1887

Journal of General Virology header logo

Volume 92, Issue 6

Single cell detection of latent cytomegalovirus reactivation in host tissue Free

Abstract

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor–GFP reporter mice, we never observed reactivation originating from CX3CR1 monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b and CX3CR1 subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

  • Received:
  • Accepted:
  • Published Online:
© 2011 SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.029827-0
2011-06-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/jgv/92/6/1279.html?itemId=/content/journal/jgv/10.1099/vir.0.029827-0&mimeType=html&fmt=ahah

References

  1. Balthesen M., Messerle M., Reddehase M. J. 1993; Lungs are a major organ site of cytomegalovirus latency and recurrence. J Virol 67:5360–5366[PubMed]
     [Google Scholar]
  2. Benedict C. A., De Trez C., Schneider K., Ha S., Patterson G., Ware C. F. 2006; Specific remodeling of splenic architecture by cytomegalovirus. PLoS Pathog 2:e16 [View Article][PubMed]
     [Google Scholar]
  3. Böhm V., Simon C. O., Podlech J., Seckert C. K., Gendig D., Deegen P., Gillert-Marien D., Lemmermann N. A., Holtappels R., Reddehase M. J. 2008; The immune evasion paradox: immunoevasins of murine cytomegalovirus enhance priming of CD8 T cells by preventing negative feedback regulation. J Virol 82:11637–11650 [View Article][PubMed]
     [Google Scholar]
  4. Böhm V., Seckert C. K., Simon C. O., Thomas D., Renzaho A., Gendig D., Holtappels R., Reddehase M. J. 2009; Immune evasion proteins enhance cytomegalovirus latency in the lungs. J Virol 83:10293–10298 [View Article][PubMed]
     [Google Scholar]
  5. Borst E. M., Benkartek C., Messerle M. 2007; Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses. In Current Protocols in Immunology pp. 10.32.1–10.32.30 Edited by Coligan J. E., Bierer B., Margulies D. H., Shevach E. M., Strober W., Coico R. New York: John Wiley & Sons;
     [Google Scholar]
  6. Brune W., Hengel H., Koszinowski U. H. 1999; A mouse model for cytomegalovirus infection. In Current Protocols in Immunology pp. 19.7.1–19.7.13 Edited by Coligan J. E., Bierer B., Margulies D. H., Shevach E. M., Strober W., Coico R. New York: John Wiley & Sons;
     [Google Scholar]
  7. Bubić I., Wagner M., Krmpotić A., Saulig T., Kim S., Yokoyama W. M., Jonjić S., Koszinowski U. H. 2004; Gain of virulence caused by loss of a gene in murine cytomegalovirus. J Virol 78:7536–7544 [View Article][PubMed]
     [Google Scholar]
  8. Chatellard P., Pankiewicz R., Meier E., Durrer L., Sauvage C., Imhof M. O. 2007; The IE2 promoter/enhancer region from mouse CMV provides high levels of therapeutic protein expression in mammalian cells. Biotechnol Bioeng 96:106–117 [View Article][PubMed]
     [Google Scholar]
  9. Cheung K. S., Lang D. J. 1977; Transmission and activation of cytomegalovirus with blood transfusion: a mouse model. J Infect Dis 135:841–845[PubMed] [CrossRef]
     [Google Scholar]
  10. Cook C. H., Trgovcich J., Zimmerman P. D., Zhang Y., Sedmak D. D. 2006; Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1β triggers reactivation of latent cytomegalovirus in immunocompetent mice. J Virol 80:9151–9158 [View Article][PubMed]
     [Google Scholar]
  11. Grzimek N. K., Dreis D., Schmalz S., Reddehase M. J. 2001; Random, asynchronous, and asymmetric transcriptional activity of enhancer-flanking major immediate-early genes ie1/3 and ie2 during murine cytomegalovirus latency in the lungs. J Virol 75:2692–2705 [View Article][PubMed]
     [Google Scholar]
  12. Held H. D., Martin C., Uhlig S. 1999; Characterization of airway and vascular responses in murine lungs. Br J Pharmacol 126:1191–1199 [View Article][PubMed]
     [Google Scholar]
  13. Henjaković M., Sewald K., Switalla S., Kaiser D., Müller M., Veres T. Z., Martin C., Uhlig S., Krug N., Braun A. 2008; Ex vivo testing of immune responses in precision-cut lung slices. Toxicol Appl Pharmacol 231:68–76 [View Article][PubMed]
     [Google Scholar]
  14. Henry S. C., Schmader K., Brown T. T., Miller S. E., Howell D. N., Daley G. G., Hamilton J. D. 2000; Enhanced green fluorescent protein as a marker for localizing murine cytomegalovirus in acute and latent infection. J Virol Methods 89:61–73 [View Article][PubMed]
     [Google Scholar]
