Fig. 1. Systematic identification of cccDNA-associated proteins.

A Schematic of the methodology to identify cccDNA-associated proteins. MCP MS2 coat protein. B Western blotting (WB) analysis with antibodies against dCas9. β-actin was used as a loading control. C The level of dCas9 associated with cccDNA was examined by ChIP-qPCR assay. Cross-linked chromatin was immunoprecipitated with specific anti-dCas9 antibody or corresponding IgG, followed by PCR quantification of HBV cccDNA using specific primers. D APEX2-mediated biotinylation of endogenous proteins associated with cccDNA, as detected by WB with streptavidin-HRP (SRP). E Mass spectrometry results were verified by immunoprecipitation and WB assays. HBV-infected cells were collected, lysates were immunoprecipitated with streptavidin agarose, and the products were separated by SDS–PAGE. GAPDH antibody was used as a negative control.