DNA binding and modulation of gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. T he latency-associated nuclear antigen (LANA) is highly expressed in these m alignancies and has been shown to play an important role in episomal mainte nance, presumably by binding to a putative oriP. In addition, LANA modulate s cellular and viral gene expression and interacts with the cellular tumor suppressors p53 and retinoblastoma suppressor protein. Many of these featur es are reminiscent of Epstein-Barr virus nuclear antigens (EBNAs), a family of six proteins expressed during latency. EBNA-1 is required for episome m aintenance, binds to oriP, and strongly activates transcription from two pr omoters, including its own. We have previously shown that LANA can transact ivate its own promoter and therefore asked whether LANA, like EBNA-1, activ ates transcription by direct binding to DNA. By using recombinant LANA expr essed from vaccinia virus vectors for electrophoretic mobility shift assays , we found that LANA does not bind to its own promoter. In contrast, LANA b inds specifically to sequences containing an imperfect 20-by palindrome in the terminal repeat (TR) of KSHV. We further show that the C-terminal domai n of LANA is sufficient for site-specific DNA binding. Unlike EBNA-1, which activates transcription through binding of oriP, we found that LANA inhibi ts transcription from a single TR binding site. A multimerized TR as found in the viral genome results in strong transcriptional suppression when link ed to a heterologous promoter. These data suggest that LANA, although fulfi lling functions similar to those of EBNA-1, does so by very different mecha nisms.