A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA Proteins

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA ‘nuclear speckles’ and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence.

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

“…In addition, work found that the peripheral patch mutation K1109A and the central patch mutations K1138A or K1140A each reduced BRD2 and BRD4 binding and that combined K1109A/K1138A mutations completely abolished BRD2 and BRD4 binding. The central patch mutation K1141A also reduced BRD2 and BRD4 binding, although to a lesser extent (47). The peripheral patch mutation K1113A reduced BRD2, but not BRD4 binding (47).…”

“…1). Here, we also assessed the lateral (47). Although K1109A and K1138A did not reduce replication in isolation, a mutant with both these substitutions was deficient.…”

“…These findings suggest that DNA replication and episome maintenance deficiencies may underlie the observed reductions in viral latency after infection of mice with MHV68 containing LANA peripheral positive patch mutations. Alanine or glutamate substitution mutations of the corresponding residues in the MHV68 LANA positive patch resulted in substantial reduction in expansion of latent infection when the peripheral patch was mutated but not when the central patch was mutated (45,47). Because MHV68 LANA mediates MHV68 episome persistence using mechanisms similar to KSHV LANA (58), it is likely that these MHV68 LANA mutants are also deficient for DNA replication and episome persistence.…”

“…We and others recently solved the x-ray crystal structure of the murine gamma herpesvirus 68 (MHV68) or KSHV LANA C-terminal DNA binding domain (45)(46)(47)(48). The LANA DBD shares common structural features with the Epstein-Barr virus EBNA1 and papillomavirus E2 episome maintenance proteins; however, LANA contains a unique positive electrostatic charged patch on its dorsal surface, opposite the DNA binding interface, which is absent from the EBNA1 and E2 proteins.…”

“…The LANA DBD shares common structural features with the Epstein-Barr virus EBNA1 and papillomavirus E2 episome maintenance proteins; however, LANA contains a unique positive electrostatic charged patch on its dorsal surface, opposite the DNA binding interface, which is absent from the EBNA1 and E2 proteins. The positively charged LANA dorsal face has an important role in MHV68 establishment of latent infection in mice, as substitution mutations that altered the peripheral, but not central dorsal positive patch charge resulted in substantial deficiency in expansion of latent infection (45,47). The dorsal positive patch is also notable for being a site of interaction with BET (bromodomain and extra terminal domain) family proteins (21, 45-47, 49 -51), including BRD2, BRD3, and BRD4, which interact with acetylated histones.…”