How oral pathogenic bacteria stimulate Epstein-Barr virus (EBV) reactivation and commensal bacteria suppress EBV reactivation

Objective: Epstein-Barr virus (EBV) is associated with human oral and gastric cancers. Lytic reactivation from latency contributes to EBV spread. Reactivation is dependent on the initial expression of two immediate-early genes, BRLF1 and BZLF1. Increased oral EBV load correlates with periodontitis severity suggesting bacterial pathogens contribute to reactivation. This study sought to develop a stable cell line containing the luciferase gene under the transcriptional control of the promoters for the EBV immediately-early genes, BRLF1 and BZLF1, to study bacterial products involved in lytic reactivation. Methods: The latently-infected EBV+ human gastric carcinoma cell line, AGS-EBV, was treated with bacterial spent media (BSM) from anaerobic cultures of F. nucleatum (Fn) or P. gingivalis (Pg). The oral commensal bacteria, S. sanguinis (Ss), was used as a negative control. Lytic reactivation was determined by immunoblot to detect immediate-early viral lytic proteins. The promoters of the EBV immediate-early genes, were PCR-amplified and cloned into the reporter plasmid, pGL2-Basic to produce pEBVRpLuc and pEBVZpluc, respectively. EBV promoter/luciferase cassettes were cloned into the lentiviral vector, pLKO.1. A promoterless luciferase was constructed as a negative control.Results: Treatment of latently-infected EBV cells treated with Fn or Pg BSM resulted in expression of the immediate-early genes as determined by western blot analysis. No increase in viral proteins was seen in Ss-treated cells. PCR products were obtained using primers specific for the promoters of EBV BRLF1 and BZLF1. Plasmids were generated and restriction digest analysis confirmed the presence of each viral promoter/luciferase cassette in the lentiviral vector.Conclusion: BSM-induced EBV reactivation suggested that transcription occurred at the viral immediate-early lytic genes. Stable cell lines containing viral promoter/luciferase cassettes will be useful in elucidating mechanisms of viral reactivation by oral pathogenic bacteria. This is an important connection to show that improving oral hygiene can limit EBV reactivation and improve systemic health.Bachelor of Scienc