doi: 10.1128/jvi.61.9.2947-2949.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lieberman P. M., Hardwick J. M., Hayward S. D. Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type-specific manner by two independent signal regions. J Virol. 1989 Jul;63(7):3040–3050. doi: 10.1128/jvi.63.7.3040-3050.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lieberman P. M., Hardwick J. M., Sample J., Hayward G. S., Hayward S. D. The zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions. J Virol. 1990 Mar;64(3):1143–1155. doi: 10.1128/jvi.64.3.1143-1155.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Malone C. L., Vesole D. H., Stinski M. F. 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 Apr;64(4):1498–1506. doi: 10.1128/jvi.64.4.1498-1506.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Marchetti M. E., Smith C. A., Schaffer P. A. A temperature-sensitive mutation in a herpes simplex virus type 1 gene required for viral DNA synthesis maps to coordinates 0.609 through 0.614 in UL. J Virol. 1988 Mar;62(3):715–721. doi: 10.1128/jvi.62.3.715-721.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Masse M. J., Karlin S., Schachtel G. A., Mocarski E. S. Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5246–5250. doi: 10.1073/pnas.89.12.5246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Matz B., Subak-Sharpe J. H., Preston V. G. Physical mapping of temperature-sensitive mutations of herpes simplex virus type 1 using cloned restriction endonuclease fragments. J Gen Virol. 1983 Oct;64(Pt 10):2261–2270. doi: 10.1099/0022-1317-64-10-2261. [DOI] [PubMed] [Google Scholar]
  43. Mocarski E. S., Pereira L., Michael N. Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1266–1270. doi: 10.1073/pnas.82.4.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mohr I. J., Clark R., Sun S., Androphy E. J., MacPherson P., Botchan M. R. Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator. Science. 1990 Dec 21;250(4988):1694–1699. doi: 10.1126/science.2176744. [DOI] [PubMed] [Google Scholar]
  45. Pari G. S., St Jeor S. C. Human cytomegalovirus major immediate early gene product can induce SV40 DNA replication in human embryonic lung cells. Virology. 1990 Dec;179(2):785–794. doi: 10.1016/0042-6822(90)90146-i. [DOI] [PubMed] [Google Scholar]
  46. Pizzorno M. C., Mullen M. A., Chang Y. N., Hayward G. S. The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal. J Virol. 1991 Jul;65(7):3839–3852. doi: 10.1128/jvi.65.7.3839-3852.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Purifoy D. J., Lewis R. B., Powell K. L. Identification of the herpes simplex virus DNA polymerase gene. Nature. 1977 Oct 13;269(5629):621–623. doi: 10.1038/269621a0. [DOI] [PubMed] [Google Scholar]
  48. Purifoy D. J., Powell K. L. Temperature-sensitive mutants in two distinct complementation groups of herpes simplex virus type 1 specify thermolabile DNA polymerase. J Gen Virol. 1981 May;54(Pt 1):219–222. doi: 10.1099/0022-1317-54-1-219. [DOI] [PubMed] [Google Scholar]
  49. Schickedanz J., Philipson L., Ansorge W., Pepperkok R., Klein R., Koszinowski U. H. The 89,000-Mr murine cytomegalovirus immediate-early protein stimulates c-fos expression and cellular DNA synthesis. J Virol. 1988 Sep;62(9):3341–3347. doi: 10.1128/jvi.62.9.3341-3347.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Staprans S. I., Rabert D. K., Spector D. H. Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene. J Virol. 1988 Sep;62(9):3463–3473. doi: 10.1128/jvi.62.9.3463-3473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stasiak P. C., Mocarski E. S. Transactivation of the cytomegalovirus ICP36 gene promoter requires the alpha gene product TRS1 in addition to IE1 and IE2. J Virol. 1992 Feb;66(2):1050–1058. doi: 10.1128/jvi.66.2.1050-1058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stenberg R. M., Fortney J., Barlow S. W., Magrane B. P., Nelson J. A., Ghazal P. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains. J Virol. 1990 Apr;64(4):1556–1565. doi: 10.1128/jvi.64.4.1556-1565.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tenney D. J., Colberg-Poley A. M. Expression of the human cytomegalovirus UL36-38 immediate early region during permissive infection. Virology. 1991 May;182(1):199–210. doi: 10.1016/0042-6822(91)90663-v. [DOI] [PubMed] [Google Scholar]
  54. Tenney D. J., Colberg-Poley A. M. Human cytomegalovirus UL36-38 and US3 immediate-early genes: temporally regulated expression of nuclear, cytoplasmic, and polysome-associated transcripts during infection. J Virol. 1991 Dec;65(12):6724–6734. doi: 10.1128/jvi.65.12.6724-6734.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wade M., Kowalik T. F., Mudryj M., Huang E. S., Azizkhan J. C. E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection. Mol Cell Biol. 1992 Oct;12(10):4364–4374. doi: 10.1128/mcb.12.10.4364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weller S. K., Lee K. J., Sabourin D. J., Schaffer P. A. Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein. J Virol. 1983 Jan;45(1):354–366. doi: 10.1128/jvi.45.1.354-366.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weston K., Barrell B. G. Sequence of the short unique region, short repeats, and part of the long repeats of human cytomegalovirus. J Mol Biol. 1986 Nov 20;192(2):177–208. doi: 10.1016/0022-2836(86)90359-1. [DOI] [PubMed] [Google Scholar]
  58. Wu C. A., Nelson N. J., McGeoch D. J., Challberg M. D. Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis. J Virol. 1988 Feb;62(2):435–443. doi: 10.1128/jvi.62.2.435-443.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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