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-lerminal 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 tor DNA binding, as assayed by electrophoretic mobility shift assay, and mitotic chromosome association, indicating that distinct C-terminal LANA subdomains effect BNA 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. Copyright © 2007, American Society for Microbiology. All Rights Reserved.