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. 2011 Apr;85(7):3535-45.
doi: 10.1128/JVI.02086-10. Epub 2011 Jan 19.

Cellular gene expression that correlates with EBER expression in Epstein-Barr Virus-infected lymphoblastoid cell lines

Goran Gregorovic  1 , Rachel Bosshard, Claudio Elgueta Karstegl, Robert E White, Samuel Pattle, Alan K S Chiang, Oliver Dittrich-Breiholz, Michael Kracht, Rainer Russ, Paul J Farrell
Affiliations

Cellular gene expression that correlates with EBER expression in Epstein-Barr Virus-infected lymphoblastoid cell lines

Goran Gregorovic et al. J Virol. 2011 Apr.

Abstract

Novel Epstein-Barr Virus (EBV) strains with deletion of either EBER1 or EBER2 and corresponding revertant viruses were constructed and used to infect B lymphocytes to make lymphoblastoid cell lines (LCLs). The LCLs were used in microarray expression profiling to identify genes whose expression correlates with the presence of EBER1 or EBER2. Functions of regulated genes identified in the microarray analysis include membrane signaling, regulation of apoptosis, and the interferon/antiviral response. Although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cell gene expression were greater with EBER2 deletion. In this system, deletion of EBER1 or EBER2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting LCLs. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed, but in LCLs almost all of the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not detectably changed by EBER1. The changes in LCL gene expression identified here will provide a basis for identifying the mechanisms of action of EBER RNAs.

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Figures

FIG. 1.
FIG. 1.
(A) Summary of EBER deletions introduced into EBV BAC. The relative positions of XbaI and EcoRI (E) restriction sites in the EBV genome are shown; the EcoRI sites at bases 4163 and 7315 flank the EBER genes, represented by the filled boxes. The part of the EBV genome between bases 5800 and 7600 is expanded below under a scale in kilobases. Below that, the EBV content of the targeting plasmids is shown; the deletion of EBER1 (bases 6599 to 6798) includes the EBER1 promoter and the EBER1 sequence. The EBER2 deletion (bases 6949 to 7131) just removes the EBER2 transcript sequence. In both mutants an XbaI site was introduced at the point of deletion to facilitate the analysis. (B) Restriction digestion of BAC DNA analyzed by pulsed-field gel electrophoresis. In the left panels, the XbaI site introduced at the EBER deletion point splits the largest XbaI fragment into two fragments (arrowed) in the XbaI digest; this example is EBER2 mutant and revertant BAC DNA recovered from transfected 293 cells. In the right panel, the EcoRI fragment (bases 4163 to 7315) containing the EBER genes is reduced in length in the EcoRI digest of the EBER1 deletion mutant (arrowed). The largest EcoRI and XbaI restriction fragments span the major internal repeat of EBV, and the similar sizes of these fragments in the parental (WT) and revertant (E1R and E2R) indicate that no loss of internal repeat sequences has occurred in the recombinations. (C) Northern blots of total RNA extracted from the indicated LCLs confirm the expected patterns of EBER expression. Negative control (293pHEBo) and positive control (C666.1) samples are indicated, and the corresponding ethidium bromide stain of rRNA is shown below to confirm equal RNA loading on the gels. Probe specific activities and hybridization efficiencies gave approximately equal signals for EBER1 and EBER2 on these blots.
FIG. 2.
FIG. 2.
(A) Comparison of the transformation abilities (TD50) of different types of recombinant EBV. Two independent EBVs were used per EBV type and each infection was done in triplicate. Values are given as log10 TD50 with the standard deviation. (B and C) LCL growth assay. LCLs established with the parental EBV or different EBV mutants (delEBER1, EBER1rev, delEBER2, and EBER2rev) were compared for growth rates by plating them at 5 × 104 cells/ml in 24-well plates. The number of cells was counted every 1 to 3 days in a 10-day period, and the results represent the mean values with standard deviations based on six (B) or three (C) experiments. Two independent sets of LCLs (B and C), each derived from B cells isolated from a different blood donor, were used in the assay. For each EBV mutant and the wt EBV, two independent LCLs were analyzed.
FIG. 3.
FIG. 3.
(A) Experimental design for microarrays. (B) Principal component analysis (Partek software) for differences in cell gene expression pattern comparing microarray data from LCLs containing parental EBV (WT), EBV with deletion of EBER1 (del E1), deletion of EBER2 (del E2), revertant of EBER1 deletion (E1R), or revertant of EBER2 deletion (E2R). (C) Venn diagram showing the number of EBER1 or EBER2 regulated genes.
FIG. 4.
FIG. 4.
(A) Dot plots of EBER2 regulated genes in LCLs (2010 experiment with revertants). Each point represents the level of expression in a separate LCL, plotted on a log base 2 scale. (B) Dot plots of EBER2 regulated genes in LCLs from a 2008 experiment common with a 2010 experiment on a log base 2 scale. (C) RT-PCR of selected genes shown in the dot plots from the 2010 experiment. (D) RT-PCR of selected genes shown in the dot plots from the 2008 experiment. (E) Western blot of CXCR3 showing dependence on EBER2. Protein extracts from two independent LCLs for each EBV variant are shown; actin served as a loading control.
FIG. 5.
FIG. 5.
(A) Dot plots of EBER1 regulated genes in LCLs (2010 experiment with revertants) on a log base 2 scale. (B) RT-PCR of selected genes shown in the dot plots from 2010 experiment.
FIG. 6.
FIG. 6.
(A) 293 cells infected as indicated with B95-8 BAC EBV (WT) or BAC EBV with deletion of EBER1, EBER2, or the revertant of the EBER1 deletion were stained for expression of endogenous L22 (upper panel) or transfected FLAG-L22 (lower panel). Staining of the endogenous L22 was classified as predominantly nucleolar (NR), nucleoplasmic (NP), or cytoplasmic (CP), and the percentage of cells with each type of staining, averaged from 100 fields of view, is shown above the image. (B) 293 cells or 293 cells containing the BAC EBV with EBER1 deleted were transfected with the indicated plasmids and with a FLAG-L22 expression plasmid. FLAG-L22 immunofluorescence and phase-contrast overlay images are shown. (C) LCLs stained for endogenous L22 show a predominantly cytoplasmic distribution.
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