EBV-positive tumors are dependent on EBV’s oncogenic latency proteins therefore, targeting EBV is a promising therapeutic strategy. In the clinic, however, EBV-positive patients are treated the same as patients with EBV-negative disease, and while these therapies can certainly manage disease, there is significant associated morbidity. There is therefore a need for therapeutic strategies that efficiently and specifically target and treat EBV-associated malignancies.
Lytic induction therapy is a therapeutic strategy that specifically targets EBV-infected tumor cells and uses properties of EBV infection against itself. EBV persists latently in hosts meaning only a small fraction of viral genes are expressed, many of which are immunoevasive. The virus can escape latency and enter the lytic cascade whereby the full repertoire of viral genes is expressed, and viral particles are produced. The lytic phase is toxic for the cell and during this stage, the virus expresses antigens that are more easily detectable by the immune system. Lytic induction therapy utilizes small molecules to reactivate EBV-infected cells from latency into the lytic phase. This process diminishes expression of EBV’s oncogenic latency proteins, increases immunogenicity of the tumor, and ultimately leads to tumor cell death. The limitation with this strategy, however, is that reactivation is a poorly efficient process and the small molecule lytic induction agents currently available are also poorly efficient. The full potential of this therapeutic approach, however, requires the development of more effective second-generation lytic induction agents that induce a higher percentage of EBV-positive tumor cells. Further understanding the mechanisms of EBV-s latent-lytic switch, and the cellular mechanisms that govern it, are crucial for this initiative and are the focus of this project.
EBV entry into the lytic phase is controlled by two viral transcription factors, Rta and Zta, but much is still unclear as to what induces their primary induction. We seek to utilize novel reporter viruses to discover agents that successfully induce Zta and Rta to study how they might lend to the development of lytic induction therapies. Using these methods, we also hope to elucidate the degree to which Rta and Zta lead to reactivation in EBV-positive B-cell malignancies.
The initial switch into the lytic cycle is controlled by two viral transcriptional activators, Rta and Zta. Rta and Zta are both crucial for lytic reactivation and different cell types vary in their dependance on one activator verses the other. We hypothesize that to induce the greatest proportion of cells into the lytic cycle across diverse cell types requires an induction regimen capable of strongly activating both Rta and Zta. To investigate which viral transcriptional activator is induced by various small molecules, we have constructed a novel reporter virus, the Rta/Zta Induction Reporter virus (RZIR-EBV). This reporter virus overcomes many limitations facing previous studies and will be used as a tool to assess potential therapeutic agents.
Many known lytic induction agents exist, but several are not therapeutically relevant and can’t be used to treat patients. We are using the RZIR-EBV reporter virus, and another reporter virus we created, DFLR-EBV, to screen FDA-approved drugs and identify compounds capable of reactivating EBV and classifying if they induce via Rta or Zta.
~ Emilie Greene
Rta, as one of the two essential viral transcriptional activators, is a crucial player in lytic reactivation. Recently, it has been shown that Rta transcripts are degraded by the host regulatory process nonsense-mediated decay (NMD). NMD identifies and degrades mRNA transcripts with premature stop codons and long 3’ UTRs, of which Rta has both. Our goal is to identify how NMD impacts EBV lytic reactivation and to make an important distinction: does EBV antagonize NMD or does EBV only reactivate in a setting where NMD is downregulated by non-viral mechanisms?
~ Emilie Greene
