Investigating nonsense-mediated decay (NMD) as a restriction factor for EBV-reactivation

Approximately 0.01% of adults infected with Epstein-Barr virus (EBV) will develop an EBV-associated cancer which equates to 200,000 annual cases globally. EBV malignancies are dependent on EBV latency gene products for continued growth and survival, making the disruption of latency a promising therapeutic approach. Despite this vulnerability, EBV-targeting strategies have yet to be successfully translated into therapy for EBV-associated cancers. “Lytic induction therapy” combines small molecules capable of reactivating EBV from latency with antiviral drugs to increase toxicity to tumor cells and prevent EBV particle production. Although this therapeutic strategy has been well tolerated in clinical trials, current induction agents display low efficacy. The full potential of this approach will require the development of second-generation induction agents, necessitating a greater understanding of EBV biology and host-pathogen interactions. Recent studies have identified nonsense-mediated decay (NMD), a host regulatory pathway, is a key restriction factor for EBV reactivation. The EBV transcript encoding Rta, one of the two viral transcription factors responsible for entry into the lytic cycle, contains multiple splice junctions downstream of its stop codon. Consequently, the Rta transcript is rapidly degraded by NMD. This previously unrecognized post-transcriptional regulation revealed that strategies inducing the lytic cycle cannot focus solely on activating Rta and Zta transcription; they must include approaches to stabilize the Rta transcript, likely by NMD inhibition. To investigate this phenomenon, we developed an NMD reporter based on a previously described dual-fluorophore system. By combining this reporter with an EBV fluorescent reporter virus that indicates Zta (red) and Rta (green) promoter activity, we can assess the effects of NMD on Rta expression and lytic activation (measured as the change in blue and far-red fluorescence). This system enables us to determine whether EBV can only reactivate under NMD-low conditions and evaluate how various induction agents influence NMD activity. We can also identify EBV gene products that may act as NMD antagonists, which would establish a positive feedback loop for promoting lytic cycle entry. The insights gained from these studies will provide a deeper understanding of the mechanisms governing viral reactivation and inform the development of next generation lytic induction therapies.