Hypoxia induced Epstein-Barr virus lytic infection. What can be learned from a drug screen?

摘要

Epstein-Barr virus (EBV) is a member of human gamma-herpesvirus subfamily. It is associated with many malignancies. Lytic infection may play a role in the pathogenesis of these tumors, and it's also proposed as a novel therapy since lytic induction specifically kills the tumor cells. We have developed two assays to screen for lytic induction drugs of EBV using a luciferase assay and a recombinant GFP-virus system, and screened a clinical compound based library to find EBV lytic induction reagents among currently used drugs. The screening identified many FDA approved agents with potent lytic induction activity. Some of the hits were previously recognized, others were not known before. The proteasome inhibitor Bortezomib turns out to be the most prominent hit in both assays, and it can induce EBV lytic infection in many EBV+ cell lines. By analyzing the possible mechanisms of its induction of EBV reactivation, we suggest a new regulatory pathway of EBV promoter induction by hypoxia inducible factor (HIF-1alpha). Further study found a hypoxia responsive element (HRE) in the zta promoter -81-76 region, and later experiments proved that hypoxia can reactivate EBV in many EBV positive B cell lines.; Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA-1) is required for the persistence of extrachromosomal KSHV DNA. We characterized the effects of LANA suppression by siRNA in KSHV(+) cell lines. Neither the absolute KSHV viral load nor the KSHV/EBV ratio was affected by LANA suppression, the cell cycle and replication were not affected either. EBV nuclear antigen1 (EBNA1) is expressed in all EBV associated tumors. It is required to maintain EBV episomes in cells and has been suggested to have an anti-apoptotic effect. We showed that siRNA mediated inhibition of EBNA1 expression induced cell apoptosis in a gastric cancer cell line SNU719 as well as in Namalwa cells. This result indicated that EBNA1 plays a direct role in EBV associated tumor growth and progression besides episomal maintenance.