Separating late gene transcription from viral DNA replication in KSHV - Project Summary: Human oncogenic viruses are a major cause of cancer, with recent estimates that 15% of all cancers are associated with a viral infection. One such oncovirus is Kaposi’s sarcoma-associated herpesvirus (KSHV), which primarily affects untreated AIDS patients and other immunocompromised individuals. Like most viruses, KSHV relies both on host and unique viral processes for infection, each representing potential therapeutic vulnerabilities. One of these is an unusual link between the expression of an essential class of viral “late” genes and the replication of the viral dsDNA genome (vDNA). Though long recognized, the mechanism driving this link remains unknown. Here, I will use a multi-pronged functional genomics approach to identify and test new models for this process. The dependency of viral late gene transcription on vDNA replication depends on both the trans and cis viral components supporting vDNA replication. The trans component consists of the virally encoded vDNA replication factors. In Aim 1, I will use a method I have previously developed in my postdoc for high-throughput mutagenesis and phenotyping of viral mutants to identify mutations in these viral components that prevent late gene expression but still support vDNA replication. Preliminary work has already demonstrated that such mutations exist, and by expanding on this we can identify the role these viral proteins play in enabling late gene expression. In Aim 2, I will examine the viral origin of lytic replication, which is required in cis for both vDNA replication and late gene expression. While previous studies have been limited technically, new use of dCas9 will allow me to directly perturb the functional elements of this regulatory sequence and identify the distinct molecular events required to support vDNA replication and/or late gene expression. In my final aim, I will extend my work beyond the virus to identify and characterize the host components of this process. I propose to use genetic interactions to identify the host factors hijacked to support the viral life cycle. Specifically, by leveraging the mutants and functional elements discovered in my previous aims, I can further identify how the host supports or antagonizes this process both at the cis and trans levels. Each of these proposed aims will reveal new knowledge about vDNA replication and late gene expression, allow us to test models for how these processes are linked, and finally represent powerful functional genomic approaches that can be applied to many problems in KSHV and related dsDNA viruses.
