1887

Abstract

Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.83286-0
2008-02-01
2019-11-14
Loading full text...

Full text loading...

/deliver/fulltext/jgv/89/2/359.html?itemId=/content/journal/jgv/10.1099/vir.0.83286-0&mimeType=html&fmt=ahah

References

  1. Adler, B., Scrivano, L., Ruzcics, Z., Rupp, B., Sinzger, C. & Koszinowski, U. ( 2006; ). Role of human cytomegalovirus UL131A in cell type-specific virus entry and release. J Gen Virol 87, 2451–2460.[CrossRef]
    [Google Scholar]
  2. Akter, P., Cunningham, C., McSharry, B. P., Dolan, A., Addison, C., Dargan, D. J., Hassan-Walker, A. F., Emery, V. C., Griffiths, P. D. & other authors ( 2003; ). Two novel spliced genes in human cytomegalovirus. J Gen Virol 84, 1117–1122.[CrossRef]
    [Google Scholar]
  3. Allal, C., Buisson-Brenac, C., Marion, V., Claudel-Renard, C., Faraut, T., Dal Monte, P., Streblow, D., Record, M. & Davignon, J. L. ( 2004; ). Human cytomegalovirus carries a cell-derived phospholipase A2 required for infectivity. J Virol 78, 7717–7726.[CrossRef]
    [Google Scholar]
  4. Baldanti, F., Paolucci, S., Campanini, G., Sarasini, A., Percivalle, E., Revello, M. G. & Gerna, G. ( 2006; ). Human cytomegalovirus UL131A, UL130 and UL128 genes are highly conserved among field isolates. Arch Virol 151, 1225–1233.[CrossRef]
    [Google Scholar]
  5. Bentz, G. L., Jarquin-Pardo, M., Chan, G., Smith, M. S., Sinzger, C. & Yurochko, A. D. ( 2006; ). Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV. J Virol 80, 11539–11555.[CrossRef]
    [Google Scholar]
  6. Borst, E. M., Hahn, G., Koszinowski, U. H. & Messerle, M. ( 1999; ). Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J Virol 73, 8320–8329.
    [Google Scholar]
  7. Brune, W., Menard, C., Hobom, U., Odenbreit, S., Messerle, M. & Koszinowski, U. H. ( 1999; ). Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis. Nat Biotechnol 17, 360–364.[CrossRef]
    [Google Scholar]
  8. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. & Thompson, J. D. ( 2003; ). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31, 3497–3500.[CrossRef]
    [Google Scholar]
  9. 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]
  10. 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]
  11. Gerna, G., Percivalle, E., Baldanti, F. & Revello, M. G. ( 2002; ). Lack of transmission to polymorphonuclear leukocytes and human umbilical vein endothelial cells as a marker of attenuation of human cytomegalovirus. J Med Virol 66, 335–339.[CrossRef]
    [Google Scholar]
  12. Hahn, G., Khan, H., Baldanti, F., Koszinowski, U. H., Revello, M. G. & Gerna, G. ( 2002; ). The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics. J Virol 76, 9551–9555.[CrossRef]
    [Google Scholar]
  13. Hahn, G., Revello, M. G., Patrone, M., Percivalle, E., Campanini, G., Sarasini, A., Wagner, M., Gallina, A., Milanesi, G. & other authors ( 2004; ). Human cytomegalovirus UL131–128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. J Virol 78, 10023–10033.[CrossRef]
    [Google Scholar]
  14. Hall, T. ( 2001; ). BioEdit, 5.0.6 edn. Raleigh: Department of Microbiology, North Carolina State University.
  15. Hertel, L., Lacaille, V. G., Strobl, H., Mellins, E. D. & Mocarski, E. S. ( 2003; ). Susceptibility of immature and mature Langerhans cell-type dendritic cells to infection and immunomodulation by human cytomegalovirus. J Virol 77, 7563–7574.[CrossRef]
    [Google Scholar]
  16. Hirt, B. ( 1967; ). Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26, 365–369.[CrossRef]
    [Google Scholar]
  17. Homman-Loudiyi, M., Hultenby, K., Britt, W. & Soderberg-Naucler, C. ( 2003; ). Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-golgi network 46, and mannosidase II. J Virol 77, 3191–3203.[CrossRef]
    [Google Scholar]
  18. Jarvis, M. A. & Nelson, J. A. ( 2002; ). Mechanisms of human cytomegalovirus persistence and latency. Front Biosci 7, d1575–d1582.[CrossRef]
    [Google Scholar]
  19. Kilpatrick, B. A. & Huang, E. S. ( 1977; ). Human cytomegalovirus genome: partial denaturation map and organization of genome sequences. J Virol 24, 261–276.
    [Google Scholar]
  20. King, C. A., Baillie, J. & Sinclair, J. H. ( 2006; ). Human cytomegalovirus modulation of CCR5 expression on myeloid cells affects susceptibility to human immunodeficiency virus type 1 infection. J Gen Virol 87, 2171–2180.[CrossRef]
