Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Aug;70(8):5272–5281. doi: 10.1128/jvi.70.8.5272-5281.1996

The variable 3' ends of a human cytomegalovirus oriLyt transcript (SRT) overlap an essential, conserved replicator element.

L Huang 1, Y Zhu 1, D G Anders 1
PMCID: PMC190484  PMID: 8764037

Abstract

The genetically defined human cytomegalovirus (HCMV) lytic-phase replicator, oriLyt, comprises more than 2 kb in a structurally complex region that spans a variety of potential transcription control signals. Several transcripts originate within or cross oriLyt, and we are studying these oriLyt transcription units to determine whether they participate in initiating or regulating lytic-phase DNA synthesis. Results presented here establish the temporal accumulation and structure of the smallest replicator transcript, which we call SRT, and identify a single-sequence element essential to replicator function. SRT was detected as early as 2 h after HCMV infection of human fibroblast cells; transcript levels increased by 24 h and continued to increase thereafter. Consistent with its early appearance, treatment of HCMV-infected cells with the viral DNA polymerase inhibitor phosphonoformic acid had no effect on SRT accumulation; however, no SRT was detected in RNA preparations from cycloheximide-treated infected cells. Additional Northern (RNA) analysis localized the 0.2- to 0.25-kb SRT to an apparently noncoding segment near the center of the oriLyt core region. Reverse transcriptase PCR (rapid amplification of cDNA 5' ends [5'-RACE]) identified a single 5' end. In transient-transfection assays, the sequence immediately upstream of SRT functioned as a promoter responsive to HCMV infection when placed upstream of a reporter gene, suggesting that SRT is the product of a discrete transcription unit. RNA ligase-mediated 3'-RACE showed that SRT is not polyadenylated and has heterogeneous 3' ends within a roughly 45-nucleotide window overlapping an oligopyrimidine sequence having counterparts in the lytic-phase replicators of several herpesviruses. Mutation of the oligopyrimidine element showed that it is essential to oriLyt replicator function; it is the only essential single-sequence HCMV oriLyt replicator element described to date. Collectively, the location of SRT near the center of the oriLyt core region, its early expression, its overlapping relationship with a sequence element essential to replicator function, and its similarities to replicator transcripts in other systems suggest the possibility that SRT plays a role in initiating or regulating HCMV lytic-phase DNA synthesis.

Full Text

The Full Text of this article is available as a PDF (545.9 KB).

