Molecular basis and regulation of the DNA binding activity of KSHV LANA

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DESCRIPTION (provided by applicant): Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of several human diseases, including Kaposi's sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman's disease. KSHV infections exist in two states: a non-productive latent state and a proliferative lytic state. During latency, viral gene expression focuses on those genes that encode products required for preventing a host immune response and maintaining a state of latency. To ensure that viral infection persists after cell division, the viral genome is circularized as an episome and tethered to the host chromosome by the protein latency-associated nuclear antigen (LANA). LANA is a multi-functional protein that plays a pivotal role in maintaining passage of the episome as well as repressing lytic reactivation and host immune response. The N-terminus of LANA interacts with a host nucleosome while the C-terminus binds sequence specifically to a portion of the viral episome. The structure of an N-terminal peptide bound to the nucleosome has previously been solved; however, no structural information is available on the C-terminus. To understand how LANA mediates this sequence specific interaction we propose to determine the three-dimensional crystal structure of the LANA DNA binding domain both alone and bound to its DNA binding site. Utilizing this structural information we will then create mutations in regions that we identify to e important for sequence specificity and DNA binding function. Also, we have identified novel serine phosphorylation sites within the C-terminus that have been suggested to play a role in interactions with cellular proteins and may have an effect on DNA binding. We propose to make mutations of these serines to either abrogate or mimic a constitutive state of phosphorylation. We will utilize previously developed assays to test these mutations for DNA binding, episome maintenance, and genome replication. The information obtained from these studies will provide a foundation for the development of small-molecule inhibitors of LANA as well as an understanding of the role of post-translational modifications in DNA binding and the viral life cycle. PUBLIC HEALTH RELEVANCE: Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is a human viral pathogen that is the causative agent of several lymphomas. The KSHV genome is persistent in infected cells and is directly attached to the host chromosome. The goal of this project is to determine the three-dimensional structure of the viral genome binding portion of the protein LANA, which is responsible for attaching the viral genome to the host chromosome and to understand how modifications to this protein affect its DNA binding function.