title: The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence.
abstract: Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.
authors.affiliation: Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115.
authors.forename: Shijun
authors.initials: S
authors.lastname: Li
authors.affiliation: Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115.
authors.forename: Min
authors.initials: M
authors.lastname: Tan
authors.affiliation: Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115.
authors.forename: Franceline
authors.initials: F
authors.lastname: Juillard
authors.affiliation: Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Ponnusamy
authors.affiliation: Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
authors.forename: Bruno
authors.initials: B
authors.lastname: Correia
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors.forename: J Pedro
authors.initials: JP
authors.lastname: Simas
authors.affiliation: Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Carrondo
authors.affiliation: Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
authors.forename: Colin E
authors.initials: CE
authors.lastname: McVey
authors.affiliation: Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115. Electronic address: kkaye@bwh.harvard.edu.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Kaye
mesh_terms: D000956:Antigens, Viral / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D004261:DNA Replication; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000502:physiology*; D055672:Static Electricity; D020079:Terminal Repeat Sequences; D017735:Virus Latency
chemical_list: D000956:Antigens, Viral; D009687:Nuclear Proteins; C109540:latency-associated nuclear antigen
keywords: DNA viruses; Kaposi sarcoma-associated herpesvirus (KSHV),; cancer; episome; herpesvirus; latency-associated nuclear antigen (LANA); site-directed mutagenesis; structure-function; tumor virus; viral protein