Selected References

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

  1. Alford C. A., Stagno S., Pass R. F., Britt W. J. Congenital and perinatal cytomegalovirus infections. Rev Infect Dis. 1990 Sep-Oct;12 (Suppl 7):S745–S753. doi: 10.1093/clinids/12.supplement_7.s745. [DOI] [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., 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]
  4. Arlt H., Lang D., Gebert S., Stamminger T. Identification of binding sites for the 86-kilodalton IE2 protein of human cytomegalovirus within an IE2-responsive viral early promoter. J Virol. 1994 Jul;68(7):4117–4125. doi: 10.1128/jvi.68.7.4117-4125.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arrand J. R., Rymo L. Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells. J Virol. 1982 Feb;41(2):376–389. doi: 10.1128/jvi.41.2.376-389.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Belyavsky A., Vinogradova T., Rajewsky K. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells. Nucleic Acids Res. 1989 Apr 25;17(8):2919–2932. doi: 10.1093/nar/17.8.2919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Birchmeier C., Schümperli D., Sconzo G., Birnstiel M. L. 3' editing of mRNAs: sequence requirements and involvement of a 60-nucleotide RNA in maturation of histone mRNA precursors. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1057–1061. doi: 10.1073/pnas.81.4.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bruckner R. C., Crute J. J., Dodson M. S., Lehman I. R. The herpes simplex virus 1 origin binding protein: a DNA helicase. J Biol Chem. 1991 Feb 5;266(4):2669–2674. [PubMed] [Google Scholar]
  10. Cha T. A., Tom E., Kemble G. W., Duke G. M., Mocarski E. S., Spaete R. R. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol. 1996 Jan;70(1):78–83. doi: 10.1128/jvi.70.1.78-83.1996. [DOI] [PMC free article] [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. 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]
  13. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  14. Clayton D. A. Replication and transcription of vertebrate mitochondrial DNA. Annu Rev Cell Biol. 1991;7:453–478. doi: 10.1146/annurev.cb.07.110191.002321. [DOI] [PubMed] [Google Scholar]
  15. DePamphili M. L. How transcription factors regulate origins of DNA replication in eukaryotic cells. Trends Cell Biol. 1993 May;3(5):161–167. doi: 10.1016/0962-8924(93)90137-p. [DOI] [PubMed] [Google Scholar]
  16. Elias P., Lehman I. R. Interaction of origin binding protein with an origin of replication of herpes simplex virus 1. Proc Natl Acad Sci U S A. 1988 May;85(9):2959–2963. doi: 10.1073/pnas.85.9.2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Elias P., O'Donnell M. E., Mocarski E. S., Lehman I. R. A DNA binding protein specific for an origin of replication of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6322–6326. doi: 10.1073/pnas.83.17.6322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fixman E. D., Hayward G. S., Hayward S. D. Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays. J Virol. 1995 May;69(5):2998–3006. doi: 10.1128/jvi.69.5.2998-3006.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fowlkes D. M., Shenk T. Transcriptional control regions of the adenovirus VAI RNA gene. Cell. 1980 Nov;22(2 Pt 2):405–413. doi: 10.1016/0092-8674(80)90351-7. [DOI] [PubMed] [Google Scholar]
  20. Frederiksen S., Hellung-Larsen P., Gram Jensen E. The differential inhibitory effect of alpha-amanitin on the synthesis of low molecular weight RNA components in BHK cells. FEBS Lett. 1978 Mar 15;87(2):227–231. doi: 10.1016/0014-5793(78)80338-x. [DOI] [PubMed] [Google Scholar]
  21. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  23. Gruffat H., Renner O., Pich D., Hammerschmidt W. Cellular proteins bind to the downstream component of the lytic origin of DNA replication of Epstein-Barr virus. J Virol. 1995 Mar;69(3):1878–1886. doi: 10.1128/jvi.69.3.1878-1886.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gupta G., García A. E., Hiriyanna K. T. Sampling of the conformations of the d(CGCTGCGGC) hairpin in solution by two-dimensional nuclear magnetic resonance and theoretical methods. Biochemistry. 1993 Jan 26;32(3):948–960. doi: 10.1021/bi00054a028. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  28. Inoue N., Dambaugh T. R., Rapp J. C., Pellett P. E. Alphaherpesvirus origin-binding protein homolog encoded by human herpesvirus 6B, a betaherpesvirus, binds to nucleotide sequences that are similar to ori regions of alphaherpesviruses. J Virol. 1994 Jul;68(7):4126–4136. doi: 10.1128/jvi.68.7.4126-4136.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Inoue N., Pellett P. E. Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence. J Virol. 1995 Aug;69(8):4619–4627. doi: 10.1128/jvi.69.8.4619-4627.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Iskenderian A. C., Huang L., Reilly A., Stenberg R. M., Anders D. G. Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes. J Virol. 1996 Jan;70(1):383–392. doi: 10.1128/jvi.70.1.383-392.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jat P., Arrand J. R. In vitro transcription of two Epstein-Barr virus specified small RNA molecules. Nucleic Acids Res. 1982 Jun 11;10(11):3407–3425. doi: 10.1093/nar/10.11.3407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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. Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection. J Virol. 1996 Jan;70(1):373–382. doi: 10.1128/jvi.70.1.373-382.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Knitt D. S., Narlikar G. J., Herschlag D. Dissection of the role of the conserved G.U pair in group I RNA self-splicing. Biochemistry. 1994 Nov 22;33(46):13864–13879. doi: 10.1021/bi00250a041. [DOI] [PubMed] [Google Scholar]
