Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization
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.