Recombinant baculovirus expressing anti‐HBsAg shRNA inhibits HBV cccDNA amplification in HepG2 cells

Abstract

A recombinant baculovirus (bac) expressing U6 promoter-encoded anti-HBV surface antigen (sAg) short hairpin RNAs (U6S) was generated. HepG2 cells were transduced with U6S 24hrs prior to bac-mediated HBV infection and the effect of U6S on HBV replicative intermediates (RI), extracellular DNA (EC), and covalently closed circular DNA (cccDNA) was analyzed. U6S significantly inhibited HBV RI, EC, and cccDNA levels through day 6 post-HBV infection compared to control treated cells. The effect of U6S on a persistent HBV infection was analyzed using a novel HBV infected HepG2 cell subculture system. HBV infected HepG2 cells subcultured 1:4 10 days post bac-mediated HBV infection, a time in which HBV transcription is primarily driven off cccDNA, continue to produce constant levels of HBV RI, EC, and cccDNA through 20 days post-subculture while retaining the ability to be reproducibly transduced at 100% efficiency by recombinant bac. HBV RI and EC were significantly inhibited through day 8 post-U6S treatment in HBV infected HepG2 cells transduced with U6S 24 hrs post-subculture; however, HBV cccDNA levels were unaffected compared to control treated cells. Bac expressed shRNA U6S is capable of reducing HBV RI, EC and cccDNA levels when administered prior to HBV infection; however, during a persistent infection, U6S can reduce only HBV RI and EC and not cccDNA levels. Research supported by RO1-CA 023931 to H.C.I. from N.I.H.