Abstract
The cell cycle position at the time of infection has a profound influence on human cytomegalovirus (HCMV) gene expression and therefore needs consideration in the design and control of HCMV experiments. While G0/G1 cells support the immediate onset of viral transcription, cells progressing through the S and G2 cell cycle phases prevent HCMV from entering the lytic replication cycle. Here, we provide two fast and reliable protocols that allow one to determine the cell cycle distribution of the designated host cells and monitor viral protein expression as a function of the cell cycle state. Both protocols make use of the thymidine analogue 5-ethynyl-2′-deoxyuridine and “click” chemistry to label HCMV-non-permissive S phase cells in a gentle and sensitive way.
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References
Fortunato EA, Sanchez V, Yen JY, Spector DH (2002) Infection of cells with human cytomegalovirus during S phase results in a blockade to immediate-early gene expression that can be overcome by inhibition of the proteasome. J Virol 76(11):5369–5379
Zydek M, Hagemeier C, Wiebusch L (2010) Cyclin-dependent kinase activity controls the onset of the HCMV lytic cycle. PLoS Pathog 6(9):e1001096. doi:10.1371/journal.ppat.1001096
Oduro JD, Uecker R, Hagemeier C, Wiebusch L (2012) Inhibition of human cytomegalovirus immediate-early gene expression by cyclin A2-dependent kinase activity. J Virol 86(17):9369–9383. doi:10.1128/JVI.07181-11, JVI.07181-11 [pii]
Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105(7):2415–2420. doi:10.1073/pnas.0712168105, 0712168105 [pii]
Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed Engl 40(11):2004–2021, doi:10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5 [pii]
Vindelov LL, Christensen IJ (1994) Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis. Methods Cell Biol 41:219–229
Strang BL, Boulant S, Chang L, Knipe DM, Kirchhausen T, Coen DM (2012) Human cytomegalovirus UL44 concentrates at the periphery of replication compartments, the site of viral DNA synthesis. J Virol 86(4):2089–2095. doi:10.1128/JVI.06720-11, JVI.06720-11 [pii]
Lu M, Shenk T (1996) Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S. J Virol 70(12):8850–8857
Neef AB, Luedtke NW (2011) Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc Natl Acad Sci U S A 108(51):20404–20409. doi:10.1073/pnas.1101126108, 1101126108 [pii]
Bain M, Sinclair J (2007) The S phase of the cell cycle and its perturbation by human cytomegalovirus. Rev Med Virol 17(6):423–434. doi:10.1002/rmv.551
Jault FM, Jault JM, Ruchti F, Fortunato EA, Clark C, Corbeil J, Richman DD, Spector DH (1995) Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest. J Virol 69(11):6697–6704
Antonsson A, Johansson PJ (2001) Binding of human and animal immunoglobulins to the IgG Fc receptor induced by human cytomegalovirus. J Gen Virol 82(Pt 5):1137–1145
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This work has been supported by the DFG grant WI2043/3-1 to L.W. and C.H.
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Wiebusch, L., Hagemeier, C. (2014). Use of 5-Ethynyl-2′-Deoxyuridine Labelling and Flow Cytometry to Study Cell Cycle-Dependent Regulation of Human Cytomegalovirus Gene Expression. 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_9
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DOI: https://doi.org/10.1007/978-1-62703-788-4_9
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Publisher Name: Humana Press, Totowa, NJ
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