  15. Hogquist K. A., Jameson S. C., Heath W. R., Howard J. L., Bevan M. J., Carbone F. R. 1994; T cell receptor antagonist peptides induce positive selection. Cell 76:17–27 [View Article][PubMed]
     [Google Scholar]
  16. Hummel M., Abecassis M. M. 2002; A model for reactivation of CMV from latency. J Clin Virol 25:Suppl. 2S123–S136 [View Article][PubMed]
     [Google Scholar]
  17. Hummel M., Yan S., Li Z., Varghese T. K., Abecassis M. 2007; Transcriptional reactivation of murine cytomegalovirus ie gene expression by 5-aza-2′-deoxycytidine and trichostatin A in latently infected cells despite lack of methylation of the major immediate-early promoter. J Gen Virol 88:1097–1102 [View Article][PubMed]
     [Google Scholar]
  18. Jarvis M. A., Nelson J. A. 2002; Human cytomegalovirus persistence and latency in endothelial cells and macrophages. Curr Opin Microbiol 5:403–407 [View Article][PubMed]
     [Google Scholar]
  19. Jordan M. C., Mar V. L. 1982; Spontaneous activation of latent cytomegalovirus from murine spleen explants. Role of lymphocytes and macrophages in release and replication of virus. J Clin Invest 70:762–768 [View Article][PubMed]
     [Google Scholar]
  20. Jung S., Aliberti J., Graemmel P., Sunshine M. J., Kreutzberg G. W., Sher A., Littman D. R. 2000; Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20:4106–4114 [View Article][PubMed]
     [Google Scholar]
  21. Koffron A. J., Hummel M., Patterson B. K., Yan S., Kaufman D. B., Fryer J. P., Stuart F. P., Abecassis M. I. 1998; Cellular localization of latent murine cytomegalovirus. J Virol 72:95–103[PubMed]
     [Google Scholar]
  22. Koffron A., Varghese T., Hummel M., Yan S., Kaufman D., Fryer J., Leventhal J., Stuart F., Abecassis M. 1999; Immunosuppression is not required for reactivation of latent murine cytomegalovirus. Transplant Proc 31:1395–1396 [View Article][PubMed]
     [Google Scholar]
  23. Kurz S. K., Reddehase M. J. 1999; Patchwork pattern of transcriptional reactivation in the lungs indicates sequential checkpoints in the transition from murine cytomegalovirus latency to recurrence. J Virol 73:8612–8622[PubMed]
     [Google Scholar]
  24. Kurz S. K., Rapp M., Steffens H. P., Grzimek N. K., Schmalz S., Reddehase M. J. 1999; Focal transcriptional activity of murine cytomegalovirus during latency in the lungs. J Virol 73:482–494[PubMed]
     [Google Scholar]
  25. Lemmermann N. A., Gergely K., Böhm V., Deegen P., Däubner T., Reddehase M. J. 2010; Immune evasion proteins of murine cytomegalovirus preferentially affect cell surface display of recently generated peptide presentation complexes. J Virol 84:1221–1236 [View Article][PubMed]
     [Google Scholar]
  26. Lenac T., Arapović J., Traven L., Krmpotić A., Jonjić S. 2008; Murine cytomegalovirus regulation of NKG2D ligands. Med Microbiol Immunol (Berl) 197:159–166 [View Article][PubMed]
     [Google Scholar]
  27. Livet J., Weissman T. A., Kang H., Draft R. W., Lu J., Bennis R. A., Sanes J. R., Lichtman J. W. 2007; Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56–62 [View Article][PubMed]
     [Google Scholar]
  28. Mocarski E. S., Shenk T., Pass R. F. 2007; Cytomegaloviruses. In Fields Virology, 5 edn. vol. 2 pp. 2702–2772 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
     [Google Scholar]
  29. Polić B., Hengel H., Krmpotić A., Trgovcich J., Pavić I., Luccaronin P., Jonjić S., Koszinowski U. H. 1998; Hierarchical and redundant lymphocyte subset control precludes cytomegalovirus replication during latent infection. J Exp Med 188:1047–1054 [View Article][PubMed]
     [Google Scholar]
  30. Presti R. M., Pollock J. L., Dal Canto A. J., O’Guin A. K., Virgin H. W. IV 1998; Interferon γ regulates acute and latent murine cytomegalovirus infection and chronic disease of the great vessels. J Exp Med 188:577–588 [View Article][PubMed]
     [Google Scholar]
  31. Reddehase M. J., Balthesen M., Rapp M., Jonjić S., Pavić I., Koszinowski U. H. 1994; The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease. J Exp Med 179:185–193 [View Article][PubMed]
     [Google Scholar]
  32. Reddehase M. J., Podlech J., Grzimek N. K. 2002; Mouse models of cytomegalovirus latency: overview. J Clin Virol 25:Suppl. 2S23–S36 [View Article][PubMed]
     [Google Scholar]
