Abstract: This R15 AREA grant application proposes a Course-based Undergraduate Research Experience (CURE) to investigate RTA-induced ubiquitination in the context of Kaposi's Sarcoma Herpesvirus (KSHV) latency and lytic replication to identify cellular targets for intervention while providing authentic research experience to undergraduate students. Kaposi’s sarcoma is the most common cancer in individuals diagnosed with HIV/AIDS. While antiretroviral therapy has reduced incidence, according to the WHO, 35,813 cases occurred in 2022, with 74% of cases occurring in Africa. Additionally, people living with HIV are living longer, resulting in increased incidence in KSHV-associated cancers due to the confluence of HIV status and age. KSHV is also associated with primary effusion lymphoma and multicentric Castleman's disease in addition to inflammatory syndromes. With no vaccine and limited treatment options available, studying novel virus-host interactions represents a strategy for identifying targets for intervention. Ubiquitin and ubiquitin-like proteins regulate diverse cellular processes, and KSHV interacts with the cellular ubiquitin-proteasome system, influencing transcriptional regulation, signaling, apoptosis, immune response, and cell cycle control to name a few. KSHV RTA, the major lytic switch, manipulates the cellular ubiquitinated proteome. Through a comparative proteomics analysis, we identified 40 RTA-induced ubiquitination targets. Over the past four years, undergraduate and master's students validated 25% of the targets and characterized a novel mechanism of immune modulation involving TAP2 degradation. Our long-term goal is to identify and characterize cellular processes targeted by KSHV, contributing to the understanding of KSHV biology and aiding in the identification of targets for intervention. We hypothesize that RTA-induced substrate ubiquitination is crucial for the latency-to-lytic replication transition and that the KSHV ubiquitome represents a tool for identifying small molecule inhibitors. Here we propose a rigorous examination of our hypothesis through two aims: (1) Evaluate the impact of RTA target modification/degradation on latency and lytic replication; (2) Assess small molecule modulation based on the ubiquitome dataset. The proposed studies aim to identify intervention targets and provide research experiences for ~120 undergraduate students over three years. Understanding KSHV infection biology advances cell biology knowledge and can identify potential therapeutic targets translatable to related viruses of biomedical importance like Epstein Barr virus. Engaging diverse groups of undergraduate students in research is expected to have lasting benefits, including inspiring the pursuit of graduate education and supporting individuals from underrepresented groups in science.

Project Narrative We propose to examine how KSHV manipulates the ubiquitin and ubiquitin-like modification systems to regulate the transition from latency to lytic replication. Kaposi’s sarcoma herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma and is associated with primary effusion lymphoma and multicentric Castleman’s disease as well as two inflammatory diseases: KSHV inflammatory cytokine syndrome and immune reconstitution syndrome-KS. The results of the proposed experiments have the potential to inform the development of novel chemotherapeutics.