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

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 DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 üüÅ 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 üüÅÅ 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. Significance KSHV is the etiological agent of Kaposi sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. During latency, this dsDNA tumor virus expresses only a small subset of its more than 90 ORFs. Among these is ORF73/latency-associated nuclear antigen (LANA), which acts as the origin binding protein and chromatin anchor of the extrachromosomal viral genome. This work provides detailed structural insights into the DNA-binding characteristics of LANA. We also report a previously unrecognized, third LANA binding site within the minimal replicator of Kaposi sarcoma herpesvirus. In addition to its mechanistic implications for latent viral persistence, the X-ray crystal structure of LANA bound to LANA binding site 1 DNA may assist in the tailored development of therapeutic LANA inh