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Difference in cytokine production and cell cycle progression induced by Epstein-Barr virus Lmp1 deletion variants in Kmh2, a Hodgkin lymphoma cell line

Charlotte Sueur12347*, Julien Lupo1234, Philippe Mas35, Patrice Morand1234 and Véronique Boyer1236

Author Affiliations

1 Université Grenoble Alpes, UVHCI, F-38000 Grenoble, France

2 CNRS, UVHCI, F-38000 Grenoble, France

3 Unit for Virus Host-Cell Interactions, Univ. Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, Grenoble 38042, France

4 CHU de Grenoble, CS 10217, 38043 Grenoble, Cedex 9, France

5 EMBL Grenoble Outstation, F-38000 Grenoble, France

6 UMR 912 SESSTIM, 23 rue S. Torrents, 13006 Marseille, France

7 Hopitaux Universitaires de Strasbourg, Plateau Technique de Microbiologie, Laboratoire de Virologie, 1, place de l’hôpital, BP426 67091 Strasbourg, Cedex, France

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Virology Journal 2014, 11:94  doi:10.1186/1743-422X-11-94

Published: 19 May 2014

Abstract

Background

Epstein-Barr virus (EBV) is associated with 20-40% of Hodgkin’s Lymphoma (HL) cases. EBV-encoded latent membrane protein 1 (LMP1) is a well-known oncogenic protein and two C-terminal deletion variants, del30-LMP1 and del69-LMP1, have been described in animal models to be more tumorigenic than the wild-type form. This work aims to detail the implication of LMP1 in the development of HL and to characterize the particular effects of these variants.

Methods

We established HL-derived cell lines stably transfected with the pRT-LMP1 vector coding for the EBNA1 gene and allowing expression of the different LMP1 variants under the control of a doxycyclin-inducible promoter. Communication between cells was assessed by measuring the expression of various pro-inflammatory cytokines by flow cytometry after intracellular LMP1 and cytokine double staining. Proliferative properties of LMP1 variants were also compared by studying the repartition of cells in the different phases of the cell cycle after EdU incorporation combined to LMP1 and DAPI staining.

Results

All LMP1 proteins induced the expression of several pro-inflammatory cytokines such as TNF-α, TNF-β, IL-6, RANTES/CCL5 and IFN-γ. However, the del30-LMP1 variant induced cytokine expression at a lower level than the other variants, especially IFN-γ, while the del69-LMP1 variant stimulated greater cytokine expression. In addition, we measured that all LMP1 proteins greatly impacted the cell cycle progression, triggering a reduction in the number of cells in S-phase and an accumulation of cells in the G2/M phase compared to the HL-non induced cells. Interestingly, the del30-LMP1 variant reduced the number of cells in S-phase in a significantly greater manner and also increased the number of cells in the G0/G1 phase of the cell cycle.

Conclusion

Weak IFN-γ expression and specific alteration of the cell cycle might be a way for del30-LMP1 infected cells to escape the immune anti-viral response and to promote the development of cancer. The differences observed between the LMP1 variants reflect their own oncogenic properties and eventually impact the development of HL.

Keywords:
EBV; LMP1; Variant; Hodgkin’s Lymphoma; Cytokine; Cell cycle