Differential regulation of LMP-1 and CXCR7 mRNA by ER-EBNA-2 shows that type 1 EBNA-2 induces LMP-1 and CXCR7 to a higher level and more rapidly than type 2 EBNA-2

We previously identified CXCR7 as the most differentially regulated type-specific EBNA-2-target gene in the human genome, being more strongly induced by type 1 EBNA-2 in a BL cell background [26]. RNAi experiments demonstrated that CXCR7 is required for proliferation of EBV-LCLs [26]. We also showed, in an EBV-positive BL cell line with oestrogen-regulated EBNA-2 proteins, that type 1 EBNA-2 induced LMP-1 protein more rapidly and to a higher extent compared to the type 2 [26]. This has been now verified at the RNA level using the same system. Briefly, this consists of Daudi cells (which lack the EBNA-2 locus [83] and therefore expression of LMP-1) engineered to stably express type 1 or type 2 chimaeric EBNA-2 proteins fused to the hormone-binding domain of the oestrogen receptor (ER-EBNA-2) [26]. In the absence of oestrogen, the ER-tagged EBNA-2 proteins are localized to the cytoplasm and therefore inactive [84]. Addition of oestrogen results in normal localization of the EBNA-2 fusion proteins to the nucleus and therefore in activation of the LMP-1 gene from the resident EBV genome. In a time-course experiment of oestrogen stimulation over 48 hours, total cell RNA from the cell lines containing type 1 or type 2 ER-EBNA-2 was analyzed by ribonuclease protection assay (RPA) using an LMP-1 specific probe (Figure 1 A). When type 1 EBNA-2 function was activated, LMP-1 mRNA levels were strongly induced as soon as 8 hours after the stimulation and were maintained thereafter throughout the time-course. In contrast, after induction of type 2 EBNA-2 function, markedly lower levels of LMP-1 transcripts were detected. Analysis by qRT-PCR for LMP-1 mRNA gave a similar result (Figure 1 B) and qRT-PCR analysis was also performed for CXCR7 transcripts levels in the same experiment (Figure 1 C). These were found to be significantly higher in type 1 EBNA-2 cell lines stimulated with oestrogen than in type 2 cells that had undergone the same treatment. Agarose-gel electrophoresis analysis of total cell RNA used for the RPA analysis confirmed equal loading and no degradation of the samples (data not shown). The somewhat higher expression of type 2 ER-EBNA-2 in the Daudi cell line (Figure S1 and [26]) further emphasizes the superior induction of target genes by type 1 ER-EBNA-2.

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Figure 1. LMP-1 and CXCR7 are differentially induced by type 1 and type 2 EBNA-2 in Daudi:ER-EBNA-2 T1/T2 and P3HR1 cl.16:ER-EBNA-2 T1/T2 stable cell lines.

(A) (B) (C) 20×10⁶ Daudi cells stably expressing type 1 (T1) or type 2 (T2) ER-EBNA-2 were induced with 1 µM oestrogen (+) or ethanol (-) as a solvent control, for 8, 20 or 48 hours. (A) RPA for LMP-1 mRNA. 20 µg of total cell RNA were used. The doublet protected fragment of around 175 nt, is marked by arrows. M: size marker, the 179 nt band is shown; P: undigested probe; Y: yeast RNA (RPAIIIkit, Ambion). 1 representative experiment of 3 is shown. (B) and (C) LMP-1 and CXCR7 mRNA qRT-PCR analysis. 1 µg of total cell RNA was used to synthesize cDNA and qPCR was performed to quantify LMP-1 and CXCR7 transcripts. For LMP-1 qRT-PCR, values for each sample are expressed as the ratio LMP-1/GAPDH mRNA relative to the sample T1 stimulated with oestrogen for 20 hours, whereas for CXCR7 qRT-PCR values shown represent the ratio between CXCR7 and GAPDH mRNA levels, relative to the sample T1 stimulated with oestrogen for 48 hours. Each time-point corresponds to the average from 2 independent experiments both performed in duplicate. Error bars represent standard errors. Data shown in panels (B) and (C) and results presented in panel (A) are from different experiments. (D) P3HR1 cl.16 cells stably expressing type 1 (T1) or type 2 (T2) ER-EBNA-2 were induced with 1 µM oestrogen (+) or ethanol (-) as a solvent control, for 4, 8, 12, 20, 24 or 48 hours. At these time-points proteins were extracted and analyzed by western blotting for EBNA-2 (PE2 antibody), LMP-1 (CS 1-4 antibody) and β-actin, as a loading control. h p.i.: hours post-induction; est: oestrogen.

https://doi.org/10.1371/journal.ppat.1002164.g001

The more rapid induction of LMP-1 by type 1 EBNA-2 was also confirmed in the type 2 EBV P3HR1 cl.16 BL cell line. The resident EBV genome in P3HR1 cl.16 line also lacks the EBNA-2 locus and the Y1Y2-coding region of EBNA-LP, having a deletion similar but not identical to that present in Daudi cells [83], [85]. Plasmids expressing type 1 or type 2 ER-EBNA-2 (p554-4 and its type 2 derivative, described in Materials and Methods and in [26], [86]) were transfected into P3HR1 cl.16 cells and stable cell lines were selected with G418. In P3HR1 cl.16 cells, despite the absence of EBNA-2, basal expression of LMP-1 can still be detected, albeit at very low levels (Figure 1 D). Nevertheless, when a time-course experiment of oestrogen stimulation was performed, a clear increase of LMP-1 expression levels above background was detected by western blotting when type 1 EBNA-2 function was activated (Figure 1 D). LMP-1 induction was detected as soon as 4 hours after oestrogen stimulation; after 8 hours LMP-1 expression level reached its maximum and was thereafter maintained throughout the time-course. Conversely, when type 2 EBNA-2 function was activated by oestrogen, LMP-1 levels were induced only moderately above the background, with a modest peak at 20 hours followed by a slight decrease (Figure 1 D).

Weaker induction of LMP-1 by type 2 EBNA-2 is not affected by the EBNA-LP type

As EBNA-LP is important in cooperation with EBNA-2 for induction of LMP-1 [28], [29], we determined which isoforms of EBNA-LP are expressed in normal Daudi and P3HR1 cl.16 cells and in the derivative stable cell lines bearing the oestrogen-inducible EBNA-2 proteins (Figures S1 and S2). In several previous reports, depending on the antibodies used, one or two species of EBNA-LP (around 31 and 37 kDa) have been detected in Daudi cells, both lacking the Y1Y2 domain [87]–[90]. Here we used the 4D3 antibody and observed the 37 kDa isoform (Figure S1, lane 4), which corresponds to a 4-repeat species, as determined by comparison with the EBNA-LPs expressed in B95-8 and in 293 cells transiently transfected with plasmids coding for 3- or 7- repeat EBNA-LP as standards (Figure S1, lanes 1, 2 and 3). P3HR1 cl.16 cells have been reported to express Y1Y2-deleted EBNA-LP species migrating as a major band of around 28–30 kDa and a minor band of about 48–50 kDa on a SDS-PAGE [91] and this was confirmed by our western blot analysis with the 4D3 antibody (Figure S2, lane 4). We identified these species as containing 3 and 6 W repeats, respectively (by comparison with B95-8). In both cell lines these truncated EBNA-LP proteins are type 2, since they were detected with the 4D3 antibody, which detects both type 1 and type 2 EBNA-LP [89], but not with the JF186 antibody, which is type 1-specific [92]. In the stable cell lines expressing ER-EBNA-2 proteins several EBNA-LP isoforms were detected, with 2 to 4 repeats in Daudi:ER-EBNA-2 T1/T2 (Figure S1, lanes 5 to 8) and with 2 to 7 repeats in P3HR1 cl.16:ER-EBNA-2 T1/T2 (Figure S2, lanes 5 to 8). These species are type 1, since they can be detected with the JF186 antibody, and are expressed from the plasmids that code for the ER-EBNA-2 proteins (p554-4 and its type 2 derivative [26], [86]). In fact, these vectors contain not only the ER-EBNA-2 open reading frame, but also 2 W repeats and the Y fragment, both of type 1 sequence. The expression of EBNA-LP species that are larger in size than that predicted simply from the number of W fragments has been already reported by others [92]-[94] and may indicate splicing of RNA transcribed more than once around the plasmid. In both systems (Daudi and P3HR1 cl.16:ER-EBNA-2 T1/T2) several isoforms of full-length type 1 EBNA-LP are present but no full-length type 2 EBNA-LP is available.

