Identification of Slug and SOX7 as transcriptional repressors binding to the hepatitis B virus core promoter

Hui Ling Ko; Tze Hau Lam; Huijin Ng; Jiaying Toh; Liang Wei Wang; Ee Chee Ren

doi:10.1016/j.jhep.2017.08.033

Identification of Slug and SOX7 as transcriptional repressors binding to the hepatitis B virus core promoter

J Hepatol. 2017 Sep 5:S0168-8278(17)32276-6. doi: 10.1016/j.jhep.2017.08.033. Online ahead of print.

Authors

Hui Ling Ko¹, Tze Hau Lam¹, Huijin Ng², Jiaying Toh³, Liang Wei Wang⁴, Ee Chee Ren⁵

Affiliations

¹ Singapore Immunology Network, 8A Biomedical Grove, #03-06 Immunos, Singapore 138648, Singapore.
² Singapore Immunology Network, 8A Biomedical Grove, #03-06 Immunos, Singapore 138648, Singapore; Oxford Center for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Headington OX3 7LE, United Kingdom.
³ Singapore Immunology Network, 8A Biomedical Grove, #03-06 Immunos, Singapore 138648, Singapore; Department of Microbiology & Immunology, Stanford University, 300, Palo Alto, CA 94304, United States.
⁴ Singapore Immunology Network, 8A Biomedical Grove, #03-06 Immunos, Singapore 138648, Singapore; Division of Medical Sciences, Virology Program, Harvard Medical School, 260 Longwood Ave, Boston, MA 02115, United States.
⁵ Singapore Immunology Network, 8A Biomedical Grove, #03-06 Immunos, Singapore 138648, Singapore; Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 119260, Singapore. Electronic address: ren_ee_chee@immunol.a-star.edu.sg.

PMID: 28887167
DOI: 10.1016/j.jhep.2017.08.033

Abstract

Background & aims: The Hepatitis B Virus (HBV) may gain entry into non-liver cells but does not actively replicate in them. We investigated the possibility that these cells possess mechanisms that block HBV core promoter (HBVCP) transcription, specifically absent in liver cells, which together with other liver-specific mechanisms, such as sodium-taurocholate cotransporting polypeptide-mediated entry, enable liver cells to effectively produce HBV.

Methods: Liver and non-liver cell lines were screened for their capacity to activate the HBVCP and synthesize pre-genomic RNA (pgRNA). Transcription regulators differentially expressed between cells with active or inactive HBVCP were determined by human transcriptome array. Slug (SNAI2) and SRY-related HMG box 7 (SOX7) transcriptional repressors were identified and shown to bind specifically to the HBVCP by electrophoretic mobility shift assay. The resultant inhibitory effect on HBVCP transcription was validated using luciferase reporter and assays for pgRNA, HBcAg and cccDNA accumulation in cells with HBV replicon and HBV infection models. To further confirm their specific activity, short peptide mimetics generated from Slug zinc-finger domains and SOX7 HMG-box were generated.

Results: The HBVCP was found to be active in liver and selected non-liver cells. These cells have low/negligible expression of Slug and SOX7, which inhibit HBVCP transcription specifically by binding at the pgRNA initiator site and competitively displacing hepatocyte nuclear factor 4α, respectively. Overexpression of Slug and/or SOX7 specifically reduced HBVCP transcription, significantly diminishing pgRNA synthesis, HBcAg and cccDNA accumulation in HBV-infected primary human hepatocytes. Similar results were obtained with Slug and SOX7 stapled peptides individually, which were even more potent in combination.

Conclusions: Slug and SOX7 are transcriptional repressors that bind specifically to the HBVCP. Their absence or weak expression in liver cells contribute to the favorable host environment for the active and efficient production of HBV.

Lay summary: Hepatitis B virus (HBV) replication occurs efficiently in human liver because of the specificity of viral uptake receptors and presence of numerous liver-enriched transcription activators. Herein, we show that the specific lack of transcriptional inhibitory mechanisms in liver cells also contribute to effective HBV production. HBV replication is kept low in non-liver cells as transcriptional repressors Slug and SRY-related HMG box 7 (SOX7) actively bind to the transcriptional initiator and displace transcription activators, respectively, within the HBV core promoter.

Keywords: HBV; HNF4α; Hepatocytes; Liver; Promoter; Repression; SOX7; Slug; Transcription; cccDNA; pgRNA.