Transcription of HBV cccDNA is synchronized at single-cell level and orchestrated by expression of HBx

Chronic infection of Hepatitis B virus (HBV) remains a public health problem worldwide. HBV infection relies on the persistence of covalently closed circular DNA (cccDNA) in the nucleus and actively cccDNA transcription. To understand HBV cccDNA transcription regulation at single cell level, we isolated primary human hepatocytes from liver humanized FRG mice infected by one or more (two or three) HBV genotypes, and we quantified transcripts of HBV structural genes in single cells. HBV transcripts were ascribed to the transcription of individual HBV genes by 5’ end sequencing thus avoiding the ambiguity caused by the overlap of viral genome coding at the 3’ ends. Transcripts from different cccDNA in single cells were separated according to the single-nucleotide polymorphism (SNP) among different HBV genotypes. We found that the transcription of HBV follows “all-or-none” pattern in single cells: either all of the individual cccDNA molecules actively transcribe simultaneously, or, none of them generates transcripts of the structural genes. In vitro cell infection assays with recombinant HBV are consistent with the sequencing results of ex vivo samples from natural HBV infection, and also confirm that such a pattern is apparently controlled by the expression of HBx protein. These results strongly support a synchronized transcription model of HBV cccDNA molecules in single hepatocytes, and provide new insight helpful for developing HBV cure strategy. 02 humanized liver samples treated with HBV for 150days, 18 humanized liver samples treated with HBV for 180days, 492 humanized liver samples treated with Genotype A and D HBV for 77 days, 907 humanized liver samples treated with Genotype A,C and D HBV for 119 days, HBV X Minus PHH sample treated with HBV for 7days were collected for single cell RNA sequencing by 10X Genomics) 18 humanized liver samples treated with HBV for 180days, 993 humanized liver samples treated with HBV for 193days were collected for DNA sequencing