To exclude the possibility that type 1 EBNA-LP proteins are able to enhance the ability of type 1 ER-EBNA-2 to induce LMP-1 but not that of the type 2 ER-EBNA-2, we performed an assay for EBNA-LP function, similar to that used in earlier studies [17], [29], whereby the effect of EBNA-LP on EBNA-2-induced expression of LMP-1 from the resident EBV genome can be examined. This assay was adapted to compare the cooperative effect of type 1 and type 2 EBNA-LP on type 1 and type 2 EBNA-2-induced expression of LMP-1 in Daudi cells. Transfection of vectors expressing type 1 or type 2 EBNA-2 alone did not produce any significant induction of LMP-1, despite the strong expression of both types of EBNA-2 proteins, as assessed by immunoblotting (Figure 2). Likewise, no LMP-1 was detected when plasmids coding for either type 1 or type 2 EBNA-LP bearing 3 repeats were transfected, even though both EBNA-LP species were clearly detected on a western blot as an approximately 30 kDa band. Co-transfection of type 1 EBNA-2 and type 1 EBNA-LP plasmids produced a clear induction of LMP-1 protein. A similar, but slightly lower level of induction was observed when the type 2 EBNA-LP was co-expressed with type 1 EBNA-2. In contrast, up-regulation of LMP-1 induced by the type 2 EBNA-2, in the presence of either type of EBNA-LP, was very modest and equal with either type 1 or type 2 EBNA-LP (Figure 2). These results indicate that the weaker LMP-1 up-regulation induced by type 2 EBNA-2 can be ascribed to the EBNA-2 type and is not affected by the type of EBNA-LP present. Type 1 EBNA-2 up-regulates LMP-1 better than type 2 EBNA-2, in the presence of either type 1 or type 2 EBNA-LP. These observations support the conclusion that, in the Daudi and P3HR1 cl.16:ER-EBNA-2 T1/T2 cell lines, the impaired ability of type 2 EBNA-2 to induce LMP-1 is not due to sub-optimal cooperation with the full-length type 1 EBNA-LP isoforms present in the systems, but is rather due to the type 2 EBNA-2 protein.

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Figure 2. In a transient transfection assay in Daudi cells, the weaker induction of LMP-1 by type 2 EBNA-2, compared to the type 1 EBNA-2, is not affected by the EBNA-LP type.

2×10⁶ Daudi cells were transiently transfected with an array of combinations of EBNA-2-expressing plasmids (OriP-p294) and EBNA-LP-expressing plasmids (pSNOC) of type 1 (T1) and type 2 (T2). The Neon transfection system (Invitrogen) was used. 24 hours after transfection, cells were lysed in RIPA lysis buffer and analyzed by SDS-PAGE followed by immunoblotting for LMP-1 (CS 1-4 antibody), EBNA-2 (PE2 antibody), EBNA-LP (4D3 antibody) and β-actin (to ensure equal loading of the proteins). The pSNOC plasmids express EBNA-LP proteins with 3 W repeats (3R). Daudi cells already express an endogenous species of EBNA-LP which bears 4 repeats and lacks the Y1Y2 domains (4R ΔY1Y2). Numbers next to the EBNA-LP blot indicate protein molecular weight in kDa. 1 representative experiment of 2 is shown.

https://doi.org/10.1371/journal.ppat.1002164.g002

Differential regulation of LMP-1 and CXCR7 genes during early stages of infection of primary B cells with BAC-derived EBV expressing type 1 EBNA-2 or type 2 EBNA-2

Having showed, in our previous report [26] and in the present study in assays in cell lines, that type 1 and type 2 EBNA-2 differentially regulate LMP-1 and CXCR7, we examined whether this phenomenon occurs also during infection of primary B cells with BAC-derived EBV recombinant viruses, expressing either type 1 or type 2 EBNA-2. Early stages of infection were studied since the differences in kinetics of gene regulation induced by the two types of EBNA-2 appeared rapidly in our cell line-based systems. Normalized amounts of the EB viruses expressing type 1 or type 2 EBNA-2 (described in Materials and Methods) were used to infect aliquots of 2×10⁶ human primary B cells, purified from peripheral blood. Samples were taken at 1, 2, 4 and 8 days post-infection and examined by western blotting and quantitative RT-PCR (qRT-PCR) for EBNA-2-target gene expression. Similar amounts of type 1 and type 2 EBNA-2 proteins were expressed, as determined by the EBNA-2 immunoblot (Figure 3 A). In type 1 EBV-infected cells, LMP-1 transcripts were detected as soon as 2 days post-infection and gradually increased thereafter reaching typical LCL levels of expression by day 8, whereas in type 2-infected cells LMP-1 induction was delayed, with overall lower levels than those observed in type 1 infection (Figure 3 B). These different kinetics of LMP-1 mRNA induction were consistently observed in 4 independent infection experiments, although the overall level of expression and precise timing varied slightly in different experiments, this variability perhaps being due to different B cell donors being used each time. As we were able to perform these experiments only on a small scale, LMP-1 protein could not be detected by western blotting at these early times after infection.

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Figure 3. Differential regulation of LMP-1 and CXCR7 genes during early stages of infection of primary B cells with BAC-derived EBV expressing type 1 EBNA-2 or type 2 EBNA-2.

Primary B cells were isolated from peripheral blood by negative selection using RosetteSep (Stemcell Technologies). 2×10⁶ cells were infected with 5000 GRUs of either T1 E2 EBV-BAC (expressing type 1 EBNA-2) or T2 E2 EBV-BAC (expressing type 2 EBNA-2) recombinant viruses. Cells were harvested at 1, 2, 4 and 8 days post-infection and processed for protein and total RNA extraction. Uninfected cells, harvested at day 0, were included in the analysis. (A) Whole protein extracts from each well of infected cells were loaded on an SDS-PAGE gel and subjected to western blot analysis using the anti-EBNA-2 antibody (PE2 clone). The blots were re-probed for GAPDH to show the total amount of protein recovered from each sample. GAPDH levels at 4 and 8 days post-infection were lower in type 2-infected cells compared to type 1-infected cells, due to faster proliferation of the latter. (B) (C) and (D) qRT-PCR analysis of total cell RNA samples using LMP-1, CXCR7 and CXCR4 specific primers. RNAs from LCLs established with T1 and T2 EBNA-2 EBV-BAC were included in the analysis (LCL T1 E2 and LCL T2 E2). For LMP-1 qRT-PCR (B), values for each sample are expressed as the ratio LMP-1/GAPDH mRNA relative to the sample T1 day 8; for CXCR7 qRT-PCR (C), values shown represent the ratio between CXCR7 and GAPDH mRNA levels relative to the sample T1 day 1; for CXCR4 qRT-PCR (D), values are reported as the ratio CXCR4/GAPDH mRNA relative to mock infected cells. The qRT-PCR experiment was performed in duplicate and error bars represent standard errors. 1 representative experiment of 4 is shown. p.i.: post-infection.

https://doi.org/10.1371/journal.ppat.1002164.g003

qRT-PCR analysis for CXCR7 mRNA revealed that uninfected resting B cells (harvested at day 0) already expressed substantial basal amounts of mRNA (Figure 3 C), an observation consistent with other reports [76], [78]. Following viral infection, CXCR7 mRNA increased up to 2 days after infection and then settled back to the LCL level (Figure 3 C). On the other hand, in type 2 infection the mRNA for CXCR7 was consistently at about a 4-fold lower level than in type 1 virus-infected cells (Figure 3 C). This trend in CXCR7 mRNA levels was observed in 3 independent experiments, with minor differences in the overall levels of expression.

