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.
Highlights
Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) binds virus DNA to mediate episome maintenance
C-terminal LANA directly binds a specific sequence in terminal repeat (TR) DNA, allowing LANA to mediate DNA replication and to tether KSHV episomes to mitotic chromosomes
Because the dorsal positive electrostatic patch is a distinctive LANA feature that is absent from the structurally homologous EBV EBNA1 gamma-1 herpesvirus DNA binding domain, we asked whether these residues are important for LANA’s episome maintenance function
Summary
Results: Mutating the novel LANA DNA binding domain (DBD) positive electrostatic patch engenders replication and episome persistence deficiencies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. 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 electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell. LANA mediates episome persistence by promoting KSHV DNA replication before each cell division and ensuring segregation of episomes to progeny nuclei. Some mutations in the LANA dorsal patch reduced BET protein binding; the reduction in binding did not correlate with the deficiencies in episome persistence
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