Domains of the Epstein--Barr virus nuclear antigen 2 (EBNA2) involved in the transactivation of the latent membrane protein 1 and the EBNA Cp promoters

Abstract

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) is one of the first EBV-encoded gene products expressed after infection of primary B lymphocytes. EBNA2 is essential for the growth-transforming potential of the virus and it functions as a transcriptional activator of a set of viral and cellular genes. Sequencespecific DNA-binding by EBNA2 has not been demonstrated but the molecule is targeted to specific DNA regions by a cellular protein, RBP-JK, which recognizes the GTGGGAA sequence present in the regulatory region of all EBNA2-responsive promoters defined so far. We have determined the contribution of a RBP-Jx recognition sequence, an adjacent interferon-stimulated response element (ISRE) motif and a PU.l-binding site in the LMP1 regulatory sequence (LRS) to EBNA2-induced transactivation of the promoter by sitedirected mutagenesis of LRS-carrying reporter plasmids. EBNA2 responsiveness was reduced by approximately twofold when either or both of the RBP-JK-binding and ISRE sequences were mutated. ISRE seemed to function as an EBNA2-independent positive element. On the other hand, mutation of the PU box resulted in a drastic reduction of EBNA2 responsiveness, irrespective of whether the RBP-JK site or the ISRE motif was present. A comparative study by deletion mutation identified regions of EBV B95-8 EBNA2 involved in the transactivation of the LMP1 and the EBNA Cp promoters. Two domains of EBNA2 defined by deletion of amino acids 247-337 and 437-476 were found to be important for the activation of both promoters, while two different domains corresponding to residues 4-18 and 118-198 were required solely for the LMP1 promoter. Thus, EBNA2 must activate the LMP1 and Cp promoters by different mechanisms. All deletions involved in transcriptional activation of the two promoters contained regions that are conserved in EBNA2 of B95-8 EBV (type 1), AG876 EBV (type 2) and herpesvirus papio origin.

Interferon Regulatory Factor 5 Represses Expression of the Epstein-Barr Virus Oncoprotein LMP1: Braking of the IRF7/LMP1 Regulatory Circuit

“…essential for EBV's hallmark function, growth transformation of B lymphocytes (21,35,36). The finding that IRF7 induces expression of LMP1 provides a possible partial explanation for how LMP1 is expressed, although at low levels, in type II cells in the absence of EBNA2 (28) (Fig.…”

“…The other two pathways, ubiquitination and CDC42 activation, are mediated by LMP1 N-terminal and transmembrane domains, respectively (reviewed in references 9 and 20). Induction of expression of LMP1 depends on several virus-specific factors, the best characterized of which is the transcriptional activator EBV nuclear antigen 2 (EBNA2) (16,34,35,38,40).Our previous work uncovered the intimate involvement of IRF7 in EBV latency and showed first that IRF7, along with IRF2, represses the promoter used for expression of EBNA1 in type I latency in which latent gene expression is most restricted (45). We then detailed a special relationship between IRF7 and LMP1, which is capable of both inducing expression of and activating IRF7 (46, 49).…”

“…The other two pathways, ubiquitination and CDC42 activation, are mediated by LMP1 N-terminal and transmembrane domains, respectively (reviewed in references 9 and 20). Induction of expression of LMP1 depends on several virus-specific factors, the best characterized of which is the transcriptional activator EBV nuclear antigen 2 (EBNA2) (16,34,35,38,40).…”

The Latency-Associated Nuclear Antigen Encoded by Kaposi's Sarcoma-Associated Herpesvirus Activates Two Major Essential Epstein-Barr Virus Latent Promoters

“…The transforming ability of LMP1 is tightly linked to its association with the tumor necrosis factor family receptor-associated factors and may function similarly to the tumor necrosis factor family receptor, aggregating in the plasma membrane, where it is capable of binding tumor necrosis factor family receptor-associated factors (15,48). Numerous studies of LMP1 regulation have demonstrated that expression of LMP1 is dependent on regulation of its promoters by viral and cellular transcription factors (20,21,30,42,64,65,76).…”

“…EBNA2 lacks the ability to directly bind DNA but targets and activates EBV promoter elements through its association with RBP-J and other cellular transcription factors, including PU.1 and AML1 (21,23,30,42,(64)(65)(66)81). EBNA2 also associates with the basal transcription machinery and other coactivators of transcription and has been shown to transactivate the major EBV latent promoters and other responsive cellular promoters (73)(74)(75).…”