Wednesday, January 15, 2025
1/15/2025
Project Summary The Kaposi's sarcoma-associated herpesvirus (KSHV) causes the AIDS-defining B cell malignancy, primary effusion lymphoma (PEL). KSHV is found in all tumor cells and is tightly latent in >95% of infected cells. Latency is an intricately organized program that highly restricts gene expression to a handful of genes (i.e. latency genes). PEL cell lines require these latency genes to be constitutively expressed while the lytic genes are silenced for survival. Thus, both expression of the latency genes and maintenance of the latency program are critical for PEL cells. However, the contributions of most viral genes to the survival and proliferation of PEL-derived cell lines are unknown. Our long-term goal is to accelerate our understanding of the biology of this AIDS-defining lymphoma by comprehensively analyzing viral factors required for PEL tumor cell survival. We, therefore, performed an RNA-targeting CRISPR/CasRx tiled screen to identify KSHV transcripts required for the survival of PEL cells. Our results reveal that the left-hand origin of lytic replication (OriLytL) encodes a previously unannotated RNA critical for the survival of PEL cell lines. The OriLytL serves as one of two initiation sites crucial for viral DNA replication during reactivation from latency. In this lytic phase, the OriLytL produces two long noncoding RNAs required for lytic viral DNA replication. However, our data suggest that the latency-specific OriLytL (OriLytLlat) RNA is distinct and functions differently from the lytic OriLytL RNAs. Our central hypothesis is that the OriLytLlat RNA acts to maintain the latency program. Since latency is crucial for the survival of tumor cells, the OriLytLlat RNA is a critical oncogenic driver of KSHV-associated cancers. The over-all objective is to characterize this latency-specific OriLytLlat RNA and determine its role in latency maintenance in PEL. In Aim 1, we will characterize the latent viral transcriptome, including the full-length OriLytLlat RNA using long-read RNA sequencing. In Aim 2, we will define the role of the OriLytLlat RNA in the recruitment of chromatin binding factors and resulting epigenetic changes in the viral genome that contribute to establish and maintain the latency program. Together, this work will provide a new model on how viral latency is influenced by a latent RNA from the lytic origin of replication. Importantly, our studies will also shed light on the biology of other KSHV-associated malignancies, including Kaposi’s sarcoma.
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