Viral modulators of KSHV lytic replication

Kaposi's Sarcoma and the B-cell derived primary effusion lymphoma and multicentric Castleman's Disease are all outcomes of infection with Kaposi's Sarcoma-- associated Herpesvirus (KSHV). A predominantly latent virus, KSHV also requires a subpopulation of cells to undergo lytic reactivation in order to generate an environment conducive to neoplastic growth. The replication and transcription activator (RTA, Orf50) is necessary and sufficient to initiate the lytic cascade. We have studied two modulators of lytic replication encoded by KSHV Orf49 and Orf64 that support RTA during lytic reactivation.;KSHV Orf49 lies adjacent to and in the opposite strand as Orf50. Upon ectopic expression, Orf49 was found to encode a 30 kDa protein that localized to the cytoplasm and nucleus. The Orf49 transcript displayed early lytic kinetics and was shown to enhance RTA-mediated transactivation of lytic promoters including vGPCR, K8 and Orf57. Orf49 induced c-Jun, JNK and p38 kinase activation. Pharmacologic inhibition demonstrated that activation of the c-Jun and p38 signaling pathways are required for the completion of the lytic cycle, underscoring the importance of KSHV Orf49 during lytic replication.;KSHV Orf64 encodes a large, 2636 amino acid tegument protein. We have found that Orf64 encodes deubiquitinase (DUB) activity in its amino-terminal 200 amino acids in vitro and in vivo. Whereas all other herpesviral DUBs cleave Lysine 48 (K48)-linked ubiquitin, KSHV Orf64 was capable of cleaving both K48 and K63-linked ubiquitin chains. Cleavage of K63-linked ubiquitin is associated with functional regulation whereas K48-ubiquitination leads to proteosomal degradation. Mutation of a cysteine residue at amino acid position 29 in the catalytic core of the DUB ablated DUB activity. Orf64 also synergized with RTA to augment transcription from RTA-responsive promoters. These data suggest that the deubiquitination function of Orf64 may play an important role in viral replication.;The role of NFkappaB pathway activation on KSHV pathogenesis in unclear. Various members of this pathway are regulated by ubiquitination and represent potential targets of Orf64. Reactivating KSHV-293 cells selectively activated the canonical NFkappaB pathway. We discovered that Orf64 induced degradation of IkappaBalpha and IKKgamma suggesting that it can induce the canonical pathway, highlighting the multiple roles and functions of Orf64.