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Comparative Study
. 2017 Feb 7;8(6):10238-10254.
doi: 10.18632/oncotarget.14380.

The biological properties of different Epstein-Barr virus strains explain their association with various types of cancers

Ming-Han Tsai  1   2 , Xiaochen Lin  1   2 , Anatoliy Shumilov  1   2 , Katharina Bernhardt  1   2 , Regina Feederle  1   2   3 , Remy Poirey  1   2 , Annette Kopp-Schneider  4 , Bruno Pereira  5 , Raquel Almeida  5 , Henri-Jacques Delecluse  1   2
Affiliations
Comparative Study

The biological properties of different Epstein-Barr virus strains explain their association with various types of cancers

Ming-Han Tsai et al. Oncotarget. .

Abstract

The Epstein-Barr virus (EBV) is etiologically associated with the development of multiple types of tumors, but it is unclear whether this diversity is due to infection with different EBV strains. We report a comparative characterization of SNU719, GP202, and YCCEL1, three EBV strains that were isolated from gastric carcinomas, M81, a virus isolated in a nasopharyngeal carcinoma and several well-characterized laboratory type A strains. We found that B95-8, Akata and GP202 induced cell growth more efficiently than YCCEL1, SNU719 and M81 and this correlated positively with the expression levels of the viral BHRF1 miRNAs. In infected B cells, all strains except Akata and B95-8 induced lytic replication, a risk factor for carcinoma development, although less efficiently than M81. The panel of viruses induced tumors in immunocompromised mice with variable speed and efficacy that did not strictly mirror their in vitro characteristics, suggesting that additional parameters play an important role. We found that YCCEL1 and M81 infected primary epithelial cells, gastric carcinoma cells and gastric spheroids more efficiently than Akata or B95-8. Reciprocally, Akata and B95-8 had a stronger tropism for B cells than YCCEL1 or M81. These data suggest that different EBV strains will induce the development of lymphoid tumors with variable efficacy in immunocompromised patients and that there is a parallel between the cell tropism of the viral strains and the lineage of the tumors they induce. Thus, EBV strains can be endowed with properties that will influence their transforming abilities and the type of tumor they induce.

Keywords: Epstein-Barr virus strains; carcinoma and lymphoma; host-virus interactions; human tumor viruses; viral infection and transformation.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no potential conflicts of interests.

