Noncanonical circRNA biogenesis driven by alpha and gamma herpesviruses

Herpesviruses require the host transcriptional machinery, inducing significant changes in gene expression to prioritize viral transcripts. We examined alpha- and gamma-herpesvirus alterations to a type of alternative splicing, namely circular RNA (circRNA) synthesis. We developed "Circrnas in Host And viRuses anaLysis pIpEline" (CHARLIE) to facilitate viral profiling. This method identified thousands of back-splicing variants, including circRNA common to lytic and latent phases of infection. Ours is the first report of Herpes Simplex Virus-1 circRNAs, including species derived from ICP0 and the latency-associated transcript. We characterized back-splicing cis- and trans-elements, and found viral circRNAs resistant to spliceosome perturbation and lacking canonical splice donor-acceptors. Subsequent loss-of-function studies of host RNA ligases (RTCB, RLIG1) revealed instances of decreased viral back splicing. Using eCLIP and 4sU-Sequencing, we determined that the KSHV RNA-binding protein, ORF57, enhanced synthesis for a subset of viral and host circRNAs. Our work explores unique splicing mechanisms driven by lytic infection, and identifies a class of transcripts with the potential to function in replication, persistence, or tumorigenesis.