Expression of the chemokine receptor CXCR4, which shares the ligand CXCL12/SDF-1 with CXCR7, has been shown to be down-regulated in primary B cells upon infection by EBV [95]-[97]. Uninfected primary B cells were found to express high levels of CXCR4 mRNA (Figure 3 D), in agreement with other reports [98]. Upon viral infection, CXCR4 transcript levels were effectively repressed in cells infected with both type 1 and type 2 EBNA-2 recombinant EB viruses (Figure 3 D).

In order to establish LCLs, serial dilutions of preparations of EBV-BACs expressing type 1 or type 2 EBNA-2 were used to infect primary B lymphocytes (10⁶ cells) and their outgrowth was monitored over time. As expected, type 1 EBV-BAC-infected cells grew very rapidly, leading to LCLs within approximately 1 month post-infection, whereas type 2 transformants were much more difficult to expand and yielded cell lines only after approximately 4 months of culture. The growth phenotype observed with our recombinant type 1 and type 2 EBNA-2-expressing viruses is consistent with the growth behaviour originally reported for the natural strains of EBV B95-8 (type 1 prototype) and AG876 (type 2 prototype) [19]. These differences in growth kinetics, which were revealed by microscopic observation of the cells in culture, were mirrored by the counts of proliferating cells assessed at a single time-point after infection, for each of the cell lines originally infected with serial dilutions of virus (Figure S3). For example, the amount of live cells counted in type 1 LCLs originally infected with either 1600 or 320 GRUs of viral preparation was increased compared to the corresponding type 2 transformants by 53 and 22-fold, respectively (Figure S3). Once LCLs were established, hygromycin selection was applied to ensure the cells contained the BAC-EBV constructs, which bear the hygromycin-resistance gene, and not an EBV genome that might potentially have been endogenous at a low level in the donor B cells. The fully established type 1 and type 2 EBNA-2 LCLs were then further checked by examining the pattern of expression of all the EBV latent antigens, using western blotting (Figure S4). The EBV latent antigens examined are expressed at similar levels and display the expected size. EBNA-LP isoforms with slightly variable number of W repeats (from 3 to 5) were detected in type 1 and type 2 LCLs, but this is well above the minimum required for cooperation with EBNA-2 [17], [60]. Some differences were observed in EBNA-3A and -3C expression levels among the several clones analyzed, but do not seem to be consistently associated with one specific type of LCL (Figure S4).

Mapping the regions of type 1 EBNA-2 which confer higher B cell immortalization efficiency compared to type 2 EBNA-2

We previously developed a functional assay that distinguishes the ability of type 1 and type 2 EBNA-2 to maintain B cell proliferation using the EREB2.5 cell line [26], [86]. EREB2.5 cells contain a P3HR1 EBV genome lacking the EBNA-2 locus and a separate plasmid (p554-4) expressing the ER-EBNA-2 fusion protein. Hence, EBNA-2 activity and therefore survival and proliferation of the cells are dependent on oestrogen. In the absence of oestrogen, EBNA-2 function is inhibited by retention of the conditional ER-EBNA-2 protein in the cytoplasm but cell proliferation can be rescued if an OriP plasmid expressing constitutive type 1 EBNA-2 protein is transfected into the cells. In this situation the wild-type type 1 EBNA-2 functionally replaces the ER-EBNA-2 fusion protein. In contrast, if a wild-type type 2 EBNA-2-expressing OriP vector is transfected into oestrogen-starved cells, the cells undergo growth arrest. In this assay only type 1 but not type 2 constitutive EBNA-2 protein can provide essential EBNA-2 functions in trans. The trans-complementation assay was used in the present study to identify the regions of the type 1 EBNA-2 protein that can complement the deficiency of the type 2 protein to maintain proliferation of LCLs. For this, several type 1/type 2 EBNA-2 chimaeras were generated (by swapping sequences between the two types of protein) and these were tested for their ability to sustain cell growth of oestrogen-starved EREB2.5 cells. The complete set of chimaeras tested and the respective growth phenotype observed in the trans-complementation assay are shown in Figure 4 B. The chimaeras were precise swaps of EBNA-2 sequence without extraneous amino acids at the point of fusion. OriP-p294 plasmids expressing the chimaeric or wild-type type 1 or type 2 EBNA-2 proteins were Amaxa-transfected into EREB2.5 cells that had been growing normally in the presence of oestrogen. Immediately after transfection, oestrogen was withdrawn from the culture medium and cell proliferation was monitored over time by counting numbers of live cells (Figure 4 C). When the C-terminal region of type 1 EBNA-2 protein spanning from the WWPP motif (codons 323–326), within the RBP-Jk binding domain, to the end of the protein was swapped at homologous amino acid positions into the type 2 EBNA-2 protein (chimaera 2), live cell counts were similar to those obtained with wild-type type 1 EBNA-2 in the EREB2.5 growth assay (Figure 4 C). However, a null growth phenotype, similar to wild-type type 2 or empty vector control, was observed with the converse chimaera (chimaera 1), generated by joining together the type 1 sequence from codon 1 to the WWPP motif with type 2 sequences spanning from the WWPP motif to the last codon of the protein (Figure 4 C). Therefore, the C-terminus of type 1 EBNA-2 is able to confer a type 1 growth phenotype to type 2 EBNA-2 in the EREB2.5 trans-complementation assay. The C-terminus of the EBNA-2 protein includes the RG motif, the conserved region 7 (CR7), the transactivation domain (TAD) and the nuclear localization signal (NLS) (Figure 4 A). To further map the minimum type 1 sequences within the C-terminus of EBNA-2 responsible for the higher B cell immortalization efficiency, additional chimaeras were constructed by swapping different combinations of the RG, CR7, TAD and NLS regions from the type 1 into the type 2 protein (chimaeras 3 to 7). Only when the type 2 RG, CR7 and TAD regions were together replaced with homologous type 1 sequences (chimaera 7), accumulation of proliferating cells was restored to almost wild-type type 1 levels (Figure 4 C). In contrast, chimaeras from 3 to 6 behaved like wild-type type 2 EBNA-2, as they were unable to sustain proliferation (Figure 4 C). Long-term culture of oestrogen-independent EREB2.5 cells expressing the different EBNA-2 chimaeras and wild-type proteins resulted in establishment of continuously proliferating LCLs only in the case of chimaeras 2 and 7 and wild-type type 1 EBNA-2. These results demonstrate that, within the C-terminus of the type 1 EBNA-2, the RG, CR7 and TAD are the minimum type 1 sequences required to complement the lack of ability of type 2 EBNA-2 to rescue growth of oestrogen-depleted EREB2.5 cells.

