All Title Author
Keywords Abstract

PLOS ONE  2012 

Tracking Murine Gammaherpesvirus 68 Infection of Germinal Center B Cells In Vivo

DOI: 10.1371/journal.pone.0033230

Full-Text   Cite this paper   Add to My Lib


Infection of mice with murine gammaherpesvirus 68 (MHV68) provides a tractable small animal model to study various aspects of persistent gammaherpesvirus infection. We have previously utilized a transgenic MHV68 that expresses enhanced yellow fluorescent protein (EYFP) to identify infected cells. While this recombinant MHV68 has been useful for identifying infected cell populations by flow cytometry, it has been suboptimal for identification of infected cells in tissue sections due to the high solubility of EYFP. Efficient detection of EYFP expressed from the MHV68 genome in tissue sections requires fixation of whole organs prior to sectioning, which frequently leads to over-fixation of some cellular antigens precluding their detection. To circumvent this issue, we describe the generation and characterization of a transgenic MHV68 harboring a fusion gene composed of the EYFP coding sequence fused to the histone H2B open reading frame. Because the H2bYFP fusion protein is tightly bound in nucleosomes in the nucleus it does not freely diffuse out of unfixed tissue sections, and thus eliminates the need for tissue fixation. We have used the MHV68-H2bYFP recombinant virus to assess the location and distribution of virus infected B cells in germinal centers during the peak of MHV68 latency in vivo. These analyses show that the physical location of distinct populations of infected germinal center B cells correlates well with their surface phenotype. Furthermore, analysis of the distribution of virus infection within germinal center B cell populations revealed that ca. 70% of MHV68 infected GC B cells are rapidly dividing centroblasts, while ca. 20% have a clear centrocyte phenotype. Finally, we have shown that marking of infected cells with MHV68-H2bYFP is extended long after the onset of latency – which should facilitate studies to track MHV68 latently infected cells at late times post-infection.


[1]  Barton E, Mandal P, Speck SH (2011) Pathogenesis and host control of gammaherpesviruses: lessons from the mouse. Annu Rev Immunol 29: 351–397.
[2]  Marques S, Efstathiou S, Smith KG, Haury M, Simas JP (2003) Selective gene expression of latent murine gammaherpesvirus 68 in B lymphocytes. J Virol 77: 7308–7318.
[3]  Willer DO, Speck SH (2003) Long-term latent murine Gammaherpesvirus 68 infection is preferentially found within the surface immunoglobulin D-negative subset of splenic B cells in vivo. J Virol 77: 8310–8321.
[4]  Collins CM, Boss JM, Speck SH (2009) Identification of infected B-cell populations by using a recombinant murine gammaherpesvirus 68 expressing a fluorescent protein. J Virol 83: 6484–6493.
[5]  Liang X, Collins CM, Mendel JB, Iwakoshi NN, Speck SH (2009) Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes. PLoS Pathog 5: e1000677.
[6]  Krug LT, Collins CM, Gargano LM, Speck SH (2009) NF-kappaB p50 plays distinct roles in the establishment and control of murine gammaherpesvirus 68 latency. J Virol 83: 4732–4748.
[7]  Liang X, Paden CR, Morales FM, Powers RP, Jacob J, et al. (2011) Murine gamma-herpesvirus immortalization of fetal liver-derived B cells requires both the viral cyclin D homolog and latency-associated nuclear antigen. PLoS Pathog 7: e1002220.
[8]  Richner JM, Clyde K, Pezda AC, Cheng BY, Wang T, et al. (2011) Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency. PLoS Pathog 7: e1002150.
[9]  Dauner JG, Chappell CP, Williams IR, Jacob J (2009) Perfusion fixation preserves enhanced yellow fluorescent protein and other cellular markers in lymphoid tissues. J Immunol Methods 340: 116–122.
[10]  Virgin HW, Presti RM, Li XY, Liu C, Speck SH (1999) Three distinct regions of the murine gammaherpesvirus 68 genome are transcriptionally active in latently infected mice. J Virol 73: 2321–2332.
[11]  Virgin HW, Latreille P, Wamsley P, Hallsworth K, Weck KE, et al. (1997) Complete sequence and genomic analysis of murine gammaherpesvirus 68. J Virol 71: 5894–5904.
[12]  Foudi A, Hochedlinger K, Van Buren D, Schindler JW, Jaenisch R, et al. (2009) Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 27: 84–90.
[13]  Fraser ST, Hadjantonakis AK, Sahr KE, Willey S, Kelly OG, et al. (2005) Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice. Genesis 42: 162–171.
[14]  Schaniel C, Moore KA (2009) Genetic models to study quiescent stem cells and their niches. Ann N Y Acad Sci 1176: 26–35.
[15]  Moser JM, Upton JW, Allen RD 3rd, Wilson CB, Speck SH (2005) Role of B-cell proliferation in the establishment of gammaherpesvirus latency. J Virol 79: 9480–9491.
[16]  Nutt SL, Tarlinton DM (2011) Germinal center B and follicular helper T cells: siblings, cousins or just good friends? Nat Immunol 12: 472–477.
[17]  Victora GD, Schwickert TA, Fooksman DR, Kamphorst AO, Meyer-Hermann M, et al. (2010) Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143: 592–605.
[18]  Weck KE, Kim SS, Virgin HW, Speck SH (1999) B cells regulate murine gammaherpesvirus 68 latency. J Virol 73: 4651–4661.
[19]  Weck KE, Kim SS, Virgin HW, Speck SH (1999) Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells. J Virol 73: 3273–3283.
[20]  Schwickert TA, Alabyev B, Manser T, Nussenzweig MC (2009) Germinal center reutilization by newly activated B cells. J Exp Med 206: 2907–2914.
[21]  Hughes DJ, Kipar A, Milligan SG, Cunningham C, Sanders M, et al. (2010) Characterization of a novel wood mouse virus related to murid herpesvirus 4. J Gen Virol 91: 867–879.
[22]  Hughes DJ, Kipar A, Sample JT, Stewart JP (2010) Pathogenesis of a model gammaherpesvirus in a natural host. J Virol 84: 3949–3961.
[23]  Bowden RJ, Simas JP, Davis AJ, Efstathiou S (1997) Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency. J Gen Virol 78(Pt 7): 1675–1687.
[24]  Hughes DJ, Kipar A, Leeming GH, Bennett E, Howarth D, et al. (2011) Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host. PLoS Pathog 7: e1001321.
[25]  Clambey ET, Virgin HW, Speck SH (2000) Disruption of the murine gammaherpesvirus 68 M1 open reading frame leads to enhanced reactivation from latency. J Virol 74: 1973–1984.
[26]  Gierasch WW, Zimmerman DL, Ward SL, Vanheyningen TK, Romine JD, et al. (2006) Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS. J Virol Methods 135: 197–206.
[27]  Weck KE, Barkon ML, Yoo LI, Speck SH, Virgin HW (1996) Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68. J Virol 70: 6775–6780.


comments powered by Disqus

Contact Us


微信:OALib Journal