Human Herpesvirus 8 (KSHV or HHV-8) is a gamma-2 herpesvirus that is present in all cases of Kaposi's sarcoma, primary effusion lymphoma, and some cases of multicentric Castleman's disease, with increased incidence in HIV positive individuals. KSHV- associated neoplasia occurs, in part, due to latently expressed genes in infected cells. Many of the proteins encoded by latent genes are viral homologs of cellular angiogenic, prosurvival and anti-apoptotic factors. Viral FLICE inhibitory protein (vFLIP) is a latently expressed gene that is homologous to cellular inhibitors of apoptosis. vFLIP has been shown to inhibit death receptor induced apoptosis. In addition, vFLIP has been shown to activate NF?B through activation of the IKK complex. NF?B activation is essential for maintaining latency, with inhibitors of this pathway resulting in enhanced lytic reactivation and apoptosis. We recently reported that the activator of lytic replication, RTA, induces proteasome dependent degradation of vFLIP thereby inhibiting NF?B early in lytic reactivation. Our preliminary data suggests that the Itch/A20 ubiquitin editing complex is required for RTA induced degradation of vFLIP. This ubiquitin editing complex is a well characterized negative regulator of NF?B signaling. We and others have reported that vFLIP induces A20 expression. A20 plays an important role in preventing an inflammatory phenotype, however to maintain latency, the virus must somehow control the activity of A20, as inhibition of NF?B enhances lytic reactivation. We have observed vFLIP interaction with the Itch/A20 ubiquitin editing complex in the absence of RTA. In preliminary studies, we have identified a SUMO interaction motif (SIM) in vFLIP and SUMO 2/3 modification of A20. The SIM in vFLIP is necessary for both NF?B activation and RTA induced degradation of vFLIP. We propose that vFLIP maintains latency, in part, by interfering with the function of the Itch/A20 complex, allowing for persistent NF?B signaling. Upon lytic reactivation, RTA interacts with Itch and A20, targeting vFLIP for degradation, resulting in NF?B downregulation. The interactions between vFLIP and RTA appear to be SUMO dependent. We propose that vFLIP interacts with the Itch/A20 complex via a SIM and that RTA interacts with a SUMO modified form of this complex as it has been shown to be a SUMO targeting ubiquitin ligase (See model below). The findings of this project have broad implications in KSHV virology, viral oncology and immunology. Understanding the processes that govern the latent to lytic transition will aid in the identification of novel target for drug development for treatment of KSHV associated cancers. Characterization of the interaction of vFLIP and RTA with the Itch/A20 ubiquitin editing complex will contribute to our understanding of how this complex is regulated which will increase our understanding of how the NF?B pathway is regulated. We are the first to our knowledge to identify SUMO2/3 modified A20. SUMO modification of the Itch/A20 ubiquitin editing complex, implies a novel mechanism of complex regulation. Our findings have the potential to impact broad areas of immunology, from cancer biology to autoimmunity and inflammatory disease research.
Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi's Sarcoma (KS) and is associated with primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV associated neoplasia is associated with genes expressed during the latent phase of the viral lifecycle that allow the infected cell to survive despite harboring a viral genome. We have recently reported that early in lytic replication, the major lytic switch, RTA, targets the latent protein, vFLIP, for proteasomal degradation. Here we propose a mechanism by which vFLIP and RTA coopt the cellular protein degradation machinery in both latency and lytic replication; in latency to promote NF?B activation and in lyti to inhibit NF?B. Since vFLIP is a potent activator of NF?B signaling, our results have the potential to impact NF?B biology in addition to increasing our understanding of the latent to lyti transition in KSHV.
| Chmura, Jennifer C; Herold, Kevin; Ruffin, Ayana et al. (2017) The Itch ubiquitin ligase is required for KSHV RTA induced vFLIP degradation. Virology 501:119-126 |
| Jankowsky, Eckhard; Harris, Michael E (2017) Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing. Methods 118-119:111-118 |