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Figure 4. Type 1/type 2 EBNA-2 chimaeras tested in the EREB2.5 growth assay.

The carboxyl-terminal region of type 1 EBNA-2 complements the deficiency of type 2 EBNA-2 in the EREB2.5 growth assay. RG, CR7 and TAD domains are the minimum type 1 sequences required. (A) Structure of EBNA-2 protein. Type 1 EBNA-2 protein of the prototype B95-8 strain is 487 amino acids long, whereas the type 2 protein from the strain AG876 comprises 455 amino acids. The two types of EBNA-2 proteins share around 50% of sequence identity, which consists mainly of 9 short stretches of homology, named conserved regions (CR1 - CR9). Characteristic parts of the EBNA-2 protein are: two N-terminal self-association domains (SAD1 and SAD2); a poly-proline region (PPR); a diversity region, a sequence with low similarity between EBV types; a region interacting with the cell DNA-binding protein RBP-Jk (RBP-Jk); a short sequence rich in arginine and glycine residues (RG); a transactivation domain (TAD); a carboxyl-terminal nuclear localisation signal (NLS). EBNA-LP indicates regions involved in cooperation between EBNA-2 and EBNA-LP in transcriptional activation. (B) Panel of chimaeric proteins (C1 - C7) tested for their ability to rescue proliferation of oestrogen-starved EREB2.5 cells. The growth phenotype was scored as “++” (type 1 and chimaera 2) and “+” (chimaera 7), when cell proliferation was maintained over time after transfection and oestrogen withdrawal and LCLs were successfully established, or as “–” (type 2, chimaeras 1, 3, 4, 5 and 6), when proliferation ceased after transfection. (C) Live cell counts of EREB2.5 cells expressing wild-type type 1/type 2 or chimaeric EBNA-2 proteins. EREB2.5 cells were transfected with OriP-p294 plasmids expressing wild-type type 1 (T1), type 2 (T2) EBNA-2, chimaeras 1 to 7 (C1 - C7) or with empty vector (e.v.). Oestrogen was removed from the culture medium and accumulation of proliferating cells was assessed at 1, 2, 3 and 4 weeks after transfection by counting cells that exclude Trypan Blue on a haemocytometer. Data from 1 representative experiment of at least 4 is shown. Transfections were performed in duplicate and for each replica, triplicate wells of cells were counted. Error bars indicate standard deviations. (D) Western blot analysis of protein extracts from EREB2.5 cells transfected with wild-type type 1 (E2T1) and type 2 (E2T2) and chimaeric (C1 - C7) EBNA-2 proteins. Cells were harvested 4 days after transfection. EBNA-2 was detected using the PE2 antibody, which recognizes both types. All the chimaeric EBNA-2 proteins were expressed at levels comparable to wild-type type 1 and type 2 and displayed the expected size, which corresponds to 85 kDa (similar to type 1) for chimaera 1 and to 75 kDa (similar to type 2) for all the other chimaeras. ER-EBNA-2 fusion protein (120 kDa) is also detected in every transfection. Triplicate samples were analyzed for each transfection. B95-8 and AG876: positive controls for expression of EBNA-2 type 1 (85 kDa) and type 2 (75 kDa), respectively; E2.5 + est: non-transfected EREB2.5 cells normally grown in medium supplemented with oestrogen. Immunoblotting for β-actin was performed as a loading control.

https://doi.org/10.1371/journal.ppat.1002164.g004

To ensure that the lack of growth of oestrogen-depleted EREB2.5 cells was not due to either lack of expression or inappropriate protein localization of the chimaeras, western blot and immunofluorescence experiments were performed. Western blot analysis for EBNA-2 on extracts from transfected EREB2.5 cells grown without oestrogen revealed that all the chimaeric proteins display the expected size and are expressed at equal levels, similar to wild-type type 1 and type 2 EBNA-2 proteins (Figure 4 D). Levels of EBNA-2 expressed from the OriP plasmids was similar to those normally found in LCLs (Figure 4 D). For the analysis, the PE2 antibody was used, which was previously shown to detect the type 1 and the type 2 EBNA-2 with equal efficiency on a western blot [26]. Immunofluorescence analysis of HeLa cells transiently transfected with OriP-p294 plasmids, expressing the chimaeric and wild-type EBNA-2 proteins, confirmed that all the chimaeras are appropriately localized to the nucleus of the cells with exclusion from nucleoli (Figure S5A), with a pattern similar to that of wild-type type 1 and type 2 EBNA-2 (Figure S5B and [99]).

Expression of the EBNA-2-direct viral target and major EBV oncogene LMP-1 was assessed in the EREB2.5 growth assay. Western blot analysis revealed that at 3 and 5 days after transfection, in oestrogen-starved cells expressing type 1 EBNA-2 and chimaeras 2 and 7, LMP-1 protein expression was maintained at levels similar to those detected in EREB2.5 cells normally grown in the presence of oestrogen (Figure 5 A). In contrast, LMP-1 expression was lost in type 2 EBNA-2 or empty vector-transfected cells (Figure 5 A). qRT-PCR analysis for the mRNA of the cell gene CXCR7 showed increased levels following expression of either type 1 EBNA-2 or chimaeras 2 and 7, with a 14-fold increase (type 1 EBNA-2) or approximately 7-fold increase (chimaeras 2 and 7), compared to non-transfected cells, at 1 week after transfection (Figure 5 B). In contrast, when type 2 EBNA-2-expressing vector was transfected, CXCR7 mRNA was maintained at the same levels normally observed in non-transfected cells (fold-increase of 1) up to 5 days after transfection but after this time-point it was not further induced and the cells died (Figure 5 B). These results demonstrate that LMP-1 protein and CXCR7 mRNA expression levels, induced by type 1 and type 2 EBNA-2 and chimaeras 2 and 7, correlate with the growth phenotype in the EREB2.5 trans-complementation system. Loss of LMP-1 and CXCR7 expression in type 2 EBNA-2-expressing cells is rapidly followed by growth arrest of the cells, whereas in cells expressing type 1 EBNA-2, chimaera 2 or 7, maintenance of LMP-1 and CXCR7 expression is accompanied by sustained proliferation of the cells. Therefore the C-terminus of type 1 EBNA-2 in chimaeras 2 and 7 is sufficient to confer induction of type 1 growth phenotype and approximately type 1 expression levels of LMP-1 and CXCR7.

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Figure 5. LMP-1 and CXCR7 expression induced by type 1 and type 2 EBNA-2 and chimaeras 2 and 7 correlates with the growth phenotype in the EREB2.5 growth assay.

