title: Determination of Kaposi's sarcoma-associated herpesvirus C-terminal latency-associated nuclear antigen residues mediating chromosome association and DNA binding.
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.
authors.affiliation: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Brenna
authors.initials: B
authors.lastname: Kelley-Clarke
authors.forename: Mary E
authors.initials: ME
authors.lastname: Ballestas
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Srinivasan
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Barbera
authors.forename: Takashi
authors.initials: T
authors.lastname: Komatsu
authors.forename: Te-Ana
authors.initials: TA
authors.lastname: Harris
authors.forename: Mia
authors.initials: M
authors.lastname: Kazanjian
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Kaye
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D002877:Chromosomes, Human / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D019288:Herpesvirus 8, Human / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D008938:Mitosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014779:Virus Replication / Q000502:physiology
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C109540:latency-associated nuclear antigen; D004247:DNA