Phase separation and DAXX redistribution contribute to LANA nuclear body and KSHV genome dynamics during latency and reactivation

Abstract

Liquid-liquid phase separation (LLPS) can drive formation of diverse and essential macromolecular structures, including those specified by viruses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) genomes associate with the viral encoded Latency-Associated Nuclear Antigen (LANA) to form stable nuclear bodies (NBs) during latent infection. Here, we show that LANA-NB formation and KSHV genome conformation involves LLPS. Using LLPS disrupting solvents, we show that LANA-NBs are partially disrupted, while DAXX and PML foci are highly resistant. LLPS disruption altered the LANA-dependent KSHV chromosome conformation but did not stimulate lytic reactivation. We found that LANA-NBs undergo major morphological transformation during KSHV lytic reactivation to form LANA-associated replication compartments encompassing KSHV DNA. DAXX colocalizes with the LANA-NBs during latency but is evicted from the LANA-associated lytic replication compartments. These findings indicate the LANA-NBs are dynamic super-molecular nuclear structures that partly depend on LLPS and undergo morphological transitions corresponding the different modes of viral replication.

A Panel of Kaposi’s Sarcoma-Associated Herpesvirus Mutants in the Polycistronic Kaposin Locus for Precise Analysis of Individual Protein Products

“…LANA forms homo-oligomers and binds the terminal repeat (TR) regions of the KSHV genome while simultaneously binding histones H2A and H2B of host chromatin, thereby tethering the viral DNA to host chromosomes ( 8 , 10 , 11 , 103 ). This binding leads to the formation of structures called LANA NBs that colocalize with viral genomes ( 10 , 104 , 105 ). The number of LANA-positive puncta in an infected cell was reported to correlate with the number of viral episomes ( 13 ).…”

“…However, we and others have observed heterogenous LANA NB size within and between latent infected cells, but the significance of this size variability is unclear. Because LANA can bind to nucleosomes in cellular chromosomes, as well as those bound to viral episomes ( 15 ), LANA NBs are classified as supramolecular structures that cluster multiple viral episomes together ( 12 , 105 ). Genome clustering was proposed to be an effective evolutionary strategy that allows KSHV to rapidly increase viral genome copy number per cell at the expense of total number of cells infected ( 15 ).…”

A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

“…LANA forms homo-oligomers and binds the terminal repeat (TR) regions of the KSHV genome while simultaneously binding histones H2A and H2B of host chromatin, thereby tethering the viral DNA to host chromosomes (8, 10, 11, 96). This binding leads to the formation of structures called LANA NBs that colocalize with viral genomes (10, 97, 98). The number of LANA-positive puncta in an infected cell correlates with the number of viral episomes (13).…”

“…We and others have observed heterogenous LANA NB size within and between latent infected cells; however, the significance of this size variability is less clear. Because LANA can bind to nucleosomes in cellular chromosomes as well as those bound to viral episomes (15), LANA NBs are classified as supramolecular structures that cluster multiple viral episomes together (12, 98). Genome clustering was proposed to be an effective evolutionary strategy that allows KSHV to rapidly increase viral genome copy number per cell at the expense of total number of cells infected (15).…”

Deconstructing virus condensation

“…The Epstein–Barr virus proteins EBNA2 and EBNALP, with roles in viral and cellular gene transcription, have been shown to mediate the formation of liquid-like condensates at superenhancer sites of cellular genes through their IDRs [ 105 ]. Another interesting example is the viral latency-associated nuclear antigen (LANA) from Kaposi’s sarcoma-associated herpesvirus, which associates with the viral genome to form dynamic LANA-nuclear bodies implicated in episome maintenance, through a mechanism partially mediated by LLPS [ 106 ].…”