No abstract

Epstein–Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced NF-κB activation is important for infected cell survival. LMP1 activates NF-κB, in part, by engaging tumor necrosis factor (TNF) receptor-associated factors (TRAFs), which also mediate NF-κB activation from LTβR and CD40. LTβR and CD40 activation of p100/NF-κB2 is now known to be NIK/IKKα-dependent and IKKβ/IKKγ independent. In the experiments described here, we found that EBV LMP1 induced p100/NF-κB2 processing in human lymphoblasts and HEK293 cells. LMP1-induced p100 processing was NIK/IKKα dependent and IKKβ/IKKγ independent. Furthermore, the LMP1 TRAF-binding site was required for p100 processing and p52 nuclear localization, whereas the LMP1 death domain-binding site was not. Moreover, the LMP1 TRAF-binding site preferentially caused RelB nuclear accumulation. In murine embryo fibroblasts (MEFs), IKKβ was essential for LMP1 up-regulation of macrophage inflammatory protein (MIP)-2, TNFα, I-TAC, ELC, MIG, and CXCR4 RNAs. Interestingly, in IKKα knockout MEFs, LMP1 hyperinduced MIP-2, TNFα, and I-TAC expression, consistent with a role for IKKα in down-modulating canonical IKKβ activation or its effects. In contrast, LMP1 failed to up-regulate CXCR4 and MIG RNA in IKKα knockout MEFs, indicating a dependence on noncanonical IKKα activation. Furthermore, LMP1 up-regulation of MIP-2 RNA in MEFs was both IKKβ- and IKKγ-dependent, whereas LMP1 upregulation of MIG and I-TAC RNA was fully IKKγ independent. Thus, LMP1 induces typical canonical IKKβ/IKKγ-dependent, atypical canonical IKKβ-dependent/IKKγ-independent, and noncanonical NIK/IKKα-dependent NF-κB activations; NIK/IKKα-dependent NF-κB activation is principally mediated by the LMP1 TRAF-binding site.

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP, enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1-89 and 323-386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379-641), which includes the nuclear localization signal and DNAbinding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1-378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1-90 or histone H1-2 could substitute for EBNA-1 amino acids 1-378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60% of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells. Materials and Methods Cell Lines, Transfection, and Immunoblot Analysis. BJAB, an EBVnegative Burkitt lymphoma cell line, was cultured, transfected, used to derive cell lines stably expressing FLAG-tagged proteins, and analyzed by immunoblot for protein expression as described (23). Plasmids. Plasmids carrying the expression cassettes for enhanced green fluorescent protein (EGFP) fused EBNA-1 derivatives described in Fig. 1 were derived from pEGFPc1 (CLONTECH) and the BamHI-K fragment of EBV strain B95-8 DNA. The GE expression plasmid was constructed by inserting the 2.6-kb TfiI-HindIII fragment of BamHI-K between the BspEI and Abbreviations: EBV, Epstein-Barr virus; EBNA-1, EBV-encoded nuclear antigen-1; HMG-I, high-mobility group-I; NLS, nuclear localization signal; EGFP, enhanced green fluorescent protein; FISH, fluorescent in situ hybridization; FE, FLAG-tagged EBNA-1; FC, FLAG-tagged EBNA-1 C terminus; FHMC, FLAG-tagged HMG-I 1-90 fused to the EBNA-1 C terminus; FH1C, FLAG-tagged histone H1 fused to the EBNA-1 C terminus.