CREB/PKA sensitive signalling pathways activate and maintain expression levels of the hepatitis B virus pre-S2/S promoter

Abstract

Background and aims: CREB (cAMP response element binding protein) transcription factors are key regulators of homeostatic functions in the liver, and CRE binding is increased in hepatic inflammation. During chronic hepatitis B virus (HBV) infection, mutations or deletions in the pre-S region are frequently observed. These mutations can affect the pre-S2/S promoter controlling HBV envelope protein expression (hepatitis B surface antigen (HBsAg)) and have been associated with worsened clinical outcome. We aimed to test if CREB activation impacts on HBsAg expression. Methods: The effect of the CREB inducer protein kinase A (PKA) was tested by coexpression with HBV wild-type vector in vitro. Luciferase reporter gene constructs were cloned to identify novel regulatory regions for the HBV pre-S2/S promoter. Electrophoretic mobility shift assay (EMSA) gelshift and supershift experiments were conducted to confirm DNA transcription factor binding. Results: Coexpression of HBV and PKA resulted in HBV-S mRNA induction and enhanced small envelope protein expression. We identified a CREB binding motif in the transcribed part of the pre-S2 region, contributing to basal S promoter activity via binding of activating transcription factor 2 (ATF2). A second CREB motif closely linked to the S-ATG showed a similar binding pattern involving ATF2 and CREB1, without appearing essential for basal promoter activity. Moreover, a sequence in the pre-S2 region is responsible for further transcriptional induction via CREB activators such as PKA and forskolin. EMSA experiments indicate that CREB1 and ATF4 are involved in complex formation conferring PKA dependent promoter activation. Conclusions: Our data suggest a novel mechanism by which HBV may utilise CREB/PKA signal transduction pathways of hepatocytes to enhance its HBsAg expression during homeostasis and hepatic inflammation.