Zinc Finger Proteins Designed To Specifically Target Duck Hepatitis B Virus Covalently Closed Circular DNA Inhibit Viral Transcription in Tissue Culture

Abstract

Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV reemergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After the binding kinetics were assessed by using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3 and 40.2 nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and cotransfected into longhorn male hepatoma cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced, and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes.