PLoS Pathog. 13, e1006517 (2017)

Therapies against Epstein-Barr Virus (EBV) have focused on activating the virus from its latent state to enter a lytic cycle, where it becomes susceptible to treatment by antivirals. The small molecule C60 can induce the expression of markers of the lytic cycle, ZTA and EA-D, in EBV-infected cells. To determine its mechanism of action, Tikhmyanova et al. used an affinity selection approach in parallel with an shRNA screen to identify genes that restrict spontaneous lytic reactivation. Both approaches converged on CAND1, a component of the ubiquitin–proteasome system that acts as an exchange factor for complexes containing Cullin-type E3 ligases. Further mechanistic studies of C60 included mRNA expression profiling, which indicated regulation of a DNA damage checkpoint and of viral infection pathways. Immunoprecipitation experiments revealed that C60 destabilized the interaction between CAND1 and Cullin 1, increased the abundance of ubiquitylated proteins, and selectively stabilized ZTA and the tumor suppressor p53. C60 treatment showed distinct effects compared to histone deacetylase (HDAC) or proteasome inhibition and may provide a novel pathway for reactivation of EBV from latency. These results suggest that C60 targets the CAND1–Cullin pathway, ultimately preventing the degradation of such proteasome substrates as p53 and EBV lytic factor ZTA.