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Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA

DOI: 10.1016/j.jbc.2021.100589 DOI Help

Authors: Vanessa Meier-Stephenson (University of Lethbridge; University of Calgary) , Maulik D. Badmalia (University of Lethbridge) , Tyler Mrozowich (University of Lethbridge) , Keith C. K. Lau (University of Calgary) , Sarah K. Schultz (University of Lethbridge) , Darren L. Gemmill (University of Lethbridge) , Carla Osiowy (Public Health Agency of Canada) , Guido Van Marle (University of Calgary) , Carla S. Coffin (University of Calgary) , Trushar R. Patel (University of Lethbridge; University of Calgary; University of Alberta)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 61

State: Published (Approved)
Published: March 2021
Diamond Proposal Number(s): 22113

Open Access Open Access

Abstract: Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome, and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved amongst nearly all genotypes. This region is central to critical steps in the viral life-cycle, including the generation of pre-genomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism, and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pre-genomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Diamond Keywords: Hepatitis B Virus (HBV); Viruses; Liver Disease

Subject Areas: Biology and Bio-materials

Instruments: B21-High Throughput SAXS

Added On: 24/03/2021 22:25

Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Biochemistry Chemistry Drug Discovery Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)