Figures

Figure 1
Figure 1. EBV strains transform B cells with variable efficiency
A. This dot plot shows the efficiency of EBV-mediated transformation in infected primary B cell populations from 5 independent blood samples and seeded at 3 EBNA2-positive cells per well in 96 well cluster plates. The values given represent the percentage of outgrown wells. B. These curves show the increase in trypan blue- negative live cell numbers over time after infection of B cells from 7 independent blood samples with different EBV strains at high cell density. C. The bar graph shows the expression levels of all 4 members of the BHRF1 miRNA cluster in LCLs generated with 3 independent blood samples. D. We determined the efficiency of B cell infection by staining five different blood samples with an EBNA2-specific antibody 3 days after infection with the indicated viruses at the same multiplicities of infection as defined by qPCR. The graph gives the percentage of infected cells. E. The efficiency of B cell binding was assessed by qPCR after exposure of naïve B cells isolated from 5 independent blood samples to the same number of viral genomes. The dot plot shows the number of viruses bound per B cell. The p-values in (A), (B), (D), and (E) give the results of global test analyses by using global mixed linear model analyses with random effect. Error bars represent the mean with s.d.
Figure 2
Figure 2. Lytic replication in LCLs infected with multiple EBV strains
A. Representative BZLF1 and gp350 immunofluorescence stains performed on one set of LCLs generated with the same blood sample. B. The percentage of BZLF1- or gp350-positive cells in LCLs generated by the infection of 7 independent blood samples with 6 viruses. The 2 dot plots give the percentage of positive cells 4 weeks after infection. C. Representative BZLF1 and gp350-specific immunoblots performed on one set of LCLs generated with the same blood sample. Actin served as loading control. The 220kDa signals observed in the gp350-specific immunoblots are generated by a gp350 splice variant. The dot plot summarizes the BZLF1 protein levels observed after infection of five individual blood samples with the virus panel 1 month post-infection. The results are given relative to signals obtained with M81. The p-values shown in (B) give the results of global test analyses by using global mixed linear model analyses with random effect. In (C) we applied log-transformation of fold-changes for the analysis of normalized data without Bonferroni correction. Error bars represent the mean with s.d.
Figure 3
Figure 3. Survival in mice infected with various EBV strains
The schematic shows Kaplan-Meier survival curves at multiple time intervals after infection with B cells exposed to one of 6 EBV strains. Each group included 7 mice. The Log-rank test shows a significant difference in survival (p=0.0487). Please also see the Supplementary Figure 5 and Supplementary Table 2.
Figure 4
Figure 4. Infection of primary cells from a respiratory epithelium with multiple EBV strains
A. We infected four different batches of primary cells from the respiratory epithelium with supernatants containing the same numbers of infectious particles from the indicated EBV strains and stained them for EBER expression 3 days post-infection. The cells were also submitted to an immunostain with an antibody specific for keratin and the nuclei were counterstained with DAPI. The percentage of infected cells is given in the adjacent graph of bars and we show the results of 4 infections performed with samples from independent individuals. The arrows point to EBER-positive cells. B. Same experiment as in (A) except that we used transfer infection. C. The primary samples used in (A) and (B) were exposed to viral supernatants. After extensive washings, we quantified the number of copies bound to the target cells. The results are given relative to signals obtained with M81. The p-values shown in (A) and (B) give the results of global mixed linear model analyses with random effect. In (C) we applied log-transformation of fold-changes for the analysis of normalized data without Bonferroni correction. Error bars represent the mean with s.d.
Figure 5
Figure 5. EBV strains infect AGS cultures with variable efficiency
A. We infected the AGS cell line with different EBV strains at the same multiplicities of infection and performed an EBER in situ hybridization 3 days post-infection, coupled to an anti-keratin immunostain. We counted the percentage of EBV-infected cells and give the results from 7 independent experiments in a dot plot. We also show a representative example of these infections. The arrows point to EBER-positive cells. B. Same as in (A) but using transfer infection. C. The results of a binding assay with viral supernatants used in (A) and (B) and the AGS cells. The results are given as the number of EBV copies bound per cell. Here we show the results from 3 independent experiments. The p-values show in (A) to (C) give the results of global test analyses using global mixed linear model analyses with random effect. Error bars represent the mean with s.d.
Figure 6
Figure 6. YCCEL1 efficiently infects gastric spheroids
A. The picture shows the characteristic morphology of gastric spheroids generated by passaging AGS cells in vitro on a Matrigel matrix (Scale bar 1 mm). B. Representative pictures of gastric spheroids exposed to virus supernatants containing M81 and YCCEL1 and subjected to in situ hybridization with an EBER-specific probe (Scale bar 100 μm). The arrows point to EBER-positive cells. C. This bar graph compiles the results of 5 independent direct infection experiments of gastric spheroids with four different viruses at the same multiplicity of infection. We calculated the percentage of gastric spheroids that contained EBER-positive cells. D. We repeated the experiments described in (B) but using EBV transfer infection. We combined the detection of EBERs with a keratin staining to ensure that the infected cells are of epithelial lineage. Two representative examples are shown (Scale bar: 100 μm). Arrows show the EBER-positive cells. E. The transfer infections of gastric spheroids were repeated 5 times and their results were plotted in the presented graph. The p-values show in (C) and (E) give the results of global test analyses using global mixed linear model analyses with random effect.
Figure 7
Figure 7. The level of gp110 within EBV viral particles partially regulates the viral tropisms toward its hosts
A. Immunoblots of viral glycoproteins gp350/220 and gp110 in pelleted viral particles. The tegument protein BNRF1 served as a loading control. B. The dot plot shows the percentage of infected B cells determined by an EBNA2-specific staining 3 dpi at the same MOI after complementation of the indicated viruses with gp110. The results were compiled from infection of five independent blood samples. C, D, E. We repeated the infection experiments of primary epithelial cells, 2D AGS and 3D AGS cultures described in Figure 4, 5 and 6, respectively, using gp110-complemented viruses. The top panels show the results of direct infections, the bottom panels those of transfer infections. The p-values show in (B) to (E) give the results of global test analyses by using global mixed linear model analyses with random effect. Error bars represent the mean with s.d.
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