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

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Date
2021
Authors
Meier-Stephenson, Vanessa
Badmalia, Maulik D.
Mrozowich, Tyler
Lau, Keith C. K.
Schultz, Sarah K.
Gemmill, Darren L.
Osiowy, Carla
van Marle, Guido
Coffin, Carla S.
Patel, Trushar R.
Journal Title
Journal ISSN
Volume Title
Publisher
Elsevier
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 among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic 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, pregenomic 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.
Description
Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) applies
Keywords
Hepatitis B virus , Pre-core promoter , cccDNA , DNA G-quadruplex , HBV surface antigen , ELISA , Microscale thermophoresis
Citation
Meier-Stephenson, V., Badmalia, M. D., Mrozowich, T., Lau, K. C. K., Schultz, S. K., Gemmill, D. L., Osiowy, C., van Marle, G., Coffin, C. S., & Patel, T. R. (2021). Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA. Journal of Biological Chemistry, 296, Article 100589.
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