Analysis of the ubiquitin-modified proteome identifies novel host factors in Kaposi’s sarcoma herpesvirus lytic reactivation

Abstract

Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and is associated with primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, RTA, interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through SILAC labeling, ubiquitin remnant enrichment and mass spectrometry, we have analyzed the RTA dependent ubiquitome. We identified RTA dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and infected cells following lytic reactivation. We observed enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a SUMO targeting ubiquitin ligase, may function to alleviate a SUMO dependent block to lytic reactivation. We report an RTA dependent mechanism of immune evasion, targeting HLA peptide transport resulting in decreased complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA dependent mechanism of immune evasion.