  34. Koff A., Schwedes J. F., Tegtmeyer P. Herpes simplex virus origin-binding protein (UL9) loops and distorts the viral replication origin. J Virol. 1991 Jun;65(6):3284–3292. doi: 10.1128/jvi.65.6.3284-3292.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Koff A., Tegtmeyer P. Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein. J Virol. 1988 Nov;62(11):4096–4103. doi: 10.1128/jvi.62.11.4096-4103.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Krieg P. A., Melton D. A. Formation of the 3' end of histone mRNA by post-transcriptional processing. Nature. 1984 Mar 8;308(5955):203–206. doi: 10.1038/308203a0. [DOI] [PubMed] [Google Scholar]
  37. LaFemina R. L., Hayward G. S. Replicative forms of human cytomegalovirus DNA with joined termini are found in permissively infected human cells but not in non-permissive Balb/c-3T3 mouse cells. J Gen Virol. 1983 Feb;64(Pt 2):373–389. doi: 10.1099/0022-1317-64-2-373. [DOI] [PubMed] [Google Scholar]
  38. Liu X., Gorovsky M. A. Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE). Nucleic Acids Res. 1993 Oct 25;21(21):4954–4960. doi: 10.1093/nar/21.21.4954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Marschalek R., Amon-Böhm E., Stoerker J., Klages S., Fleckenstein B., Dingermann T. CMER, an RNA encoded by human cytomegalovirus is most likely transcribed by RNA polymerase III. Nucleic Acids Res. 1989 Jan 25;17(2):631–643. doi: 10.1093/nar/17.2.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. McVoy M. A., Adler S. P. Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within the concatemer. J Virol. 1994 Feb;68(2):1040–1051. doi: 10.1128/jvi.68.2.1040-1051.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Murphy J. T., Burgess R. R., Dahlberg J. E., Lund E. Transcription of a gene for human U1 small nuclear RNA. Cell. 1982 May;29(1):265–274. doi: 10.1016/0092-8674(82)90111-8. [DOI] [PubMed] [Google Scholar]
  43. Olivo P. D., Nelson N. J., Challberg M. D. Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5414–5418. doi: 10.1073/pnas.85.15.5414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pari G. S., Anders D. G. Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication. J Virol. 1993 Dec;67(12):6979–6988. doi: 10.1128/jvi.67.12.6979-6988.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pari G. S., Kacica M. A., Anders D. G. Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. J Virol. 1993 May;67(5):2575–2582. doi: 10.1128/jvi.67.5.2575-2582.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Reisman D., Sugden B. trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1. Mol Cell Biol. 1986 Nov;6(11):3838–3846. doi: 10.1128/mcb.6.11.3838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ryon J. J., Fixman E. D., Houchens C., Zong J., Lieberman P. M., Chang Y. N., Hayward G. S., Hayward S. D. The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals. J Virol. 1993 Jul;67(7):4006–4016. doi: 10.1128/jvi.67.7.4006-4016.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schepers A., Pich D., Hammerschmidt W. A transcription factor with homology to the AP-1 family links RNA transcription and DNA replication in the lytic cycle of Epstein-Barr virus. EMBO J. 1993 Oct;12(10):3921–3929. doi: 10.1002/j.1460-2075.1993.tb06070.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schepers A., Pich D., Mankertz J., Hammerschmidt W. cis-acting elements in the lytic origin of DNA replication of Epstein-Barr virus. J Virol. 1993 Jul;67(7):4237–4245. doi: 10.1128/jvi.67.7.4237-4245.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schwartz R., Sommer M. H., Scully A., Spector D. H. Site-specific binding of the human cytomegalovirus IE2 86-kilodalton protein to an early gene promoter. J Virol. 1994 Sep;68(9):5613–5622. doi: 10.1128/jvi.68.9.5613-5622.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sinzger C., Grefte A., Plachter B., Gouw A. S., The T. H., Jahn G. Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J Gen Virol. 1995 Apr;76(Pt 4):741–750. doi: 10.1099/0022-1317-76-4-741. [DOI] [PubMed] [Google Scholar]
  52. Spiro C., Richards J. P., Chandrasekaran S., Brennan R. G., McMurray C. T. Secondary structure creates mismatched base pairs required for high-affinity binding of cAMP response element-binding protein to the human enkephalin enhancer. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4606–4610. doi: 10.1073/pnas.90.10.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stow N. D. Localization of an origin of DNA replication within the TRS/IRS repeated region of the herpes simplex virus type 1 genome. EMBO J. 1982;1(7):863–867. doi: 10.1002/j.1460-2075.1982.tb01261.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stow N. D., McMonagle E. C. Characterization of the TRS/IRS origin of DNA replication of herpes simplex virus type 1. Virology. 1983 Oct 30;130(2):427–438. doi: 10.1016/0042-6822(83)90097-1. [DOI] [PubMed] [Google Scholar]
  55. Strobel S. A., Cech T. R. Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site. Science. 1995 Feb 3;267(5198):675–679. doi: 10.1126/science.7839142. [DOI] [PubMed] [Google Scholar]
  56. Weir H. M., Calder J. M., Stow N. D. Binding of the herpes simplex virus type 1 UL9 gene product to an origin of viral DNA replication. Nucleic Acids Res. 1989 Feb 25;17(4):1409–1425. doi: 10.1093/nar/17.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weller S. K., Spadaro A., Schaffer J. E., Murray A. W., Maxam A. M., Schaffer P. A. Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis. Mol Cell Biol. 1985 May;5(5):930–942. doi: 10.1128/mcb.5.5.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wong S. W., Schaffer P. A. Elements in the transcriptional regulatory region flanking herpes simplex virus type 1 oriS stimulate origin function. J Virol. 1991 May;65(5):2601–2611. doi: 10.1128/jvi.65.5.2601-2611.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wysokenski D. A., Yates J. L. Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus. J Virol. 1989 Jun;63(6):2657–2666. doi: 10.1128/jvi.63.6.2657-2666.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xu B., Clayton D. A. A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence. Mol Cell Biol. 1995 Jan;15(1):580–589. doi: 10.1128/mcb.15.1.580. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES