Fig 1.
Recombinant EBVs expressing NHP EBNA2 proteins infect human B cells.
A) Relative location of the EBNA2 gene locus downstream of the major Internal Repeat (IR1) within the EBV genome and detailed description of the flanking regions within the EBV BAC (MD1). The native flanking sequences and EBNA2 open reading frame (black arrow) are shown in the wild type EBV BAC derived from B95-8 (MD1/B95-5). The EBNA2 deleted BAC (MD1ΔE2) contains the linker region from the shuttle plasmid containing two restriction enzyme sites (RE1 and 2) and a downstream scar sequence as a result of the cloning procedure. Recombinant EBVs carrying EBV E2 (MD1-E2, black arrow), chimpanzee, baboon, cynomolgus or rhesus LCV EBNA2 (MD1-chE2, -baE2, -cyE2, -rhE2; polka dots) were cloned by recombining similar DNA fragments from shuttle plasmids containing each EBNA2 species. In this manner all rEBV contained an EBNA2 species with identical upstream and downstream sequences. B) Production of rEBVs was confirmed by infection of EBV-negative Louckes cells and detection of EBNA2. The monoclonal antibody against EBNA2 (PE2) was used to detect LCV EBNA2 expression by immunoprecipitation and subsequent immunoblotting from cell lysates prepared 2 days after infection with equal volumes of rEBV. C) Relative infectious titers of rEBV supernatants were determined by infection of EBV-negative Louckes cells and puromycin selection. Louckes cells were infected with equal volumes of rEBV, distributed into 50 microtiter wells, and selected for rEBV infection using puromycin. After 3–4 weeks the number of wells with cell growth was counted, and average results for at least 2 independent supernatants are shown. +: 1–20 wells with growth/50 total wells, ++: 21-44/50, +++: 45-50/50, n.d.: not done.
Fig 2.
Maintenance of B cell immortalization in conditionally immortalized ER/EB2-5 cells by rEBVs.
A) ER/EB2-5 cells were infected with rEBVs and distributed into 100 microtiter wells. Half the wells were selected for infection by culture with medium containing puromycin and estrogen (+estrogen) and the other half of the cells underwent biological selection in medium without additives (-estrogen) to determine functionality of the EBNA2 protein. After 6 weeks the number of wells with cell growth was determined. The average result from multiple experiments is shown with 2 independent viral supernatants of each rEBV tested. +: 1–20 wells with growth/50 total wells, ++: 21-44/50, +++: 45-50/50, -: no growth. B) Four weeks after infection, selected clones growing in the absence of estrogen were used to detect EBNA2 and GAPDH as loading control by immunoblot. Cells infected with rEBV-E2, rEBV-chE2, or rEBV-baE2 express an EBNA2 species of the relative molecular weight expected for the respective rEBV.
Fig 3.
De novo immortalization of human PBMC by rEBVs.
A) Human PBMC were infected with rEBVs, cultured in 50 microtiter wells, and outgrowth of immortalized cells was quantified 6 weeks after infection. Average results for at least 2 experiments are shown using independent virus preparations of rEBV (n = 3), rEBVΔE2 (n = 3), rEBV-E2 (n = 5), rEBV-chE2 (n = 4), rEBV-baE2 (n = 3), rEBV-cyE2 (n = 2), and rEBV-rhE2 (n = 4). +: 1–20 wells with growth/50 total wells, ++: 21-44/50, -: no growth. B) Representative cell lines derived from PBMC of the same donor (D30) infected with rEBV-E2 and rEBV-baE2 were immunoblotted for detection of EBNA2, EBV LMP1, and EBV EBNA1. Detection of GAPDH controlled for loading and BJAB cells served as EBV-negative control. C) Expression of only one EBNA2 species was confirmed by species-specific PCR amplification for EBV or baboon LCV EBNA2 in rEBV immortalized cells. Wild type MD1, MD1-E2, and MD1-baE2 BAC DNAs purified from bacteria served as controls. D) Surface expression of the EBNA2-regulated marker CD23 on rEBV immortalized cells D30/rEBV-baE2 (dark grey fill), and D30/rEBV-E2 1 (black line) or the EBV negative cell line BJAB (light grey fill) was determined by flow cytometry. E) Growth curve of D30 LCLs transformed with rEBV-E2 (grey curves, line: clone 1, dashes: 2, dots: 3) or rEBV-baE2 (black line).
Fig 4.
Transactivation activity of NHP EBNA2s in human B cells.
