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EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase ␣-1 subunit, ␣-tubulin, and ␤-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, ␣-tubulin, and ␤-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins. EBNA-LP bound to two domains in the DNA-PKcs C terminus and DNA-PKcs associated with the EBNA-LP repeat domain. DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs.Epstein-Barr virus (EBV) is a human herpesvirus that initiates primary infection and replication in the oropharyngeal epithelium (62). EBV infection then spreads to B lymphocytes, which are largely nonpermissive for virus replication (47, 68). Based on in vitro studies of B-lymphocyte infection, the first EBV transcripts initiate within the viral long internal repeat (for reviews, see references 26 and 53). These transcripts are differentially spliced to encode two nuclear proteins, EBNA-LP and EBNA-2. These two proteins act in concert to activate transcription of cell and viral genes including the cellular cmyc, CD23, and cyclin D2 and the viral EBNA-3A, -3B, -3C, and -1 and latent infection membrane protein, LMP1 and -2, genes (1,16,23,49,61). These virus-encoded proteins cause the cell to enter S phase and proliferate indefinitely. In stably transformed B lymphocytes EBV expresses six EBNAs, two LMPs, two small RNA or EBERs, and transcripts from the BamHI A fragment (13,18). Recombinant EBV reverse genetic studies indicate that EBNA-LP, EBNA-2, EBNA-3A, EBNA-3C, EBNA-1, and LMP1 are critical or essential for B-lymphocyte proliferation, while EBNA-3B, LMP2, EBERs, and most of the rest of the viral genome are not critical (8, 15, 24, 25, 27, 33, 34, 38-41, 43, 54, 55, 69).The experiments described here investigate the associations of EBNA-LP with cellular proteins. EBNA-LP is unusual in that it is encoded mostly by repeating 66 and 132 b exons, which are derived from the EBV long internal repeat (10, 58, 73). The EBNA-LP open reading frame ends within 33 and 102 b exons that are transcribed from the unique DNA downstream of the long internal repeat. EBNA-LP lacks the ability to recognize specific DNA sequences and is dependent on interaction with EBNA-2 for promoter-specific transcriptional effects (16, 49). EBNA-2 has two essential domains (6, 7). One interacts with cellular, sequence-specific, DNA binding proteins, including 17,22,32,64,78). The second is an acidic transcriptional activation domain that interacts with basal and activated transcription factors, with CR...

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.