Abstract
Hepatitis B virus (HBV) X protein (HBx) plays diverse roles in both viral life cycle and HBV-related carcinogenesis. Its interaction with DNA damage-binding protein 1 (DDB1) was shown to be essential for engendering cellular conditions favorable for optimal viral transcription and replication. Previously, we described a mouse monoclonal antibody against HBx (anti-HBx 2A7) recognizing HBx encoded by representative strains from 7 of 8 known HBV genotypes. In this work, we further characterized 2A7 in order to explore its potential usefulness in HBx-targeting applications. We demonstrated that 2A7 recognizes a linear epitope mapped to L89PKVLHKR96 on HBx, a segment that is highly conserved across genotypes and coincidentally overlaps with the DDB1-interacting segment. HBx-DDB1 binding could be inhibited by 2A7 in vitro, suggesting therapeutic potential. Nucleic acid and amino acid sequences of 2A7 were then obtained, which allowed construction of recombinant antibody and single chain variable fragments (scFv). 2A7-derived recombinant antibody and scFv recapitulate 2A7’s HBx-binding capacity and epitope specificity. We also reported preliminary results using cell-penetrating peptide for delivering 2A7 antibody across cell membrane to target intracellular HBx. Anti-HBx 2A7 and 2A7-derived scFv characterized here may give rise to novel HBx-targeting diagnostics and therapeutics for HBV- and HBx-related pathologies.
Highlights
Hepatitis B virus (HBV) is the type member of Hepadnaviridae, a family of small, non-cytopathic viruses with a relaxed circular DNA genome
We described a broadly reactive mouse monoclonal antibody against HBV X (HBx) that detects HBx encoded by representative strains from 7 of 8 known HBV genotypes in Western blot and immunofluorescence assays[27]
We demonstrated that 2A7 recognizes a linear epitope encompassing amino acids 89 to 96 of HBx, which is highly conserved across genotypes and coincidentally overlaps with HBx’s DNA damage-binding protein 1 (DDB1)-interacting segment
Summary
Hepatitis B virus (HBV) is the type member of Hepadnaviridae, a family of small, non-cytopathic viruses with a relaxed circular DNA (rcDNA) genome. HBx is generally believed to be a non-structural protein and has been shown to play diverse functions in viral life cycle, virus-cell interactions and HBV-related HCC7–10. Mechanistic studies have shown that HBx acts through interacting with cellular adaptor proteins[19,20,21] and DDB1 (DNA damage-binding protein 1) is one of the earliest HBx-binding host protein identified[19]. The fact that HBx requires interaction with cellular adaptor proteins to be functional suggest the possibility of targeting such interactions for treating liver diseases associated with HBV or HBx. Previously, we described a broadly reactive mouse monoclonal antibody against HBx (anti-HBx 2A7) that detects HBx encoded by representative strains from 7 of 8 known HBV genotypes in Western blot and immunofluorescence assays[27]. We explored the possibility of delivering 2A7 into intracellular space for potential HBx-targeting applications
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