Evidence for C189- and S186- me cytosine⁻² interactions.

<p>A. Contacts between CpG motif 1 and Zta residues. B. EMSA analysis of unprogramed <i>in vitro</i> translation reaction (none), or Zta protein with the indicated probes was undertaken. The protein-DNA complex is shown. C. The ability of Zta and ZtaS186A to interact with a probe that omits the methylation of cytosine⁻² was determined by EMSA. D. The ability of Zta and ZtaC189S to interact with a probe that omits the methylation of cytosine⁻² was determined by EMSA.</p

Single amino acid in basic region of Zta blocks the ability to transactivate Rp in BL cells.

<p>A. Schematic diagram showing the relationship between Zta expression and activation of the <i>BRLF1</i> promoter. B. Expression vectors for Zta, ZtaC189S and the relevant “empty” vector (pBABE) were introduced into Raji cells and their ability to activate the endogenous <i>BRLF1</i> gene determined. 24 hours after transfection, RNA was prepared, cDNA was synthesized then amplified using quantitative PCR with specific primers for the <i>BRLF1</i> transcript and a housekeeping gene, L32. Expression of <i>BRLF1</i> mRNA, following normalization for expression of L32 is shown, relative to that seen following Zta transfection (100%). C. Expression of Zta and ZtaC89S were determined by quantitative PCR and expressed relative to expression of Zta (100%).</p

ZtaC189S binding to meZREs in Rp is compromised <i>in vitro</i>.

<p>A. EMSA analysis was undertaken for meZRE2 with the indicated proteins or an unprogrammed lysate (IVT). B. Zta, ZtaC189A and ZtaC189S were generated <i>in vitro</i> and analyzed by SDS-PAGE. C. EMSA analysis of equivalent amounts of the indicated proteins was undertaken with meZRE3 as described above.</p

Structural model of Zta bound to meZRE3.

<p>A. Alignment of Zta recognition sequences. The numbering convention is shown for base pair positions (bold italics) and individual nucleotides (plain font). Cytosines modified by methylation are indicated by a dot. B. Model of the Zta-meZRE3 complex viewed along the pseudodyad. The methylation sensitive C189 residue (red asterisk) and bidentate hydrogen bond interactions between R190^Left and Guanine^0′ (dotted black lines) are indicated. Cytosine methyl groups are semi-transparent. C. Orthogonal view. The hydrophobic contact between Cytosine^1′ and S186^Left (broken blue line) and hydrogen bond network involving S186^Left, N182^Left and Guanine2′ (dotted black lines) are shown. D. Schematic summary of contacts. van der Waals contacts involving the CpG methyl groups and Zta residues are shown as broken lines.</p

ZtaC189S binding to meZREs in Rp is compromised <i>in vivo</i>.

<p>A. Schematic representation of the <i>BRLF1</i> gene. The black arrow indicates the primary transcript. The location of primer sets used to detect sub-regions of Rp and upstream and downstream regions are indicated relative to the transcription start site. B. HisZta and HisZtaC189S were introduced into 293-BZLF1-KO cells and chromatin prepared. Chromatin affinity purification was undertaken and binding to Rp detected with the indicated primer sets by real-time PCR. The signal was set relative to the “empty vector”, pcDNA3 (striped box), and the signal for Zta (filled box) and ZtaC189S (open box) are shown together with the standard error from duplicate experiments. C. Expression of HisZta, HisZtaC189S and a loading control, PARP, were assessed by western blot analysis.</p

All four methylation sites on ZRE3 contribute to the binding by Zta.

<p>A. Schematic representation of the four methyl-cytosine residues in ZRE3. Methylation is indicated by an asterisk and the numbering system is shown. B, C. Competition EMSA reactions were undertaken with a labeled ZRE (ZIIIB) and non-labeled Zta protein. As indicated, increasing amounts (6×, 10×, 20×, 50×, 75× and 100× excess) of unlabelled competitor ZRE3 DNA (methylated or not) was included in the EMSA reaction. D–G. EMSA competition from replicate experiments showing the ability of the indicated excess of each singly methylated ZRE3 site to compete for the binding of Zta. Experiments were undertaken in duplicate and were used to calculate the standard deviation shown in the error bars.</p

Zta and ZtaC189S interact with non-methylated Rp ZREs equivalently.

