1887

Abstract

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its ORF, showed reduced virus yields (2–3 log p.f.u. ml) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. gene transcription was initiated at early times post-infection, while production of a transcript from ORF in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs and are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but is important for optimal viral growth and .

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.83133-0
2007-11-01
2019-11-13
Loading full text...

Full text loading...

/deliver/fulltext/jgv/88/11/2941.html?itemId=/content/journal/jgv/10.1099/vir.0.83133-0&mimeType=html&fmt=ahah

References

  1. Alford, C. A. & Britt, W. J. ( 1993; ). Cytomegalovirus. In The Human Herpesviruses, pp. 227–255. Edited by B. Roizman, R. J. Whitley & C. Lopez. New York: Raven Press.
  2. Bieleski, L. & Talbot, S. J. ( 2001; ). Kaposi's sarcoma-associated herpesvirus cyclin open reading frame contains an internal ribosome entry site. J Virol 75, 1864–1869.[CrossRef]
    [Google Scholar]
  3. Brocchieri, L., Kledal, T. N., Karlin, S. & Mocarski, E. S. ( 2005; ). Predicting coding potential from genome sequence: application to betaherpesviruses infecting rats and mice. J Virol 79, 7570–7596.[CrossRef]
    [Google Scholar]
  4. Cao, J. & Geballe, A. ( 1996; ). Coding sequence-dependent ribosomal arrest at termination of translation. Mol Cell Biol 16, 603–608.
    [Google Scholar]
  5. Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R., Horsnell, T., Hutchison, C. A., 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]
  6. 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]
  7. Dolan, A., Cunningham, C., Hector, R. D., Hassan-Walker, A. F., Lee, L., Addison, C., Dargan, D. J., McGeoch, D. J., Gatherer, D. & other authors ( 2004; ). Genetic content of wild-type human cytomegalovirus. J Gen Virol 85, 1301–1312.[CrossRef]
    [Google Scholar]
  8. 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]
  9. Gingras, A. C., Raught, B. & Sonenberg, N. ( 1999; ). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68, 913–963.[CrossRef]
    [Google Scholar]
  10. Ho, M. ( 1991; ). Cytomegalovirus: Biology and Infection, 2nd edn. New York: Plenum.
  11. Holtappels, R., Munks, M. W., Podlech, J. & Reddehase, M. J. ( 2006; ). CD8 T-cell-based immunotherapy of cytomegalovirus disease in the mouse model of the immunocompromised bone marrow transplant recipient. In Cytomegalovirus Molecular Biology and Immunology, pp. 383–418. Edited by M. J. Reddehase. Norfolk, UK: Caister Academic Press.
  12. Jang, S. K., Krausslich, H. G., Nicklin, M. J., Duke, G. M., Palmenberg, A. C. & Wimmer, E. ( 1988; ). A segment of the 5′ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 62, 2636–2643.
    [Google Scholar]
  13. Jonjic, S., Bubic, I. & Krmpotic, A. ( 2006; ). Innate immunity to cytomegaloviruses. In Cytomegalovirus Molecular Biology and Immunology, pp. 285–319. Edited by M. J. Reddehase. Norfolk, UK: Caister Academic Press.
  14. Kattenhorn, L. M., Mills, R., Wagner, M., Lomsadze, A., Makeev, V., Borodovsky, M., Ploegh, H. L. & Kessler, B. M. ( 2004; ). Identification of proteins associated with murine cytomegalovirus virions. J Virol 78, 11187–11197.[CrossRef]
    [Google Scholar]
  15. Li, G. P. & Rice, C. M. ( 1989; ). Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication. J Virol 63, 1326–1337.
    [Google Scholar]
  16. Lyons, P. A., Allan, J. E., Carrello, C., Shellam, G. R. & Scalzo, A. A. ( 1996; ). Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition. J Gen Virol 77, 2615–2623.[CrossRef]
    [Google Scholar]
  17. Murphy, E., Rigoutsos, I., Shibuya, T. & Shenk, T. E. ( 2003; ). Reevaluation of human cytomegalovirus coding potential. Proc Natl Acad Sci U S A 100, 13585–13590.[CrossRef]
    [Google Scholar]
  18. Pelletier, J. & Sonenberg, N. ( 1988; ). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320–325.[CrossRef]
    [Google Scholar]
  19. Rawlinson, W. D., Farrell, H. E. & Barrell, B. G. ( 1996; ). Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol 70, 8833–8849.
    [Google Scholar]
  20. Sammons, C. C. & Sweet, C. ( 1989; ). Isolation and preliminary characterization of temperature-sensitive mutants of mouse cytomegalovirus of differing virulence for 1-week-old mice. J Gen Virol 70, 2373–2381.[CrossRef]
    [Google Scholar]
  21. Shatkin, A. J. ( 1976; ). Capping of eucaryotic mRNAs. Cell 9, 645–653.[CrossRef]
    [Google Scholar]
  22. Sweet, C. ( 1999; ). The pathogenicity of cytomegalovirus. FEMS Microbiol Rev 23, 457–482.[CrossRef]
    [Google Scholar]
  23. Sweet, C., Ball, K., Morley, P. J., Guilfoyle, K. & Kirby, M. ( 2007; ). Mutations in the temperature-sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: a study of genes involved in immune evasion, DNA packaging and processing, and DNA replication. J Med Virol 79, 285–299.[CrossRef]
    [Google Scholar]
  24. Tang, Q., Murphy, E. A. & Maul, G. G. ( 2006; ). Experimental confirmation of global murine cytomegalovirus open reading frames by transcriptional detection and partial characterization of newly described gene products. J Virol 80, 6873–6882.[CrossRef]
    [Google Scholar]
  25. Wagner, M., 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]
  26. Wills, N. M., Gesteland, R. F. & Atkins, J. F. ( 1994; ). Pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2. EMBO J 13, 4137–4144.
    [Google Scholar]
  27. Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G. & Court, D. L. ( 2000; ). An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97, 5978–5983.[CrossRef]
    [Google Scholar]
  28. Yu, D., Silva, M. C. & Shenk, T. ( 2003; ). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci U S A 100, 12396–12401.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.83133-0
Loading
/content/journal/jgv/10.1099/vir.0.83133-0
Loading

Data & Media loading...

Supplements

vol. , part 11, pp. 2941 - 2951

EMBL accession numbers of the sequences reported in this paper [PDF](16 KB)



PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error