Mutation of KSHV vFLIP through CRISPR/Cas9 system in BCBL1 cells

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and is associated with other lymphatic diseases. During latency the virus expresses few viral genes such as vFLIP, which maintains this state by activating the NF?B pathway. Research in our lab has shown that the interaction of vFLIP and the Itch/A20 ubiquitin editing complex, required for induced lytic replication, may occur through a small ubiquitin-related modifier (SUMO) interaction motif (SIM) and mutation of vFLIP SIM disrupts this interaction. To explore the role of SUMOylation in latency and lytic reactivation we designed vFLIP targeting oligonucleotides and delivered a CRISPR/Cas 9 plasmid construct to cells latently infected with KSHV along with a template containing mutations for homology directed repair. We planned to examine effects of vFLIP SIM mutation on the switch from latency to lytic reactivation, but were unable to generate mutations.