<p>A. Schematic diagram showing the location of ZREs 1–3 in Rp. Transcription of this gene occurs in a leftwards direction with respect to the viral genome. The numbering relates to the type I EBV genome accession number NC_007605. Asterixes mark the methylated Cytosine residues. B. Zta and Zta C189s were generated in an <i>in vitro</i> translation system and fractionated on SDS-PAGE, together with a non-programmed translation reaction (IVT). C.–E. Equivalent amounts of the indicated proteins were subject to EMSA analysis with the probes indicated above. A reaction with no added protein was also included, indicated probe. F. The three ZREs associated with Rp are aligned and their areas of conservation indicated by boxes. The interactions of Zta and ZtaC189S with each site are summarized.</p

Quantity and phosphorylation state of the ‘pocket’ proteins and expression of E2F1-target genes in p16-null and -competent LCL 3CHT lines with or without 4HT.

<p>(<b>A</b>) Similar cell extracts to those used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003187#ppat-1003187-g001" target="_blank">Figure 1D</a> were separated by SDS-PAGE and western blotted using a pan-specific anti-Rb antibody (pan-Rb) to detect all forms of Rb from hypophosphorylated (lower bands) to hyperphosphorylated (upper bands). Anti-phospho-Rb antibody (Rb-PO₄) detects only the hyperphosphorylated forms of Rb and anti-p107 indicates the pocket protein p107. Gamma-tubulin was used as a loading control. In p16-competent LCL 3CHT lines cultured for 21 days without 4HT, Rb is down-regulated and hypophosphorylated and p107 is also down-regulated. In p16-null LCL 3CHT lines (A2 and C2 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003187#ppat-1003187-g001" target="_blank">Figure 1</a>), no difference in the quantity of Rb or p107 nor Rb phosphorylation is detectable regardless of EBNA3C activity. (<b>B</b>) Regulation of the E2F1-target EZH2. Samples of protein extracts used in (A) were separated by SDS-PAGE and western blotted with an antibody specific for the polycomb complex subunit EZH2 – a protein induced by E2F1. Again gamma-tubulin was used as a loading control. All these data are representative of two independent experiments each including at least two p16-null LCL 3CHT lines. (<b>C</b>) Heat-map visualization of microarray data from p16^INK4a-null 3CHT LCLs grown in the presence or absence of 4HT for more than 30 days. This displays relative transcript levels (i.e. raw transcript levels corrected for variation between the three cell clones and then mean-normalized for each gene) for E2F1 and 45 of its transcriptional targets. The scale indicates high (red) to low (blue) changes in transcript levels, with the extremes of the scale indicating a 0.42-fold change in expression (ie a sample on the extreme red end of the scale would show a 1.42-fold expression increase over one at the center - grey). The only gene significantly and consistently altered in its expression by the inactivation of EBNA3C is TP73 (indicated by arrow), which falls upon EBNA3C inactivation. The dendrogram (bottom) shows the ordering of the genes by their clustering into co-regulated groups. Cell line clone IDs and treatment (±4HT) are indicated to the left.</p

p53-p21^WAF1 pathway is not activated in p16-null LCL 3CHT cells following removal of 4HT and inactivation of EBNA3C.

<p>(<b>A</b>) Four p16-null LCL 3CHT lines were cultured for 21 days with (+) or without (−) 4HT. Protein extracts were separated by SDS-PAGE and western blotted for p53 or p21^WAF1. No consistent changes in expression of p53 or p21^WAF1 were observed. (<b>B</b>) qPCR of cDNAs corresponding to p21^WAF1 transcripts in the same four p16-null LCL 3CHT lines (A1, A2, C1, C2) and two p16-competent LCL 3CHT lines (A and C) cultured for 21 days with or without 4HT. The data are normalized to the expression of control RNA.</p

Cell proliferation and viability after primary infection of B cells.

<p>B cells isolated using anti-CD19 magnetic beads (<b>A and B</b>) or ficoll-purified bulk lymphocytes from a buffy-coat residue (from donor D11) (<b>C and D</b>) were infected by the viruses indicated, at day zero. The proportion of proliferating cells (<b>A and C</b>) was assessed by EdU incorporation over 16 hours. Cell viability was assessed by Live/Dead staining (<b>B and D</b>). The sampling time after infection, and the virus used to infect the cells are as indicated. Note that FACS statistics were not collected for 3CHT+HT at 27 days due to a technical failure during automated data acquisition (_*). However visual inspection indicated that the majority of the cells were viable and this was consistent with the yield of RNA recovered from these cells (which was comparable to the WT and revertant infections – see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003187#ppat-1003187-g007" target="_blank">figure 7</a>).</p