Limited disassembly of cytoplasmic hepatitis B virus nucleocapsids restricts viral infection in murine hepatic cells

Abstract

Background and Aims: Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells. Approach and Results: Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pregenomic RNA (pgRNA), covalently closed circular DNA (cccDNA), and different relaxed circular DNA (rcDNA) intermediates were produced in vitro. The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine, hepatic cells. HBV replication‐competent plasmid, cccDNA, and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo. In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein‐linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine, but not in human, hepatic cells. Conclusions: Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.

How many steps to a feasible mouse model of hepatitis B virus infection?

“…Going further, Zhao et al3 found that some rcDNA did reach the murine nucleus, but disappeared over time without cccDNA production (Figure 1B). Their most striking observation, however, was the nearly complete nuclease resistance of cytoplasmic NC‐borne rcDNA in murine cells, whereas in human cells a substantial fraction of this rcDNA was nuclease sensitive.…”

“…huNTCP enables HBV entry into murine cells (not explicitly shown), but usually not cccDNA formation. Experiments by Zhao et al [3] to identify the critical step(s) are indicated by white boxes with blue lettering. Artificial delivery of HBV plasmid vectors or cccDNA (not shown) generated all expected RNAs, proteins, and progeny NCs, ruling out transcriptional issues.…”

“…Despite these complexities, the studies by Zhao et al3 and Wei et al4 demonstrate the principal ability of murine cells to repair rcDNA into cccDNA, such that further research can fully focus on deciphering the mechanisms of intracellular HBV NC transport, disassembly, and genome uncoating in human versus murine hepatic cells.…”

Hepatitis B Virus Capsid: The Core in Productive Entry and Covalently Closed Circular DNA Formation

“…Furthermore, the ability to support HBV cccDNA formation in AML12 cells could be correlated with enhanced destabilization (uncoating) of mature HBV nucleocapsids [ 17 ]. Similarly, some evidence has been presented recently suggesting that the failure to support nucleocapsid uncoating by mouse hepatocytes may also contribute to the inability of mouse hepatocytes to support cccDNA formation during HBV infection after huNTCP expression [ 78 ]. Consistent with the expectation that the DNA repair reactions involved in rcDNA to cccDNA conversion, per se, are highly conserved during evolution, mouse, and even yeast, DNA repair factors can support cccDNA formation from rcDNA mimics [ 78 , 79 , 80 ].…”

“…Similarly, some evidence has been presented recently suggesting that the failure to support nucleocapsid uncoating by mouse hepatocytes may also contribute to the inability of mouse hepatocytes to support cccDNA formation during HBV infection after huNTCP expression [ 78 ]. Consistent with the expectation that the DNA repair reactions involved in rcDNA to cccDNA conversion, per se, are highly conserved during evolution, mouse, and even yeast, DNA repair factors can support cccDNA formation from rcDNA mimics [ 78 , 79 , 80 ]. Therefore, overcoming the failure of nucleocapsid disassembly in mouse hepatocytes should help to establish a mouse model that can support HBV cccDNA formation and infection.…”