No abstract

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) tethers viral terminal repeat (TR) DNA to mitotic chromosomes to mediate episome persistence. The 1,162-amino-acid LANA protein contains both N- and C-terminal chromosome attachment regions. The LANA C-terminal domain self-associates to specifically bind TR DNA and mitotic chromosomes. Here, we used alanine scanning substitutions spanning residues 1023 to 1145 to investigate LANA self-association, DNA binding, and C-terminal chromosome association. No residues were essential for LANA oligomerization, as assayed by coimmunoprecipitation experiments, consistent with redundant roles for amino acids in self-association. Different subsets of amino acids were important for DNA binding, as assayed by electrophoretic mobility shift assay, and mitotic chromosome association, indicating that distinct C-terminal LANA subdomains effect DNA and chromosome binding. The DNA binding domains of LANA and EBNA1 are predicted to be structurally homologous; certain LANA residues important for DNA binding correspond to those with roles in EBNA1 DNA binding, providing genetic support for at least partial structural homology. In contrast to the essential role of N-terminal LANA chromosome targeting residues in DNA replication, deficient C-terminal chromosome association did not reduce LANA-mediated DNA replication.

Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists asan episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1-and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1-and oriP-dependent transcription and do not inhibit EBNA1-and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and 486 RALL 489 and 580 KDLVM 584 as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1-and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1-and oriPCp-dependent transcription and episome persistence.Epstein-Barr virus (EBV) replicates in oropharyngeal epithelial cells (62,70,85). Subsequent latent infection of mature B lymphocytes can result in infectious mononucleosis and is essential for long-term latency and reactivation and continues to be shed in saliva (21). Latently EBV-infected B cells can emerge as Burkitt's lymphomas (BL) or Hodgkin's disease in otherwise healthy people or as lymphoproliferative disease in people who have T-lymphocyte deficiencies due to HIV infection, transplantation, advanced age, or a genetic predisposition (4,8,25,34,49,62,85). In immunocompetent people, particularly those of southern Chinese descent, latent EBV infection can also cause nasopharyngeal carcinoma, which is the most common EBV malignancy worldwide (25,34). Latent EBV infection is a significant cause of morbidity and mortality.In latent infections, including malignancies, EBV usually persists as a multicopy episome (1,47,56,60). EBV genome integration is unusual (33, 52). EBNA1 is essential for EBV episome persistence in dividing cells and enhances episome transcription (59,61,83). The EBNA1 DNA binding domain recognizes 24 cognate sites in the EBV episome OriP element, while ...