P3HR-1 cells were transfected with a Cp-Luciferase reporter, a Renilla control, and EBNA2 expression plasmids or empty control. A) 48h after transfection Luciferase and Renilla levels were measured and normalized to Renilla. Cp transactivation levels shown represent fold change over control transfection. Asterisks above standard deviation bars indicate significance relative to control, and asterisks above the dotted lines indicate significance relative to EBV E2. * p<0.05, ** p<0.01, n.s. not significant, n = 4. B) EBNA2 and LMP1 were quantified from a representative reporter assay by immunoblot, and tubulin was used as loading control. Relative expression levels of EBNA2 normalized to EBV E2 sample and LMP1 normalized to levels of control transfection are quantified under each blot. C) Cp activity in response to EBNA2 and EBNA-LP co-expression was analyzed as described for panel A) with co-transfection of an EBNA-LP expression plasmid. Luciferase levels represent fold change over LP alone (control+LP).
Fig 5.
Activity of EBV/rhLCV-EBNA2 chimeras in Cp transactivation, maintenance, and de novo immortalization.
A) Schematic representation of EBV E2 with its 10 Conserved Regions (CRs, black boxes), the poly-proline region (PPR), and diversity region (DR). Functional domains include self association domains (SAD), binding sites for SKIP, RBP-Jκ and BS69, transactivation domain (TAD) and a nuclear localization signal (NLS). B) Overview of EBNA2 chimeras used in this study showing portions derived from EBV E2 in black and those from rhE2 in grey. Relative sizes of chimeras are drawn to scale. Chimeras are labeled with the last or the first amino acid of the fragments that were fused together corresponding to the position within the wild type EBV E2 or rhE2, e.g., EBV378/rh484 is a fusion of the EBV E2 fragment 1–378 and the rhE2 fragment 484–605. For reference, position 378/379 of EBV E2 aligns with position 483/484 in rhE2. Chimeras labeled with brackets indicate internal exchanges, e.g., rh[EBV156-378] is a rhE2 mutant that expresses the EBV E2 region 156–378 in place of the corresponding rhE2 sequences. C) Transactivation activity of E2 chimeras was determined in P3HR-1 cells after transfection with expression plasmids for E2 chimeras and a Cp-Luciferase reporter plasmid. Two days after transfection Luciferase levels were measured and normalized to levels of internal Renilla controls. Activity is expressed as fold change over transfection with empty control plasmid (dashed line). All chimeras induced Cp activity significantly above background (p<0.05, n≥3). D) Recombinant EBVs expressing E2 chimeras were used to infect ER/EB2-5 cells or human PBMC as in Figs 2 and 3 to test their ability to rescue ER/EB2-5 cell growth (maintenance) or immortalize human PBMC (de novo). After infection ER/EB2-5 cells were plated into 2 microtiter plates (50 wells each) and selected with puromycin in the presence of estrogen, or cultured in medium without additives. After 6 weeks the number of wells with cell growth was counted and percent outgrowth determined (average of 2 infections using independent virus preparations). For maintenance results are relative to control infection (number of wells without estrogen/number of wells with estrogen with ‘-‘ indicating no growth without estrogen, but growth with estrogen). E) EBNA2 expression was confirmed by immunoblot with PE2 antibody from 2x105 transiently transfected P3HR-1 cells described in panel C) or selected ER/EB2-5 or LCL clones infected with rEBVs described in panel D). Uninfected ER/EB2-5 cells grown with estrogen served as a control for ER-EBNA2 expression.
Fig 6.
Interaction of EBNA2s with human RBP-Jκ.
293T cells were transfected with a myc-tagged RBP-Jκ expression plasmid. After 72h cells were lysed, and combined with comparable amounts of in vitro transcribed and translated EBNA2 proteins. The RBP-Jκ/EBNA2 complexes were isolated by co-immunoprecipitation with an anti-myc-antibody. EBNA2 and myc-RBP-Jκ were detected and quantified by immunoblot. A) Immunoblot results of a representative experiment showing EBNA2 and myc-RBP-Jκ detection from 2% of input (left lane) and 100% of precipitates (IP, right lane). EBV-E2-WW/AA is a RBP-Jκ binding mutant. Quantification of recovered EBNA2, precipitated myc-RBP-Jκ, and relative amount of bound EBNA2 in this experiment is shown below immunoblots. B) Average results for 4 independent experiments are shown with the amount of EBV E2 bound to RBP-Jκ set to 100%; for rhE2[EBV156-294] n = 3. Asterisks indicate significance relative to EBV-E2-WW/AA background. * p<0.05, ** p<0.01, *** p<0.001.