Visualization of hepatitis B virus entry – novel tools and approaches to directly follow virus entry into hepatocytes

Zhang, Zhenfeng; Zehnder, Benno; Damrau, Christine; Urban, Stephan

doi:10.1002/1873-3468.12202

Cited by 17 publications

(13 citation statements)

References 105 publications

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“…Finally, the cells allow visualization of viral replication and assembly, e.g. by FISH for genomic or antigenomic viral RNA or by fluorescent labeling of the HDAg using amber technology and click chemistry as described for the HBV capsid 36 .…”

Section: Discussionmentioning

confidence: 99%

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

et al. 2019

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Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC 50 : 1.4 nM) and interferon-α (IC 50 : 28 IU/ml, but max. 60–80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC 50 : 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

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Section: Discussionmentioning

confidence: 99%

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

et al. 2019

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“…Recent advances in imaging technologies can visualise the internalisation of virus particles (reviewed in [Wang, Burckhardt, Yakimovich, & Greber, 2018]). Several studies have reported the use of fluorescent labelled HBV viral structural proteins or sub‐viral particles (Hao et al, 2011; Zhang et al, 2016). However, these approaches come with certain constraints as the labelling can impair viral infectivity and do not always resemble natural infection.…”

Section: Discussionmentioning

confidence: 99%

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

Chakraborty

Henning

et al. 2020

Cellular Microbiology

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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.

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“…Recent advances in imaging technologies can visualise the internalization of virus particles (reviewed in 47 ). Several studies have reported the use of fluorescent labelled HBV viral structural proteins or sub-viral particles 17,48 .…”

Section: Discussionmentioning

confidence: 99%

Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle

Chakraborty

Henning

et al. 2020

Preprint

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24Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) 25 DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA 26 that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and 27 host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium 28 taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral 29 life cycle. We employed a synchronized infection protocol to quantify HBV entry kinetics. HBV attachment to cells 30 at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation 31 at 12h post-infection. HBV uptake is clathrin-and dynamin dependent with actin and tubulin playing a role in the 32 first 6h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6h of infection and 33 cccDNA was first detected at 24h post-infection. Our studies show the majority (83%) of cell bound particles enter 34 HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting 35 this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell 36 culture systems and will inform the design and evaluation of physiologically relevant models that support efficient 37 HBV replication. 39Hepatitis B Virus (HBV) infects 257 million individuals worldwide and is a major driver of end-stage liver disease, 41 cirrhosis, and hepatocellular carcinoma (HCC). HBV is an enveloped DNA and prototypic member of the 42 hepadnaviridae that establishes its genome as an episomal, covalently closed circular DNA (cccDNA) in the nucleus 43 of infected hepatocytes. Current treatments suppress viral replication but are not curative, largely due to the 44 persistence of the cccDNA transcriptional template and failure to mount an effective anti-viral immune response 1 . 45In most cases, treatment is life-long and patients may still develop HCC 2 , highlighting a clinical need for new 46 curative therapies 3 . Despite its central role in the HBV life cycle our understanding of the host factors regulating 47 cccDNA genesis and half-life is limited 4 . 49Viral entry into a host cell represents the first step in the infectious life cycle and is mediated via specific 50 interactions between virus encoded proteins and cellular receptors that define internalization pathways 5 . The 51 discovery that sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV 6,7 enabled the 52 development of in vitro culture systems that support the complete HBV replication cycle. HBV encodes three 53 envelope glycoproteins, small (S), middle (M) and large(L) 8 . The preS1 domain of the L protein binds heparan 54 sulfate proteoglycan (HSPG) 9-11 that precedes high-affinity virus interaction with NTCP....

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Visualization of hepatitis B virus entry – novel tools and approaches to directly follow virus entry into hepatocytes

Cited by 17 publications

References 105 publications

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

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

Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle

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