    [Google Scholar]
  21. Laib Sampaio, K. L., Cavignac, Y., Stierhof, Y. D. & Sinzger, C. ( 2005; ). Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements. J Virol 79, 2754–2767.[CrossRef]
    [Google Scholar]
  22. Mahy, B. W. J. & Kangro, H. O. ( 1996; ). Virology Methods Manual, pp. 35–37. San Diego: Academic Press.
  23. McVoy, M. A. & Ramnarain, D. ( 2000; ). Machinery to support genome segment inversion exists in a herpesvirus which does not naturally contain invertible elements. J Virol 74, 4882–4887.[CrossRef]
    [Google Scholar]
  24. Messerle, M., Crnkovic, I., Hammerschmidt, W., Ziegler, H. & Koszinowski, U. H. ( 1997; ). Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 94, 14759–14763.[CrossRef]
    [Google Scholar]
  25. Messerle, M., Hahn, G., Brune, W. & Koszinowski, U. H. ( 2000; ). Cytomegalovirus bacterial artificial chromosomes: a new herpesvirus vector approach. Adv Virus Res 55, 463–478.
    [Google Scholar]
  26. Moutaftsi, M., Brennan, P., Spector, S. A. & Tabi, Z. ( 2004; ). Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells. J Virol 78, 3046–3054.[CrossRef]
    [Google Scholar]
  27. Murphy, E., Rigoutsos, I., Shibuya, T. & Shenk, T. E. ( 2003a; ). Reevaluation of human cytomegalovirus coding potential. Proc Natl Acad Sci U S A 100, 13585–13590.[CrossRef]
    [Google Scholar]
  28. Murphy, E., Yu, D., Grimwood, J., Schmutz, J., Dickson, M., Jarvis, M. A., Hahn, G., Nelson, J. A., Myers, R. M. & Shenk, T. E. ( 2003b; ). Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc Natl Acad Sci U S A 100, 14976–14981.[CrossRef]
    [Google Scholar]
  29. Patrone, M., Secchi, M., Fiorina, L., Ierardi, M., Milanesi, G. & Gallina, A. ( 2005; ). Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion. J Virol 79, 8361–8373.[CrossRef]
    [Google Scholar]
  30. Pretsch, R., Kleining, V., Schierling, K., Hahn, G., Sinzger, C., Mertens, T. & Winkler, M. ( 2005; ). Human cytomegalovirus pUL24, part of a novel protein complex, is necessary for endothelial cell tropism. In 30th International Herpesvirus Workshop. Turku, Finland.
  31. Reeves, M. B., Lehner, P. J., Sissons, J. G. & Sinclair, J. H. ( 2005; ). An in vitro model for the regulation of human cytomegalovirus latency and reactivation in dendritic cells by chromatin remodelling. J Gen Virol 86, 2949–2954.[CrossRef]
    [Google Scholar]
  32. Reinhardt, B., Schaarschmidt, P., Bossert, A., Luske, A., Finkenzeller, G., Mertens, T. & Michel, D. ( 2005; ). Upregulation of functionally active vascular endothelial growth factor by human cytomegalovirus. J Gen Virol 86, 23–30.[CrossRef]
    [Google Scholar]
  33. Sinzger, C. & Jahn, G. ( 1996; ). Human cytomegalovirus cell tropism and pathogenesis. Intervirology 39, 302–319.
    [Google Scholar]
  34. Sinzger, C., Knapp, J., Plachter, B., Schmidt, K. & Jahn, G. ( 1997; ). Quantification of replication of clinical cytomegalovirus isolates in cultured endothelial cells and fibroblasts by a focus expansion assay. J Virol Methods 63, 103–112.[CrossRef]
    [Google Scholar]
  35. Sinzger, C., Schmidt, K., Knapp, J., Kahl, M., Beck, R., Waldman, J., Hebart, H., Einsele, H. & Jahn, G. ( 1999; ). Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome. J Gen Virol 80, 2867–2877.
    [Google Scholar]
  36. Sinzger, C., Kahl, M., Laib, K., Klingel, K., Rieger, P., Plachter, B. & Jahn, G. ( 2000; ). Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus. J Gen Virol 81, 3021–3035.
    [Google Scholar]
  37. Tischer, B. K., von Einem, J., Kaufer, B. & Osterrieder, N. ( 2006; ). Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40, 191–197.[CrossRef]
    [Google Scholar]
  38. Tomasec, P., Wang, E. C., Davison, A. J., Vojtesek, B., Armstrong, M., Griffin, C., McSharry, B. P., Morris, R. J., Llewellyn-Lacey, S. & other authors ( 2005; ). Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat Immunol 6, 181–188.
    [Google Scholar]
  39. Wang, D. & Shenk, T. ( 2005a; ). Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism. J Virol 79, 10330–10338.[CrossRef]
    [Google Scholar]
  40. Wang, D. & Shenk, T. ( 2005b; ). Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci U S A 102, 18153–18158.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.83286-0
Loading
/content/journal/jgv/10.1099/vir.0.83286-0
Loading

Data & Media loading...

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