Abstract
Of the many research challenges posed by human cytomegalovirus latency, perhaps the most notable is the requirement for primary hematopoietic cell culture. Culturing hematopoietic subpopulations while maintaining physiological relevance must be given utmost consideration. We describe a long-standing primary CD34+ hematopoietic progenitor cell (HPCs) system as an experimental model to study human cytomegalovirus (HCMV) latency and reactivation. Key aspects of our model include infection of primary human CD34+ HPCs prior to ex vivo expansion, maintenance of undifferentiated cells in a long-term culture with a stromal cell support, and an assay to quantitate infectious centers produced prior to and following a reactivation stimulus. Our method offers a unique way to quantitatively assess HCMV latency and reactivation to study the contribution of viral and host genes in latency and reactivation.
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References
Sinzger C, Grefte A, Plachter B, Gouw AS, The TH, Jahn G (1995) Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J Gen Virol 76:741–750
Soderberg C, Larsson S, Bergstedt-Lindqvist S, Moller E (1993) Definition of a subset of human peripheral blood mononuclear cells that are permissive to human cytomegalovirus infection. J Virol 67:3166–3175
Schrier RD, Nelson JA, Oldstone MB (1985) Detection of human cytomegalovirus in peripheral blood lymphocytes in a natural infection. Science 230:1048–1051
Boeckh M, Hoy C, Torok-Storb B (1998) Occult cytomegalovirus infection of marrow stroma. Clin Infect Dis 26:209–210
Goodrum F, Jordan CT, Terhune SS, High KP, Shenk T (2004) Differential outcomes of human cytomegalovirus infection in primitive hematopoietic subpopulations. Blood 104:687–695
Reeves MB, MacAry PA, Lehner PJ, Sissons JG, Sinclair JH (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
Sindre H, Tjoonnfjord GE, Rollag H, Ranneberg-Nilsen T, Veiby OP, Beck S, Degre M, Hestdal K (1996) Human cytomegalovirus suppression of and latency in early hematopoietic progenitor cells. Blood 88:4526–4533
von Laer D, Meyer-Koenig U, Serr A, Finke J, Kanz L, Fauser AA, Neumann-Haefelin D, Brugger W, Hufert FT (1995) Detection of cytomegalovirus DNA in CD34+ cells from blood and bone marrow. Blood 86:4086–4090
Hahn G, Jores R, Mocarski ES (1998) Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc Natl Acad Sci U S A 95:3937–3942
Kondo K, Kaneshima H, Mocarski ES (1994) Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci U S A 91:11879–11883
Kondo K, Xu J, Mocarski ES (1996) Human cytomegalovirus latent gene expression in granulocyte-macrophage progenitors in culture and in seropositive individuals. Proc Natl Acad Sci U S A 93:11137–11142
Hargett D, Shenk TE (2010) Experimental human cytomegalovirus latency in CD14+ monocytes. Proc Natl Acad Sci U S A 107:20039–20044
Smith MS, Bentz GL, Alexander JS, Yurochko AD (2004) Human cytomegalovirus induces monocyte differentiation and migration as a strategy for dissemination and persistence. J Virol 78:4444–4453
Soderberg-Naucler C, Fish KN, Nelson JA (1997) Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 91:119–126
Soderberg-Naucler C, Streblow DN, Fish KN, Allan-Yorke J, Smith PP, Nelson JA (2001) Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent. J Virol 75:7543–7554
Miller CL, Eaves CJ (2002) Long-term culture-initiating cell assays for human and murine cells. In: Klug CA, Jordan CT (eds) Hematopoietic stem cell protocols. Humana, Totowa, pp 123–141
Goodrum FD, Jordan CT, High K, Shenk T (2002) Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: a model for latency. Proc Natl Acad Sci U S A 99:16255–16260
Goodrum F, Reeves M, Sinclair J, High K, Shenk T (2007) Human cytomegalovirus sequences expressed in latently infected individuals promote a latent infection in vitro. Blood 110:937–945
Petrucelli A, Rak M, Grainger L, Goodrum F (2009) Characterization of a novel golgi-localized latency determinant encoded by human cytomegalovirus. J Virol 83:5615–5629
Umashankar M, Petrucelli A, Cicchini L, Caposio P, Kreklywich CN, Rak M, Bughio F, Goldman DC, Hamlin KL, Nelson JA, Fleming WH, Streblow DN, Goodrum F (2011) A novel human cytomegalovirus locus modulates cell type-specific outcomes of infection. PLoS Pathog 7(12):e1002444
Hu Y, Smyth GK (2009) ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347:70–78
Acknowledgments
This work was supported by Public Health Service Grants CA11343 and AI079059 to F.G. from the National Cancer Institute (NCI) and the National Institute of Allergy and Infectious Disease (NIAID), respectively.
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Umashankar, M., Goodrum, F. (2014). Hematopoietic Long-Term Culture (hLTC) for Human Cytomegalovirus Latency and Reactivation. In: Yurochko, A., Miller, W. (eds) Human Cytomegaloviruses. Methods in Molecular Biology, vol 1119. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-788-4_7
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DOI: https://doi.org/10.1007/978-1-62703-788-4_7
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