Abstract
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 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 transcription, and this correlated with the loss of a chromosome conformational DNA-loop between the TR and LANA promoter. Viral genomes with LANA oligomerization mutants were subject to genome rearrangements including the 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.
Highlights
Kaposi’s Sarcoma Associated Herpesvirus (KSHV) is a human gammaherpesvirus responsible for Kaposi’s Sarcoma (KS), Pleural Effusion Lymphoma (PEL), and multicentric Castleman’s Disease
To further investigate the formation and transmission of these Latency Associated Nuclear Antigen (LANA) structures in living cells, we fused an amino-terminal RFP to the N-terminus of LANA in the infectious KSHV bacterial artificial chromosome (BAC) clone BAC16 (Fig 1C)
While DAXX commonly colocalizes with the anti-viral protein PML and PMLnuclear bodies (PML-NBs), we found that RFP-LANA did not colocalize with PML (Fig 1G)
Summary
Kaposi’s Sarcoma Associated Herpesvirus (KSHV) is a human gammaherpesvirus responsible for Kaposi’s Sarcoma (KS), Pleural Effusion Lymphoma (PEL), and multicentric Castleman’s Disease (mCD) (reviewed in [1, 2]). KSHV is a large double-stranded DNA virus that establishes life-long latent infection in B-lymphocytes and potentially other cell types, such as mesenchymal stem cells and endothelial cells. Most KSHV-associated cancers harbor latent viral genomes in the nucleus of tumor cells. The latent genomes form covalently closed circular genomes, termed episomes, that establish a local chromatin structure similar to that of cellular genomic DNA (reviewed in [3,4,5]). Viral gene expression is restricted to a small set of viral genes. Viral DNA replication occurs coordinately with host cell division cycle utilizing host cellular DNA replication machinery
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