Functional Characterization of ORF50 Protein in the Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

Kaposis』s sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus associated with at least three human malignancies: Kaposi』s sarcoma, primary effusion lymphoma, and multicentric Castleman』s disease. Like all herpesviruses, KSHV has both a latent and a lytic phase during its life cycle. The switch between latency and lytic-cycle gene expression of KSHV is initiated with a single transactivator encoded in open reading frame 50 (ORF50) of the viral genome. The physiological signals that initiate the activation of the ORF50 gene are not clear. Although ORF50 protein possesses a DNA binding domain, direct DNA binding by ORF50 protein to its responsive promoters is not the only mechanism for transactivation. I have identified a multifunctional regulatory region in ORF50 protein, which separately controls DNA binding and protein stability of ORF50. The regulatory region contains a basic tetrapeptide (KKRK) motif that plays a critical role in regulating these two phenomena. Mutation of the KKRK motif in this regulatory region dramatically enhances DNA binding of ORF50 protein. Mutations in the KKRK motif of the full-length ORF50 also result in abundant expression of an ORF50 variant, ORF50B. The ORF50B variant appears to be an unmodified form of ORF50 protein, while hyperphosphorylation of ORF50 protein are found in form A (ORF50A). Due to the critical role of ORF50 protein in the switch from latency to lytic replication, elucidating the regulation of the expression and the structural function of ORF50 may be important not only to advance our knowledge of the molecular action of ORF50 protein, but also to provide further insight into the pathogenesis of the KSHVassociated diseases. The five specific aims of this proposed research include: (i) determining the contribution of YY1 to the KSHV reactivation; (ii) investigating the inhibitory mechanism of ORF50 DNA binding; (iii) investigating the regulatory mechanism of the protein stability of ORF50; (iv) investigating the post-translational modification of ORF50 protein; (v) identifying the proteins that interact with ORF50 protein.

Project ID:PC9706-0248
External Project ID:NSC95-2320-B182-054-MY3