KSHV encoded LANA recruits Topoisomerase II? for latent DNA replication of the terminal repeats

Abstract

The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) plays major role in maintaining latency and is critical for the perpetual segregation of viral episomes to the progeny nuclei of newly divided cells. LANA binds to KSHV terminal repeat (TR) DNA and tethers the viral episomes to host chromosomes through the association of chromatin-bound cellular proteins. Terminal Repeat (TR) elements serve as potential origin sites of KSHV replication and have been shown to play important roles in latent DNA replication and transcription of adjacent gene. Affinity chromatography and proteomics analysis using KSHV TR DNA and the LANA binding site as the affinity column identified Topoisomerase II? (TOPOII?) as a LANA interacting protein. Here, we show that TOPOII? forms complex with LANA and co-localize as punctuate bodies in the nucleus of KSHV infected cells. Specific TOPOII? binding region of LANA has been identified to its N-terminus and the first 1-32 amino acid residues containing the nucleosome-binding region crucial for binding. Moreover, this region could also act as a dominant negative to disrupt association of TOPOII? with LANA. TOPOII? plays an important role in LANA dependent latent DNA replication as addition of ellipticine, a selective inhibitor of TOPOII, negatively regulated replication mediated by the TR. DNA break labeling and ChIP assay using Biotin-16-dUTP and terminal deoxynucleotide transferase showed that TOPOII? mediate transient DNA break on viral DNA. These studies confirm that LANA recruits TOPOII? at the origins of latent replication to unwind the DNA for replication.