HCC-derived SNU cell lines as model systems to study HBV life cycle

Human SNU cell lines, derived from hepatocellular carcinomas associated with chronic hepatitis B virus (HBV) infection, were examined. The analysis of intracellular RNA and DNA markers of HBV replication and examination of HBV RNA readss coverage of selected regions on HBV-related RNAs and polyadenylation positions within HBV sequence using RNA-sequencing suggested the absence of HBV replication in SNU-423, SNU-368, SNU-398, SNU-182, SNU-449, SNU-475, SNU-354, SNU-739, and SNU-387 cells, while SNU-761 and SNU-886 still could maintain residual HBV replication. The undetectable intracellular HBV core antigen (HBcAg) and absence of significant levels of secreted core-associated and virion-associated HBV DNA confirmed the absence or profound suppression of HBV replication in parental SNU cell lines. Various 5'-human-HBV-3' and 5'-HBV-human-3' RNAs transcribed from integrated HBV DNA were found in most of the SNU cell lines. The 5'-HBV-human-3' junctions suggested that several SNU cell lines could generate 5'-HBV-human-3' RNAs encoding HBV envelope proteins. The known and novel spliced HBV RNAs were detected in SNU-886, SNU-739, SNU-387, SNU-761, and SNU-354 cells. At least some of them were generated independently of HBV replication. None of the SNU cell lines supported efficient HBV replication after transfection with the vector that initiates efficient HBV replication in Huh7 cells. This was reflected by three distinct accumulation patterns of HBV replication markers, undetectable intracellular HBcAg, and by the lack of considerable levels of secreted core-bound and virion-associated HBV DNA. Overall, SNU cell lines represent valuable model systems for detailed analysis of integrant-transcribed HBV RNAs, spliced HBV RNAs, and mechanisms of suppression of HBV genome replication.IMPORTANCESNU cell lines without ongoing hepatitis B virus (HBV) genome replication are invaluable experimental systems that allow detailed study of the biogenesis and properties of integrant-transcribed 5'-human-HBV-3' and 5'-HBV-human-3' RNAs and mechanisms generating spliced HBV-related RNA species independently of concomitant viral replication. Three unique patterns of intracellular accumulation of HBV replication markers were observed in SNU cell lines transfected with the vector that initiates efficient HBV genome replication in Huh7 cells: (i) very low levels of pre-genomic RNA (pgRNA), total HBV RNA, replication-derived RNAs (rd-RNAs), covalently closed circular DNA (cccDNA), and core-associated HBV DNA; (ii) moderate pgRNA, high total HBV RNA, rd-RNAs, and cccDNA, but very low core-associated HBV DNA; and (iii) very low pgRNA, total HBV RNA, rd-RNAs, and core-associated HBV DNA, but moderate/high cccDNA likely reflect three natural host-mediated mechanisms suppressing HBV replication, the analysis of which should advance our understanding of HBV-host interactions and could be informative for the search for novel anti-HBV interventions.