6×10⁶ EREB2.5 cells were transfected with OriP-p294 plasmids expressing type 1 EBNA-2 (E2T1), type 2 EBNA-2 (E2T1), chimaera 2 (C2) and chimaera 7 (C7) or with an empty vector (e.v.) and grown in the absence of oestrogen. At 3, 5 (A) and 7 (B) days after transfection (T), cells were harvested and lysed in either RIPA lysis buffer to extract proteins (A) or in Trizol (Invitrogen), to extract total RNA (B). (A) Protein samples were subjected to western blot analysis, probing for EBNA-2 (PE2 antibody), LMP-1 (CS 1-4 antibody) and β-actin, as loading control. Constitutive EBNA-2 proteins, wild-type type 1 (85 kDa), type 2 and chimaeric (75 kDa), as well as the conditional ER-EBNA-2 (120 kDa) are detected with the PE2 antibody. Numbers alongside the EBNA-2 immunoblot panel represent the apparent molecular weight (in kDa). (B) Total cell RNA samples were retro-transcribed into cDNA and analyzed by qPCR to quantify the levels of CXCR7 transcripts. No RNA could be extracted from type 2 EBNA-2-transfected cells at the day 7 time-point, since the majority of the cells were dead (> 70% relative to type 1 EBNA-2-transfected cells). The histograms show, for each transfection, the ratio CXCR7/GAPDH mRNA relative to non-transfected EREB2.5 cells grown in the presence of oestrogen. The analysis was performed in duplicate. Error bars represent standard errors. Data from 1 representative experiment of 2 is shown.

https://doi.org/10.1371/journal.ppat.1002164.g005

Functional differences between EBNA-2 types, mapped to the C-terminus, are typical of type sequence

In our mapping analysis performed to identify the type 1 regions of EBNA-2 important for B cell growth, EBNA-2 sequences from the EBV strains B95-8 (type 1 prototype) and AG876 (type 2 prototype) were used. To ensure that the functional differences being mapped are typical of EBV type sequences, the C-terminus of EBNA-2 from additional type 1 and type 2 isolates was sequenced and compared to the prototypes B95-8 and AG876. The EBV strains chosen for the analysis were derived from laboratory LCL, BL and NPC cell lines (Table 1). Comparison of type 1 isolates to the type 1 strain of reference B95-8 showed that, in the region examined, there is a very high percentage of identity (> 98%) with only a few nucleotide sequence variations detected. Most of the changes are silent but a few produce a change in the amino acid coded. Changes in the amino acid sequence were almost always located outside the known EBNA-2 functional domains; only in 2 strains (Mak 1 and MABA, Table 1) were there changes within a functional domain (TAD and NLS respectively). Similarly, alignment of the sequence coding for the C-terminus of EBNA-2 from several type 2 isolates against the prototype strain AG876, indicated that the sequence identity is almost 100%, with only 2 strains (Alouek and Wewak, Table 1) displaying nucleotide variations (2 silent and 1 non-silent, but localized outside functional domains). These results indicate that the functional differences between type 1 and type 2 EBNA-2, which we have mapped to this region, are representative of viral type sequence.

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Table 1. Sequence analysis of the C-terminus of EBNA-2 from type 1 and type 2 EBV isolates obtained from laboratory LCL, BL and NPC cell lines.

https://doi.org/10.1371/journal.ppat.1002164.t001

Cooperation between type 1 or type 2 EBNA-2 and EBNA-LP in the EREB2.5 growth system

EBNA-2-mediated activation of both cell (cyclin D2) and viral promoters (LMP-1/LMP-2B and Cp) can be greatly augmented by cooperation with the EBV latent protein EBNA-LP, which has been shown to be required for optimal virus-induced B cell transformation [9], [27]–[29]. Several EBNA-LP proteins, of different type and size, are believed to be present in the EREB2.5 trans-complementation system. To address whether these EBNA-LP species may influence the growth phenotype induced by the wild-type (type 1 and type 2) and chimaeric EBNA-2 proteins in oestrogen-starved EREB2.5 cells we first determined which specific EBNA-LP isoforms are expressed in the EREB2.5 cell line and in the derivative oestrogen-independent LCLs established with type 1 EBNA-2 and chimaeras 2 and 7 (Figure 6). For this, western blot analysis was performed comparing two anti-EBNA-LP antibodies: JF186 (type 1-specific) and 4D3, which recognizes both types of EBNA-LP [89], [92]. Using the 4D3 antibody, a type 2 EBNA-LP species of around 52 kDa was detected in non-transfected EREB2.5 cells, normally grown in oestrogen-supplemented medium (Figure 6 A, lane EREB2.5 + est), and in all the oestrogen-autonomous LCLs (Figure 6 A and B). This isoform is encoded by the resident P3HR1 genome and is known to lack the Y1 and Y2 domains [100]. Both antibodies detected a type 1 EBNA-LP isoform containing 3 W repeats, as determined by comparison with lysates from 293 cells transiently transfected with a plasmid expressing 3-repeat EBNA-LP (Figure 6 A, lane T1ELP3R). This EBNA-LP is encoded by the endogenous plasmid p554-4 and it was detected not only in non-transfected oestrogen-dependent EREB2.5 cells (Figure 6 A, lane EREB2.5 + est), but also in all the oestrogen-independent LCLs established by transfecting chimaeras 2 and 7 or type 1 EBNA-2, analyzed over a period of 6 and 3 months (Figure 6 A and B, respectively). This observation suggests that in these experiments the p554-4 plasmid is not completely lost from the oestrogen-free LCLs. This was corroborated by the EBNA-2 immunoblot that showed continuous expression of ER-EBNA-2 over time, although at variable levels. It is likely that some of the p554-4 plasmid has become integrated in these cells since, after extraction of low molecular weight DNA from the oestrogen-independent LCLs, only the OriP-p294 plasmid (expressing constitutive EBNA-2 proteins) but not the p554-4 vector was rescued (data not shown).

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Figure 6. Characterization of oestrogen-independent LCLs established from EREB2.5 cells expressing chimaeras 2 and 7.

EREB2.5 cells transfected with OriP-p294 plasmids coding for type 1 EBNA-2 (A) or chimaeras 2 and 7 (B) and grown in the absence of oestrogen gave rise to continuously proliferating LCLs. Protein samples were harvested from these LCLs at each month after transfection over a period of 6 months (A) or after 1.5 and 3 months (B) and analyzed by western blotting. The anti-EBNA-2 antibody (PE2), detected the transfected type 1 (E2) and chimaeric (chim E2) EBNA-2 proteins as well as the endogenous ER-EBNA-2 (ER-E2). EBNA-LP immunoblotting was performed with the antibodies JF186 (type 1-specific) and 4D3 (recognizes both types). At any time-point analyzed, both antibodies detected a type 1 3-repeat EBNA-LP species (T1 3R), expressed from the endogenous p554-4 plasmid, indicating that the plasmid was not lost from the cells. The number of repeats was judged by comparison to 293 cells transfected with a type 1 3-repeat EBNA-LP-expressing vector (T1ELP3R). A type 2 Y1Y2-truncated isoform of around 52 kDa (T2 ΔY1Y2), expressed by the resident P3HR1 genome, was also detected by the 4D3 antibody. Numbers alongside the EBNA-LP immunoblot panel represent protein molecular weight (in kDa). β-actin immunoblots ensure equal loading of the proteins.

https://doi.org/10.1371/journal.ppat.1002164.g006

Since only a type 1 full-length EBNA-LP is present in the EREB2.5 assay, it might be that a type-specific cooperation between EBNA-2 and EBNA-LP would be important for optimal proliferation in the EREB2.5 growth system and that the lack of a full-length type 2 EBNA-LP species could hamper the growth-promoting abilities of type 2 EBNA-2. We tested this issue directly by transfecting plasmids expressing either type 1 or type 2 EBNA-2 into EREB2.5 cells, in the presence or absence of a plasmid expressing a 3-repeat type 2 EBNA-LP, to check whether the presence of a full-length type 2 EBNA-LP (homologous to the endogenous type 1 3-repeat EBNA-LP) would allow the type 2 EBNA-2 to sustain cell proliferation in an oestrogen-independent fashion (Figure 7). Accumulation of proliferating cells was assessed each week after transfection and oestrogen withdrawal over a period of 3 weeks (Figure 7 A). Cells expressing type 1 or type 2 EBNA-2 alone displayed proliferation levels similar to those observed in Figure 4 C. Addition of the type 2 EBNA-LP did not produce any significant effect on growth of either type 1 or type 2 EBNA-2-expressing cells or control cells. There were no significant differences in the levels of EBNA-2 proteins comparing the type 1 and type 2 forms, as assessed by western blotting with the PE2 antibody (Figure 7 B). Immunoblotting with the 4D3 antibody confirmed good expression of the type 2 3-repeat EBNA-LP from the transfected pSNOC vector. This EBNA-LP isoform displays a slightly higher molecular weight on SDS-PAGE compared to the endogenous type 1 3-repeat EBNA-LP (Figure 7 B) because of the substantial differences (12 amino acids) in the primary amino acid sequences between type 1 and type 2 EBNA-LPs. This experiment demonstrates that the inability of type 2 EBNA-2 to rescue growth of oestrogen-depleted EREB2.5 cells is not due to the absence of a full-length type 2 EBNA-LP in the system.

