The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA

Abstract

Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNAbinding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains ("LANA speckles"), a hallmark of KSHV latency.X-ray crystallography | gammaherpesvirinae | viral latency | DNA-binding protein | KSHV LANA K aposi sarcoma herpesvirus (KSHV) is the only known γ 2 -herpesvirus of concern to human health. Apart from its involvement in two lymphoproliferative disorders, KSHV plays a vital role in the development of Kaposi sarcoma, the most common form of cancer in patients with AIDS (1, 2). After a primary infection event, the virus establishes lifelong latent persistence in the nuclei of its host cells. A molecular key player in the establishment, maintenance, and regulation of KSHV latency is the latency-associated nuclear antigen (LANA).KSHV LANA contains 1,162 amino acids in the prototype strain and exerts functions in host cell survival, transcriptional control, latent viral replication, and stable episome segregation during mitosis (3,4). Its N-terminal domain is separated from its C-terminal domain by a large internal repeat region (5, 6). Although the N-terminal domain and the internal repeat region are predicted to be only poorly structured (7), the C-terminal domain comprises a stable 3D structure with a strong hydrophobic core (8, 9). LANA's C-terminal domain binds to the LANA binding sites (LBS) within the viral terminal repeats (TRs) in a sequence-specific manner (10, 11). Therefore, this domain is referred to as the DNA-binding domain (DBD). In contrast, the N terminus of LANA binds to nucleosome...

Kaposi sarcoma–associated herpesvirus: immunobiology, oncogenesis, and therapy

“…Whereas the C-terminal end of LANA binds KSHV DNA directly, the N-terminus (and perhaps regions in the C-terminus as well) contact cellular chromosome-associated proteins, such as histones H2A and H2B and others (104). The crystal structures of the KSHV LANA and MHV-68 LANA DNA-binding domains were solved (102,103,116,117). This work identifies the DNA contact residues and reveals a folding pattern analogous to EBV EBNA1 and HPV E2.…”

“…Update on LANA function and structure LANA binds the viral terminal repeats, specifically two sequenceconserved, high-affinity binding sites (LBS1 and LBS2) and a more divergent third, low-affinity site (114)(115)(116). LANA can be thought of as a dumbbell-like structure in which a stalk of internal repeats separates the two globular terminal regions.…”

Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS

“…LANA, a multifunctional protein, is expressed in all KSHV-infected cells. LANA consists of an amino terminal domain, an extended internal repeat region, and a carboxy terminal domain involved in the binding to viral episomal DNA (4,5,25,(44)(45)(46). The internal repeat region is required for the maintenance of viral episomes (47)(48)(49).…”

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

“…The dorsal positive patch was recently shown to mediate interaction of C-terminal LANA with DNA, independent of DNA sequence (48). Therefore, it is possible that the LANA DBD can bind its specific TR recognition sequence along its ventral surface and simultaneously bind additional DNA regardless of sequence along its dorsal surface.…”

“…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.…”