Sequence and Functional Analysis of EBNA-LP and EBNA2 Proteins from Nonhuman Primate Lymphocryptoviruses

Abstract

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5-to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.Epstein-Barr virus (EBV) is a gammaherpesvirus and a preeminent tumor virus in humans. EBV is associated with a variety of cancers, including endemic Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, and lymphoma in the immunosuppressed (40). Consistent with its association with human malignancy, EBV also immortalizes human B lymphocytes with high efficiency in vitro (35). Efficient immortalization of B lymphocytes requires expression of only a subset of viral genes (22). These genes include several EBV nuclear antigens (EBNAs), EBNA1, EBNA2, EBNA3A and -C, and EBNA-LP, and an integral latent membrane protein, LMP-1. EBNA-LP is the first protein along with EBNA2 made during infection of lymphocytes by EBV (1). Despite a growing body of knowledge on the molecular mechanisms of latent protein functions, the role of EBNA-LP for EBV-induced immortalization remains enigmatic.The EBNA-LP protein (also referred to as EBNA-5 or E...

Simian homologues of Epstein–Barr virus

“…The EBNA-2 homologues are one of the least wellconserved genes among simian LCV (26.3% and 26.5% similarity for baboon and rhesus LCV, respectively, versus EBV EBNA-2) (Cho et al 1999;Ling & Hayward 1995;Peng et al 2000). Again, one of the surprising ¢ndings is that these genes are equidistant from each other, i.e.…”

“…(e) EBNA-LP An EBNA leader protein (EBNA-LP) homologue is expressed in baboon and rhesus LCV-infected B cells and both include exons derived from the major internal repeat sequences and downstream unique sequences (Peng et al 2000). The simian LCV homologues are well conserved with 61% and 64% homology, respectively, in the exons derived from the internal repeat sequence and 51% identity in the unique exons.…”

The NP9 protein encoded by the human endogenous retrovirus HERV‐K(HML‐2) negatively regulates gene activation of the Epstein‐Barr virus nuclear antigen 2 (EBNA2)

“…The corresponding EBNA2 from the baboon LCV, however, does have neither Arginines nor Lysines at this position. 26 In turn, the lower primates also do not contain endogenous retroviruses of the HERV-K family. 49 Some proteins of endogenous retroviruses have acquired essential functions in humans; for instance, syncytin encoded by HERV-W is involved in the generation of the syncytiotrophoblast layer during pregnancy.…”

“…8 The EBV-related viruses of lower primates encode EBNA2 proteins with conserved features like the aforementioned WWP-motif necessary for binding to RBPJj but show no conservation for the RG-repeat. 26,47,48 In the experiments comparing the Rhesus LCV EBNA2 to the EBV-EBNA2, the two proteins showed different abilities to activate the viral (EBV) C-or LMP2A promoters in that EBV-EBNA2 activated the C-promoter stronger that the LCV-EBNA2, while the latter was better at stimulating the LCV-C-promoter. In turn, the LCV-EBNA2 showed stronger activation of the EBV-LMP2A promoter as compared to the EBV-counterpart.…”

“…The plasmid pAG115, kindly supplied by Fred Wang, Harvard Medical School, contains the Rhesus lymphcrytopvirus (LCV) homologue of EBNA2 that can be used for in vitro transcription/translation. 26 For the luciferase assays, the LLO LMP1-promoterluciferase construct 27 was used as described. 21 The C-promoter construct (Cp-luc) which contains nucleotides from À1,425 to þ4 of the C-promoter (Cp) of EBV 28 was a gift of M. Allday (Imperial College London).…”