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Figure 7. Cooperation between type 1 and type 2 EBNA-2 and EBNA-LP in the EREB2.5 growth system: The assay functionally distinguishes between the ability of type 1 and type 2 EBNA-2 to sustain cell proliferation, regardless the type of EBNA-LP.

(A) 6×10⁶ EREB2.5 cells were transfected with OriP-p294 plasmids expressing either type 1 (E2T1) or type 2 (E2T2) EBNA-2, or with empty vector (e.v.), in the presence or absence of a pSNOC plasmid expressing a type 2 EBNA-LP with 3 repeats (T2 ELP). Oestrogen was removed immediately after transfection and outgrowth of the cells was monitored over a period of 3 weeks. Transfections were performed in duplicate and for each replica, triplicate wells of cells were counted. Error bars indicate standard deviations. (B) 4 days after transfection, proteins were extracted from the cells and fractionated by SDS-PAGE. Western blot analysis was performed using PE2 and 4D3 antibodies in order to detect EBNA-2 and EBNA-LP respectively and confirmed equal levels of expression. The PE2 antibody, detected the transfected type 1 (T1 E2) and type 2 (T2 E2) EBNA-2 proteins as well as the endogenous ER-EBNA-2 (ER-E2). EBNA-LP western blot showed that empty vector-transfected cells express the 30 kDa type 1 full-length 3-repeat EBNA-LP (T1 3R), encoded by the p554-4 plasmid, and the 52 kDa Y1Y2-truncated EBNA-LP species (T2 ΔY1Y2), expressed from the P3HR1 resident genome. The full-length type 2 3-repeat EBNA-LP protein (T2 3R), expressed from the pSNOC vector, is detected on the gel at approximately the same position of the endogenous type 1 3-repeat EBNA-LP (see main text for details). The levels of β-actin did not alter throughout the experiment. Duplicate samples for each transfection are shown. B95-8, AG876 and T2 ELP 3R (+) lanes represent positive controls for expression of EBNA-2, type 1 and type 2, and type 2 3-repeat EBNA-LP, respectively. Numbers indicate apparent molecular weight (in kDa).

https://doi.org/10.1371/journal.ppat.1002164.g007

Cell lines

P3HR1 clone 16, Daudi and Raji are EBV-positive BL cell lines [111]-[113]. B cell lines were maintained in RPMI 1640 medium (Gibco-BRL) supplemented with 10 to 20% (v/v) heat-inactivated foetal bovine serum (FBS, BioWhittaker) and antibiotics (100 units/ml penicillin and 100 units/ml streptomycin, Gibco-BRL). EREB2.5 cells [86] contain a conditional EBNA-2 protein regulated by oestrogen and were maintained in RPMI 1640 medium supplemented with 10% FBS, antibiotics and 1 µM β-oestradiol (Sigma). Daudi:ER-EBNA-2 T1/T2 [26] and P3HR1 cl.16:ER-EBNA-2 T1/T2 stable cell lines were grown in RPMI 1640 medium supplemented with 10% FBS, antibiotics and 400 or 500 µg/mL G418 (Calbiochem) respectively. 293 and HeLa cell lines were grown in Dulbecco Modified Eagle Medium (DMEM, Gibco-BRL) supplemented with 10% FBS and antibiotics. 293 cell lines stably transfected with EBV-BAC constructs were maintained in DMEM with 10% FBS, antibiotics and 100 µg/ml Hygromycin B (Roche).

Plasmids

The OriP-p294 plasmids used for expression of EBNA-2, type 1 and type 2, and the EBNA-2 chimaeras, were derived from the CMVpEBNA-1 plasmid described in [114]. The EBNA-1 coding region of the CMVpEBNA-1 plasmid was extracted and replaced with a multiple-cloning site (MCS) comprising HindIII, BamHI and NotI restriction sites, generating the OriP-p294 MCS plasmid. To generate the wild-type type 1 EBNA-2-expressing plasmid, the region comprising one BamHI W repeat and the type 1 EBNA-2 coding region from the p554 plasmid (kind gift from Bettina Kempkes, described in [86], [115]) was cloned as a BglII – NotI fragment into OriP-p294 MCS, between BamHI and NotI sites. To generate the homologous vector expressing a type 2 EBNA-2, the fragment encompassing one BamHI W repeat and the type 2 EBNA-2 sequence from the pAG1 vector (described in [26]; this is essentially homologous to p554, but carries a type 2 EBNA-2) was extracted by BglII – NotI digestion and cloned between BamHI and NotI sites in the OriP-p294 MCS vector. This procedure produced the two OriP-p294 plasmids with either type 1 or type 2 EBNA-2 sequences (OriP-p294 E2T1/T2). The expression of the EBNA-2 proteins from these plasmids is driven by the EBV Wp promoter. Additional features of these vectors include the EBV origin of replication, OriP, and a hygromycin-resistance gene.

