Synchronised infection identifies early rate‐limiting steps in the hepatitis B virus life cycle

Abstract

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double‐stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co‐transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP‐dependent and reaches saturation at 12 h post‐infection. HBV uptake is clathrin‐ and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post‐infection. Our studies show the majority (83%) of cell bound particles enter HepG2‐NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate‐limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.

Circadian control of hepatitis B virus replication

“…3a). Using a recently developed synchronized infection protocol 42 we show that SR9009 treatment reduced HBV uptake into dHepaRG cells (Fig. 3b).…”

“…To investigate a role for circadian pathways in the HBV life cycle we first assessed whether NTCP expression is rhythmic. We selected the bipotent HepaRG cell line that can be differentiated to biliary-like epithelial cells as well as hepatocyte-like cells that is frequently used for drug toxicity studies 32,33 , expresses NTCP 34 and supports HBV replication 35 . Different methods can be used to induce circadian gene expression and to reset the phase of the cellular circadian clock ex vivo 36 .…”

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint

“…We examined the impact of inhibiting ACAT on HBV replication in human HepG2 hepatocyte derived-cells engineered to express the viral entry receptor, sodium-taurocholate co-transporter polypeptide (NTCP) 44 . Building on our finding that ACAT regulates the cholesterol composition of cell membranes, we initially assessed the impact of inhibiting ACAT on HBV uptake into HepG2-NTCP cells using recently reported methodology 45 . Treating HepG2-NTCP cells with ACAT inhibition induced a modest twofold increase in internalised virus (Fig.…”

Earliest hepatitis B virus-hepatocyte genome integration: sites, mechanism, and significance in carcinogenesis

“…In yet another recent work investigating HepG2-NTCP cells, HBV DNA became detectable at 6 h p.i., whereas cccDNA at 24 h p.i. [55] . By using assay detecting HBV cccDNA by inv-PCR followed by NAH analysis of amplicons, the appearance of cccDNA was reported at 16 h p.i.…”