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 reads coverage of selected regions on HBV-related RNAs and polyadenylation positions within HBV sequence using RNA-sequencing suggested 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 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. All SNU cell lines could not support efficient HBV replication after transfection with the vector initiating 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. Overall design: Human SNU cell lines (SNU-423, SNU-368 SNU-398, SNU-182, SNU-449, SNU-475, SNU-354, SNU-739, SNU-387, SNU-761 and SNU-886) developed (by Seoul National University (SNU)) from hepatocellular carcinomas associated with chronic hepatitis B virus (HBV) infection were examined. The RNA markers of intracellular HBV replication (pre-genomic RNA (pgRNA), replication-derived RNAs generated by HBV genome replication and polyadenylated via conventional HBV polyadenylation signal (PAS) (rd-RNAs), and total HBV RNA (including rd-RNAs, cps-RNAs (HBV RNAs polyadenylated via HBV cryptic PAS) and also various RNA species transcribed from integrated HBV DNA depending on the HBV area that they bear) were measured by RT-qPCR assays. The DNA markers of intracellular HBV genome replication (core-associated HBV DNA and covalently closed circular DNA (cccDNA)) were quantified by qPCR assays. These intracellular HBV replication markers were assayed and compared between parental (untransfected) SNU cell lines and two reference cell lines, Hep3B and PLC/PRF/5 (Alexander cells), which also were derived from human HBV-related HCCs. The latter two reference cell lines contained integrated HBV DNA, and had no ongoing HBV replication in them. The intracellular expression of HBV core antigen (HBcAg) was analyzed and compared using immunofluorescence and staining with anti-HBcAg antibodies in parental SNU cell lines, and in control Huh7 cells and HepG2 cells (that were transfected with the plasmid pT-HBV1.3 and were replicating HBV genome). Huh7 cells and HepG2 cells were derived from human HCCs not related to HBV infection. The HBV DNA associated with secreted un-enveloped nucleocapsids (cores) or with HBV virions coated with HBV surface antigen (HBsAg) was assayed and compared in parental SNU cell lines and the above mentioned transfected Huh7 and HepG2 cells using immuno-precipitations (IPs) with anti-HBcAg antibodies or with anti-HBsAg antibodies (respectively) with subsequent measuring of HBV DNA in the pellet and in the supernatant using qPCR, which was designed for the analysis of the relaxed circular HBV DNA (rcDNA). The total RNA harvested from parental SNU cell lines and from Huh7 cells, which were transfected with the plasmid pT-HBV1.3 and were replicating HBV genome, were analyzed by RNA-sequencing that employed poly(A) selection. The generated RNA-seq data was analyzed (i) for HBV RNA reads coverage for the selected regions on HBV genome, which represented the unique areas of HBV genomic sequences that are expected to be present only once on rd-RNAs produced by HBV genome replication; (ii) for polyadenylation sites within HBV sequences, (iii) for 5'-human-HBV-3' RNAs and 5'-HBV-human-3' RNAs that were transcribed from integrated HBV DNA independently of HBV genome replication; and (iv) for spliced HBV RNA species as we did previously (Zaiets, I., S. Gunewardena, S. Menne, S. A. Weinman, and S. O. Gudima. 2023. Sera of individuals chronically infected with hepatitis B virus (HBV) contain diverse RNA types produced by HBV replication or derived from integrated HBV DNA. J. Virol. 97(3):e0195022. doi: 10.1128/jvi.01950-22.. PMID: 36877036. PMCID: PMC10062156). In addition, the above mentioned intracellular HBV genome replication markers, intracellular expression of HBcAg, and secreted core-bound and virion-bound HBV DNA were analyzed and compared in SNU cell lines transfected with the plasmid pT-HBV1.3 (to induce HBV genome replication) and control Huh7 cells and HepG2 cells, which were also transfected with pT-HBV1.3.