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

Kaposi's sarcoma herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and is associated with primary effusion lymphoma (PEL), multicentric Castleman's disease, 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, Replication and Transcription Activator (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 stable isotope labeling using amino acids in cell culture (SILAC) labeling, ubiquitin remnant enrichment, and mass spectrometry, we have analyzed the RTA-dependent ubiquitin-modified proteome. We identified RTA-dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a small ubiquitin-like modifier (SUMO) targeting ubiquitin ligase, may function to alleviate a SUMO-dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain-dependent mechanism as well as indirectly through cellular ubiquitin ligase RAUL. Our ubiquitome analysis revealed an RTA-dependent mechanism of immune evasion. We provide evidence of inhibition of transporter associated with antigen processing (TAP)-dependent peptide transport, resulting in decreased human leukocyte antigen (HLA) 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. IMPORTANCE Kaposi's sarcoma herpesvirus, an AIDS-associated pathogen, is associated with multiple cancers and inflammatory syndromes. This virus has a latent and lytic lifecycle, each associated with pathogenesis and oncogenesis. Here, we identify proteins that display differential abundance in different phases of the lifecycle. We provide evidence supporting a new model of viral immune evasion. These findings increase our understanding of how the virus manipulates the host cell and provides new targets for intervention.