To generate the EBNA-2 chimaeric sequences, two pBluescript plasmids carrying type 1 or type 2 EBNA-2 coding sequences between EcoRI and NotI sites, extracted from the p554 and pAG1 plasmids respectively [26], [86], were used. For chimaeras 1 and 2, a fragment spanning between two BstXI sites (one in the EBNA-2 ORF, mapping to the WWPP amino acid motif, codons 323-326, within the RBP-Jk domain, and one in the vector backbone, downstream NotI site) was swapped between the two constructs. To generate chimaera 3, the region comprising the CR7, TAD and NLS elements from type 1 EBNA-2 was Pfu-PCR amplified using a primer with overhanging SacII (5′) restriction site and the universal T3 primer. A SacII restriction site was artificially inserted by site-directed mutagenesis, using the QuickChange Site-directed mutagenesis kit (Stratagene), in type 2 EBNA-2 sequence, without altering the amino acid sequence (codons 337-338, between the RG sequence and the CR7). The type 1 PCR amplicon was then cloned as a SacII – NotI fragment at homologous positions in the type 2 EBNA-2 vector. A similar strategy was used to generate chimaera 4. Briefly, a PCR amplicon, consisting of the TAD and the NLS sequences of type 1 EBNA-2, was generated by Pfu-DNA polymerase PCR using an oligonucleotide carrying an overhanging EcoRV (5′) restriction site and the universal primer T3. This PCR product was then cloned as an EcoRV – NotI fragment into the pBluescript plasmid carrying type 2 EBNA-2 ORF, in which a silent EcoRV site had been previously introduced by in vitro mutagenesis at codons 388-389, between the CR7 and the TAD. To produce chimaera 5, the RG sequence from type 1 EBNA-2 was amplified by PCR with Pfu DNA polymerase using primers with overhanging BstXI (5′) and SacII (3′) sites and cloned between the same restriction sites into the type 2 EBNA-2 pBluescript plasmid, carrying the artificial SacII site. Likewise, to make the chimaera 6 construct, the type 1 sequences encompassing the RG and the CR7 domains were Pfu-PCR amplified with primers bearing overhanging BstXI (5′) and EcoRV (3′) restriction sites, the PCR product was BstXI – EcoRV digested and then cloned between the same positions into the type 2 EBNA-2 pBluescript vector with the engineered EcoRV sequence. For construction of chimaera 7, an artificial BstBI site was inserted in the type 2 EBNA-2 pBluescript plasmid, at the end of the TAD sequence (codons 429-430). The RG, CR7 and TAD sequences were amplified by Pfu-PCR from the type 1 EBNA-2 vector, using primers with overhanging BstXI (5′) and BstBI (3′) sites. The PCR product was then cloned into the type 2 EBNA-2 plasmid, between the same restriction sites. The identity of all the chimaeric EBNA-2 sequences was screened by restriction mapping and by sequencing (data not shown). These were subcloned as EcoRI – NotI fragments from the pBluescript background into an intermediate vector (pSuper backbone) bearing the BglII – NotI region from the pAG1 vector (described above), which consists of one BamHI W repeat and the EcoRI – NotI EBNA-2 coding sequence. The BglII – NotI sequences, comprising the BamHI W repeat and the chimaeric EBNA-2 ORFs, were then cloned into the OriP-p294 MCS expression vector, between BamHI and NotI sites. Transcription of the chimaeric EBNA-2 ORFs in these plasmids is driven by the EBV Wp promoter, similarly to the type 1 and type 2 EBNA-2 constructs.

The pSNOC vector expressing a type 1 3-repeat EBNA-LP coding sequence is described elsewhere [116]. Briefly, this OriP expression plasmid contains the G418-resistance locus and the CMV immediate early promoter and the SV-40 poly A sequences, expressing the EBNA-LP coding sequence. To generate a homologous type 2 3-repeat EBNA-LP-expressing plasmid, a cDNA clone from the type 2 cell line C2+BL16 was obtained by Pfu-PCR using oligonucleotides 5′- ATG CGG CCA TGT AGG CCC ACT T -3′ and 5′- TGC CCA ACC ACA GGT TCA GGC A-3′, complementary to sequences within the C1 and YH exons, respectively, of AG876 EBV strain (coordinates 11403-11425 and 36141-36119). The amplified product was subcloned into pCR2.1-TOPO vector (Invitrogen) via TA cloning strategy and sequenced to verify its identity to the published AG876 EBNA-LP sequence. The type 2 3-repeat EBNA-LP cDNA was then cloned as an EcoRI fragment into the pSNOC empty vector.

The p554-4 plasmid, bearing the coding sequences for a type 1 ER-EBNA-2 and 2 BamHI W repeats was a kind gift from Bettina Kempkes [86], [115]. The p554-4 derivative expressing type 2 ER-EBNA-2 is described elsewhere [26]. These two vectors were used to generate the stable cell lines P3HR1 cl.16:ER-EBNA-2 T1/T2 (described below).

Construction and characterisation of EBNA-2 EBV-BAC mutant

EBNA-2 in the type 1 B95-8 EBV-BAC [117] was substituted with the type 2 EBNA-2 gene by RecA-mediated homologous recombination. The final targeting construct used for this was cloned between the BamHI and Psp0MI sites of pKov-Kan-ΔCm p4487.1 [118] and was called p121 (Figure S6). Starting from the type 2 EBNA-2 pBluescript plasmid (described above), type 1 upstream flanking sequences were added by cloning a HindIII – EcoRI fragment from the p554 vector [86], [115] using a HindIII site in the Bluescript polylinker. Type 1 downstream flanking sequence was then added by substituting in a PmeI to NotI fragment from p554. An XhoI site in the pBluescript polylinker upstream of the HindIII site was converted into a BglII site using an oligonucleotide adaptor and then the whole assembly was cloned as a BglII – NotI fragment between the BamHI and Psp0MI sites of p4487.1. The resulting type 2 EBNA-2 with type 1 flanking sequences contained type 2 EBV from 36201 – 38008 of the AG876 sequence [119]. B95-8 EBV-BAC (p2089, chloramphenicol resistant) in E. Coli strain DH10B was kindly provided by W. Hammerschmidt [117]. For clarity, the name T1 E2 EBV-BAC is used for this plasmid in this paper. RecA recombination to produce the type 2 EBNA-2 BAC mutant used the same methods as described previously for construction of EBER mutants [120].

The integrity of the type 2 BAC was screened at each stage of recombination by miniprep DNA isolation, restriction digest and pulsed field gel electrophoresis (PFGE) in order to control for any second-site mutations that may have occurred. Substitution of the type 1 EBNA-2 ORF with the type 2 sequence introduced an additional EcoRI restriction site into the EBV-BAC, in front of the starting ATG of EBNA-2 open reading frame. Digestion with this restriction enzyme followed by PFGE analysis therefore enabled diagnosis of correct insertion of the type 2 EBNA-2 sequence (data not shown). EcoRI digestion revealed also that the type 2 EBNA-2 construct had lost 2 copies (out of 6) of the BamHI W repeat compared to the parental type 1 construct. This was confirmed by digestion with another restriction enzyme, EcoRV (data not shown) and was the only gross rearrangement that had occurred, as the pattern of the remaining bands was identical in the type 1 and the type 2 EBNA-2 BAC in both EcoRI and EcoRV digestions (data not shown). Because the number of BamHI W repeats varies considerably in different EBV strains, from 3 to 12 [94] and because the repeat number found in the type 2 BAC is within this range, this deletion was not considered to be undesirable.

Establishment of 293 stable cell lines containing EBV-BAC with type 2 EBNA-2

EBV-BAC DNA was purified using EndoFree plasmid Maxi kit (Qiagen) and 1 µg was transfected into 293 cells with Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Stable 293 cell clones were isolated under Hygromycin B selection (100 µg/ml), clonal cell lines were expanded and these were then screened for integrity of the EBV-BAC DNA by episome rescue and restriction digestion or PCR analysis (data not shown). A 293 clone containing the T1 E2 EBV-BAC, used to generate the type 2 BAC, was also produced.

Virus production and titration

Infectious virus was produced from the 293 stable cell lines containing either T1 E2 EBV-BAC (with type 1 EBNA-2) or T2 E2 EBV-BAC (with type 2 EBNA-2) by transient transfection of BZLF1 and BALF4-expressing plasmids using Lipofectamine2000 (Invitrogen) [117], [121], [122]. After 4 days, the virus-containing supernatants were harvested, filtered (0.45 µm pore size) and titred by infecting Raji cells and counting GFP-positive cells (Green Raji Units, GRU) on an inverted fluorescent microscope, following treatment with 5 nM TPA (Sigma) and 5 mM sodium butyrate (Sigma), to enhance GFP expression.

