Lytic viral genes in the pathogenesis of oncogenic gammaherpesviruses

PROJECT SUMMARY The human gammaherpesvirus (GHV) Kaposi sarcoma-associated herpesvirus (KSHV) establishes lifelong latency to cause several AIDS-associated malignancies including Kaposi sarcoma (KS) and primary effusion lymphoma (PEL). Because the virus is predominantly latent in these tumors, the prevailing model for KSHV transformation is that latency genes are the primary drivers of these viral cancers. However, published case reports in KS patients together with our preliminary data indicate that lytic gene expression may also be required for KSHV cancers. Whether lytic replication and reactivation influence tumor development and maintenance has not been directly tested. Our over-all objective is to test the hypothesis that lytic genes are drivers of GHV cancers. In this proposal, we will study how lytic gene expression is involved in the two distinct stages of GHV cancers (development and maintenance) using in vivo models. Specifically, we will inactivate the master regulators of the GHV lytic transcriptional program, ORF50 and ORF57, to prevent lytic gene expression. In Aim 1, we will test if lytic gene expression and reactivation is required for tumor initiation using conditional knockouts of ORF50 or ORF57 in the GHV murine gammaherpesvirus 68 (MHV68) in a newly developed B cell lymphomagenesis mouse model. MHV68 is a natural rodent virus that is genetically related to KSHV and provides a well-established, extensively studied, and tractable small-animal model for defining basic viral mechanisms of GHV pathogenesis. In Aim 2, we will test if lytic gene expression is required for the maintenance of KSHV lymphoma in a PEL xenograft mouse model. Using an inducible RNA-targeting CRISPR/CasRx system, we will target ORF50 or ORF57 RNA for knockdown in PEL cell lines after implantation in NOD scid gamma (NSG) mice. In addition, we will perform high-throughput targeting of the KSHV transcriptome using our newly designed CasRx tiling library in this well-established xenograft model to identify in vivo lytic drivers of PEL maintenance in an unbiased manner. Together, these complementary Aims will directly address the hypothesis that lytic gene expression is required for the development (Aim 1) and maintenance (Aim 2) of GHV cancers in vivo. We anticipate that our work will challenge the current latency-centric paradigm of GHV oncogenesis, and shed new light on roles for viral genes in pathogenesis.