Hepatitis B virus enhancer 1 activates preS1 and preS2 promoters of integrated HBV DNA impairing HBsAg secretion

Background & Aims The expression of HBsAg from integrated HBV DNA limits the achievement of functional cure for chronic hepatitis B. Thus, characterizing the unique expression and secretion of HBsAg derived from integrated HBV DNA is of clinical significance. Methods A total of 563 treatment-naïve patients and 62 functionally cured patients were enrolled, HBsAg and HBcAg immunohistochemistry of their liver biopsy tissues was conducted followed by semi-quantitative analysis. Then, based on stratified analysis of HBeAg-positive and -negative patients, long-read RNA sequencing analysis, as well as an in vitro HBV integration model, we explored the HBsAg secretion characteristics of integrated HBV DNA and underlying mechanisms. Results In contrast to the significantly lower serum HBsAg levels, no significant decrease of intrahepatic HBsAg protein was observed in HBeAg-negative patients, as compared to HBeAg-positive patients. The results of long-read RNA sequencing of liver tissues from patients with chronic HBV infection and in vitro studies using integrated HBV DNA mimicking dslDNA plasmid revealed that, the lower HBsAg secretion efficiency seen in HBeAg-negative patients might be attributed to a relative increased proportion of preS1 mRNA derived from integrated HBV DNA than covalently closed circular DNA. The latter resulted in increased L-HBsAg proportion and impaired HBsAg secretion. Enhancer 1 (EnhI) in integrated HBV DNA could retarget preS1 (SP1) and preS2 (SP2) promoters to disrupte their transcriptional activity balance. Conclusions The secretion of HBsAg originated from integrated HBV DNA was impaired. Mechanistically, functional deficiency of core promoter leads to the promoter(s) retargeting of EnhI and thus uneven activation of SP1 over SP2 promoter, resulting in an increase in proportion of L-HBsAg. Impact and implications Integrated hepatitis B virus (HBV) DNA can serve as an important reservoir for HBV surface antigen (HBsAg) expression, and this limits the achievement of a functional cure. This study uncovered that secretion efficiency is lower for HBsAg derived from integrated HBV DNA than HBsAg derived from cccDNA, as determined by the unique sequence features of integrated HBV DNA. This study can broaden our understanding of the role of HBV integration and shed new light on antiviral strategies to facilitate a functional cure. We believe our results are of great general interest to a broad audience, including patients and patient organizations, the medical community, academia, the life science industry and the public.