Purification of B cells and virus infections

Primary B cells were isolated from mixed-donors buffy coat residues by negative selection using RosetteSEP Human B cell Enrichment cocktail (Stemcell Technologies) as described by the manufacturer's instructions. The isolated cells were analyzed by flow cytometry for purity using fluorescein-isothiocyanate-conjugated anti-CD20 antibody (Dako). 2×10⁶ cells were infected with 5000 GRUs of recombinant viruses and cultured in RPMI 1640 medium supplemented with 20% FBS and antibiotics. At the desired time-points cells were harvested and processed for RNA or protein extraction. For LCL establishment, 10⁶ cells were infected with 5-fold serial dilutions of recombinant EBVs starting with 8000 GRUs. Cells were grown initially in RPMI 1640 medium with 20% FBS and antibiotics and every 2-3 days half the medium was replaced. Once LCLs had grown out in large culture volumes, the FBS level in the medium was reduced to 10% and cells were selected in Hygromycin B (Roche) at a concentration of 150 µg/ml for 3 weeks. After this time, hygromycin selection was discontinued and cells were not used for experimental investigation during hygromycin selection.

Transfections

To generate P3HR1 cl.16:ER-EBNA-2 T1/T2 stable cell lines, 2×10⁶ cells were transfected with 4 µg of p554-4 [86], [115] or pERT2 [26] plasmids expressing ER-EBNA-2 type 1 and type 2 respectively using Neon transfection system (Invitrogen) set at 1300 V, 30 pulse width, 1 pulse. 24 hours after transfection G418 (Calbiochem) selection was added at a concentration of 500 µg/ml. Selected cell lines were grown out and tested for expression of ER-EBNA-2 T1/T2 by western blotting.

For EBNA-2/EBNA-LP cooperation assays in Daudi cells, the Neon system (Invitrogen) was used to transiently transfect 2×10⁶ cells with 4 µg of the OriP-p294 plasmids expressing type 1 or type 2 EBNA-2 and 2.4 µg DNA of the pSNOC plasmids expressing either type 1 or type 2 EBNA-LP. Empty vector (OriP-p294 or pSNOC) was added to normalize the total amount of DNA transfected (6.2 µg). Electroporation conditions were 1400 V, 30 pulse width and 1 pulse, which routinely gave approximately 40% transfection efficiency (assessed as GFP-positive cells when transfecting a GFP-expressing plasmid) and 80% of cell viability.

For the EREB2.5 growth assay, 6×10⁶ EREB2.5 cells were transfected with 5 µg of OriP-p294 plasmids bearing type 1/type 2 EBNA-2 sequences, either wild-type or chimaeric, using the Amaxa system (Lonza), program A-23. β-oestradiol was withdrawn immediately after transfection in order to select for β-oestradiol – independent lymphoblastoid cell lines. Cells were incubated overnight in 2 ml of complete medium supplemented with 20% FBS and antibiotics, but without β-oestradiol, in 12-well plates. The following day each transfected sample was diluted up to 10 ml with culture medium and plated into 5 wells of a 24-well plate. At the desired time-points cell samples were harvested for cell count analysis and RNA or protein extraction.

For immunofluorescence experiments, 5×10⁶ HeLa cells, grown on coverslips (VWR) in 6-well dishes, were transfected with 4 µg of OriP-p294 plasmids encoding either type 1, type 2 EBNA-2 or the chimaeric EBNA-2 proteins using Lipofectamine2000 (Invitrogen) as recommended by the manufacturer's instructions.

RNA extraction and qRT-PCR

Total cell RNA was extracted from growing cells using Trizol reagent (Invitrogen) and then quantified by measuring its absorbance at 260 nm. RNA samples were digested with RQ1 RNase-free DNase (Promega). cDNA was prepared using Protoscript M-Mulv First strand cDNA Synthesis kit (New England Biolabs) using random primer as recommended by the manufacturer's instructions. To detect the expression of the EBNA-2-regulated genes, quantitative RT-PCR (qRT-PCR) was used with the primer pairs listed in Table S2. qRT-PCR was performed on an ABI 7900HT real-time PCR machine using Absolute QPCR SYBR Green ROX Mix (Thermo Scientific). The cycling conditions were: 95°C for 15 min, followed by 40 cycles of 15 sec at 95°C, 30 sec at 60°C and 40 sec at 72°C on a fast block. Dissociation curve analysis was performed at the end of each run to ensure absence of non-specific products. Quantification of mRNA levels was carried out using the Delta-Delta Ct method. Results were analyzed with SDS2.3 software (Applied Biosystems). mRNA levels for each target gene analyzed were normalized on the housekeeping gene GAPDH used as a loading control.

RNase protection assay (RPA)

The LMP-1-specific probe for use in RPA was generated by PCR of genomic DNA derived from Daudi cells using the following primers, designed based on the EBV genome sequence: forward 5′-CAC TCA TAA CGA TGT ACA GC-3′ (coordinates 168881 to 169000 in EBV wild-type, accession number NC007605) and reverse 5′- CAA GAA ACA CGC GTT ACT C-3′ (coordinates 169103 to 169121 in EBV wild-type, accession number NC007605). The PCR product (241 bp) was cloned into pCR2.1-TOPO vector and sequenced and the plasmid was then linearized by BamHI digestion at the 5′ end of the PCR product. 1 µg of the linearized plasmid was in vitro-transcribed with T7 enzyme using the MAXIscript SP6/T7 in vitro transcription kit (Ambion), according to the manufacturer's instructions, for production of ³²P-labelled antisense RNA probe. RPAs were carried out using RPAIII kit (Ambion) as recommended by the manufacturer's instructions. Briefly, 20 µg of total cell RNA were hybridized overnight at 42°C with 50000 cpm of the probe. As negative control, an equivalent amount of yeast RNA was included in a hybridization reaction. Single-stranded RNA was digested with an RNase A/T1 mixture for 30 min at 37°C. Protected fragments were precipitated and separated on a polyacrylamide gel and the gel was analyzed on a phosphoimager.

Immunoblotting and antibodies

Cell samples were lysed for 15 min on ice in 2 volumes of RIPA lysis buffer (150 mM NaCl, 1% v/v Nonidet-P40, 0.5% v/v deoxycholic acid, 0.1% w/v SDS, 50 mM Tris/HCl, pH 8.0) supplemented with 1 mM PMSF (Fluka) and Complete protease inhibitors (Roche). Samples were centrifuged at 16000 g for 15 min, to pellet cell debris, and the supernatant was harvested. Proteins were fractionated by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking for 1 h at room-temperature with 5% milk powder in PBS/0.1% Tween-20 (Sigma), membranes were incubated overnight at 4°C with the primary antibody diluted in blocking buffer. The primary antibodies used are listed in Table S1. The secondary antibodies were horseradish peroxidise-conjugated anti-mouse (GE Healthcare) or anti-rabbit or anti-human IgG (Dako) and were used at a dilution of 1/2000 for 1 h at room-temperature. Bound immunocomplexes were detected by using ECL western blotting detection reagents (Amersham).

Indirect immunofluorescence

24 hours after transfection, HeLa cells were fixed using 4% paraformaldehyde and permeabilized with 0.25% Triton-X 100 (Sigma) in PBS. Primary anti-EBNA-2 antibody (PE2 clone) was diluted 1/100 in blocking buffer (1% bovine serum albumin in PBS) and cells were incubated for 45 min at room-temperature. The coverslips were washed 3 times with PBS and TRITC-conjugated anti-mouse secondary antibody (Sigma) was applied at a dilution of 1/5000 in blocking buffer for 45 min at room-temperature. After 3 washes in PBS the coverslips were mounted in Mowiol and visualized using a Zeiss 510 Meta laser confocal microscope.

Accession numbers

Database entries for genes mentioned in this manuscript include: CXCR7 (RefSeq NM 020311.2), EBV type 1 (accession number AJ507799) and EBV type 2 (accession number DQ279927). Annotation in the EBV genome shows EBNA-2 and LMP-1 genes.