  33. Reddehase M. J., Simon C. O., Seckert C. K., Lemmermann N., Grzimek N. K. 2008; Murine model of cytomegalovirus latency and reactivation. Curr Top Microbiol Immunol 325:315–331 [View Article][PubMed]
     [Google Scholar]
  34. Reeves M. B., MacAry P. A., Lehner P. J., Sissons J. G., Sinclair J. H. 2005; Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers. Proc Natl Acad Sci U S A 102:4140–4145 [View Article][PubMed]
     [Google Scholar]
  35. Roizman B., Sears A. E. 1987; An inquiry into the mechanisms of herpes simplex virus latency. Annu Rev Microbiol 41:543–571 [View Article][PubMed]
     [Google Scholar]
  36. Seckert C. K., Renzaho A., Tervo H. M., Krause C., Deegen P., Kühnapfel B., Reddehase M. J., Grzimek N. K. 2009; Liver sinusoidal endothelial cells are a site of murine cytomegalovirus latency and reactivation. J Virol 83:8869–8884 [View Article][PubMed]
     [Google Scholar]
  37. Shen Z., Reznikoff G., Dranoff G., Rock K. L. 1997; Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J Immunol 158:2723–2730[PubMed]
     [Google Scholar]
  38. Simon C. O., Seckert C. K., Dreis D., Reddehase M. J., Grzimek N. K. 2005; Role for tumor necrosis factor alpha in murine cytomegalovirus transcriptional reactivation in latently infected lungs. J Virol 79:326–340 [View Article][PubMed]
     [Google Scholar]
  39. Simon C. O., Holtappels R., Tervo H. M., Böhm V., Däubner T., Oehrlein-Karpi S. A., Kühnapfel B., Renzaho A., Strand D. et al. 2006; CD8 T cells control cytomegalovirus latency by epitope-specific sensing of transcriptional reactivation. J Virol 80:10436–10456 [View Article][PubMed]
     [Google Scholar]
  40. Sinclair J. 2008; Human cytomegalovirus: latency and reactivation in the myeloid lineage. J Clin Virol 41:180–185 [View Article][PubMed]
     [Google Scholar]
  41. Smith M. S., Goldman D. C., Bailey A. S., Pfaffle D. L., Kreklywich C. N., Spencer D. B., Othieno F. A., Streblow D. N., Garcia J. V., Fleming W. H. 2010; Granulocyte-colony stimulating factor reactivates human cytomegalovirus in a latently infected humanized mouse model. Cell Host Microbe 8:284–291 [View Article][PubMed]
     [Google Scholar]
  42. Söderberg-Nauclér C., Fish K. N., Nelson J. A. 1997; Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 91:119–126 [View Article][PubMed]
     [Google Scholar]
  43. Söderberg-Nauclér C., Streblow D. N., Fish K. N., Allan-Yorke J., Smith P. P., Nelson J. A. 2001; Reactivation of latent human cytomegalovirus in CD14+ monocytes is differentiation dependent. J Virol 75:7543–7554 [View Article][PubMed]
     [Google Scholar]
  44. Srivastava M., Jung S., Wilhelm J., Fink L., Bühling F., Welte T., Bohle R. M., Seeger W., Lohmeyer J., Maus U. A. 2005; The inflammatory versus constitutive trafficking of mononuclear phagocytes into the alveolar space of mice is associated with drastic changes in their gene expression profiles. J Immunol 175:1884–1893[PubMed] [CrossRef]
     [Google Scholar]
  45. Stinski M. F., Isomura H. 2008; Role of the cytomegalovirus major immediate early enhancer in acute infection and reactivation from latency. Med Microbiol Immunol (Berl) 197:223–231 [View Article][PubMed]
     [Google Scholar]
  46. Szymczak A. L., Vignali D. A. 2005; Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin Biol Ther 5:627–638 [View Article][PubMed]
     [Google Scholar]
  47. Tannous B. A., Kim D. E., Fernandez J. L., Weissleder R., Breakefield X. O. 2005; Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo . Mol Ther 11:435–443 [View Article][PubMed]
     [Google Scholar]
  48. Vermaelen K., Pauwels R. 2004; Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights. Cytometry A 61A:170–177 [View Article][PubMed]
     [Google Scholar]
  49. Wagner M., Jonjić S., 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[PubMed]
     [Google Scholar]
  50. Wikenheiser K. A., Vorbroker D. K., Rice W. R., Clark J. C., Bachurski C. J., Oie H. K., Whitsett J. A. 1993; Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci U S A 90:11029–11033 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.029827-0
Loading
Single cell detection of latent cytomegalovirus reactivation in host tissue
J. Gen. Virol. 92, 1279 (2011); https://doi.org/10.1099/vir.0.029827-0
/content/journal/jgv/10.1099/vir.0.029827-0
/content/journal/jgv/10.1099/vir.0.029827-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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