LANA oligomeric architecture is essential for KSHV nuclear body formation and viral genome maintenance during latency

The molecular basis for the formation of functional, higher-ordered macro-molecular domains is not completely known. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) genome forms a super-molecular domain structure during latent infection that is strictly dependent on the DNA binding properties of the viral nuclear antigen LANA to the viral terminal repeats (TR). LANA is known to form oligomeric structures that have been implicated in viral episome maintenance. In this study, we show that the LANA oligomerization interface is required for the formation of higher-order nuclear bodies that partially colocalize with DAXX, EZH2, H3K27me3, and ORC2 but not with PML. These nuclear bodies assemble at the periphery of condensed cellular chromosomes during mitotic cell division. We demonstrate that the LANA oligomerization interface contributes to the cooperative DNA binding at the viral TR and the recruitment of ORC to the viral episome. Oligomerization mutants failed to auto-regulate LANA/ORF73 transcript, and this correlated with the loss of a chromosome conformation DNA-loop between the TR and LANA promoter. Viral genomes with LANA oligomerization mutants were subject to genome rearrangements including of loss of subgenomic DNA. Our data suggests that LANA oligomerization drives stable binding to the TR and formation of an epigenetically stable chromatin architecture resulting in higher-order LANA nuclear bodies important for viral genome integrity and long-term episome persistence. Author summary KSHV genomes persist in large nuclear bodies in latently infected cells. The KSHV encoded nuclear antigen LANA is required for the efficient replication and stable maintenance of viral genomes during latent infection. LANA is also known to form oligomeric structures, but it is not known how these structures contribute to LANA function in living cells. Here, we show that LANA oligomerization is required for cooperative binding to the KSHV terminal repeat (TR), and the recruitment of the Origin Recognition Complex (ORC) to viral TR. LANA oligomerization is required for a chromosome conformation DNA loop between TR and the LANA promoter implicated in LANA transcription autoregulation. LANA oligomerization is also required for formation of large nuclear bodies colocalize with DAXX, EZH2, ORC2, but not PML. LANA nuclear bodies distribute along the nuclear periphery, and their arrangement is transmitted faithfully to daughter cells during mitotic cell division. Finally, we show that KSHV genomes containing mutations in the LANA oligomerization interface fail to maintain the complete viral genome, suggesting they are defective in DNA replication or repair. These findings reveal new mechanisms of LANA episome maintenance through formation of higher-order chromosome-conformations.