Materials and Methods
Generation of EBNA3 gene-deleted and revertant EBV and infection of BL cells
The EBNA3 genes were deleted from the B95.8 EBV-BAC [p2089, (Delecluse et al ., 1998)] by RecA-based homologous recombination. Regions of the EBV genome flanking EBNA3A, EBNA3B and EBNA3C were amplified by PCR and pairs of these products were cloned contiguously into the shuttle plasmid pKovKan ΔCm [(White et al ., 2003) and Supplementary Figure 1A]. Thus each shuttle plasmid contained a fragment of the EBV genome with an internal deletion of either EBNA3A [B95.8 (Acc#:V01555) coordinates 91915 to 95428 with internal deletion 92310 to 94900], EBNA3B (B95.8 coordinates 94901 to
98826 with internal deletion 95429 to 98233) or EBNA3C (B95.8 coordinates
98234 to 101482 with internal deletion 98827 to 100998). In the case of
EBNA3C, only exon 2 (ex2) of the gene was deleted. The EBNA3 genes in the EBV-BAC were replaced by these deletion constructs by homologous recombination between the GFP-positive, hygromycin-resistant, B95.8 EBV-
BAC and these shuttle plasmids, as described previously [(White et al ., 2003);
Figure 1A and Supplementary Figure 1B]. Revertants were then made for each deletion by homologous recombination between the deletion BAC and a shuttle plasmid harbouring the appropriate EBNA3 gene, thus restoring the deleted gene. The integrity of the BACs was screened at each stage of

recombination by miniprep DNA isolation, restriction digest and pulsed field gel electrophoresis.
293 cell clones containing deletion and revertant BACs were produced: BAC
DNA was purified by maxiprep (Qiagen) and 1
µg transfected into 293 cells using an integrin-targeting peptide combined with Lipofectin (Invitrogen) as described previously (Hart et al., 1998). Cells were exposed to lipid-peptide-
DNA complexes for 4 to 6 hours in Optimem (Invitrogen). Stable 293 cell clones were isolated under hygromycin B selection (Roche; 125
µg/ml) by ring and dilution cloning. A 293 clone containing the BAC used to generate the recombinants (293-2089-F2) was also produced. Episomal BAC DNA was recovered from 293 cells grown in six-well dishes by preparation of low molecular weight DNA as described previously (Wade-Martins et al ., 1999) and 4% electroporated into El ectromax™ DH10B™ E.coli
(Invitrogen).
Several individual colonies were selected and DNA extracted by miniprep and analysed by restriction digest and pulsed field gel electrophoresis.
Infectious virus was generated from 293 producer cells by transient transfection of BZLF1 and BALF4 expression plasmids (Dirmeier et al ., 2003;
Neuhierl et al ., 2002). Supernatant was harvested and infectivity assessed by
GFP expression in Raji cells. One ml of supernatant containing EBNA3A,
EBNA3B and EBNA3C knockout and revertant viruses, and the wild type virus
(F2), was used to infect 1x10 5 EBV-negative BL cells (BL31 and BL2). After
48 hours, cells were selected in Hygromycin B (Roche) at a concentration of
200
µg/ml to produce EBV-converted BL lines. Episomal BAC DNA was recovered from 1x10 6 EBV-converted BL cells by the same method as

described for 293 cells. Integrity of the BAC was assessed by restriction digest and pulsed field gel electrophoresis.
Results
Screening of recombinant EBV-BACs
Individual EBNA3 genes were deleted from the EBV-BAC by homologous recombination with an appropriate shuttle plasmid (Figure 1A and
Supplementary Figure 1). In order to control for any second site mutations that may have occurred, each gene was replaced during a second round of recombination to generate revertant BACs. Deletion of each EBNA3 gene introduced either a Cla I or Not I restriction site into the EBV-BAC (the location of these restriction sites in the original EBV-BAC is shown in Supplementary
Figure 1A). Digestion with these restriction enzymes followed by pulsed field gel electrophoresis therefore enabled diagnosis of deletion and revertant
BACs, with the size of restriction fragments from different deletion mutants and the original EBV-BAC being sufficiently different to allow discrimination by pulsed field gel electrophoresis alone (Supplementary Figures 2A and 2B).
Two different EBNA3C-knockout BACs were generated; one in which only the expected change (deletion of EBNA3C) had taken place (3CKO), and one which had also gained DNA in the Bam W repeat region (3CKO+BamW).
Pulsed field gel analysis of 3CKO+BamW digested with various different restriction enzymes suggested that expansion of this region corresponds to addition of two copies of the Bam W repeat and was the only gross rearrangement that had occurred (data not shown). Given the considerable variation in the number of Bam W repeats in different EBV strains and the fact

that the repeat number found in 3CKO+BamW was well within this range, expansion of this region was not considered to be undesirable.
Wild type, deletion and revertant BACs were transfected into 293 cells and stable producer cell clones generated. The integrity of episomal BAC DNA recovered from 293 producer cells was analysed by restriction digest with Eco
RI (Supplementary Figure 3) and Age I (data not shown) and pulsed field gel electrophoresis. These restriction enzymes cut throughout the EBV genome allowing any gross rearrangements to be easily identified by comparison with the BAC DNA originally transfected.
Validation of recombinant viruses in BL31 cells
Infectious virus was generated from wild type, deletion and revertant BACs maintained in 293 cells and used to infect EBV-negative BL31 cells. Viruspositive converts were selected in hygromycin and analysed for EBV gene expression (shown in Figure 1C and discussed in results section). Episomal
BAC DNA was also recovered from converted BL31 cell lines and its integrity assessed by restriction digest with Eco RI and pulsed field gel electrophoresis
(data not shown). BACs rescued from many of the BL31 lines had undergone some alteration, but the vast majority of these changes were confined to either the Bam W repeat region or the terminal repeat region. It was assumed that loss or gain of repeat elements in these regions, within certain limits, would not affect the function of the EBV episome within BL31 cells, as verified by the correct expression of EBV latent genes (Figure 1C).