Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation

Wei, Lei; Ploß, Alexander

doi:10.1038/s41564-020-0678-0

Cited by 86 publications

(119 citation statements)

References 35 publications

Supporting

Mentioning

114

Contrasting

Order By: Relevance

“…However, our fractionation studies showing that up to 22% of total intracellular DNA is in the nucleus within the first 6-12h of infection suggeststhat nuclear targeting is not rate limiting for cccDNA genesis. The mechanism of rcDNA conversion to cccDNA is not fully defined and a number of host pathways have been reported 49–52 . The viral polymerase is removed by tyrosyl DNA phosphodiesterase 2 (Tdp2) 7,53,54 and this is followed by the removal of the RNA primer by a cellular flap-like structure specific endonuclease, Fen1 50,55 .…”

Section: Discussionmentioning

confidence: 99%

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

Chakraborty

Henning

et al. 2020

Preprint

View full text Add to dashboard Cite

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....

show abstract

Section: Discussionmentioning

confidence: 99%

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

Chakraborty

Henning

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…After binding to its receptor, the sodium taurocholate co-transporting polypeptide (NTCP), and uncoating, the viral capsid is transported to the nucleus where the viral genome, constituted by a relaxed circular and partially dsDNA molecule of 3.2 Kb (rcDNA), is released [6]. Conversion of rcDNA into cccDNA occurs in the nucleoplasm via the intervention of cellular enzyme [7][8][9]. It results in the establishment of a viral episome that constitutes the template for the transcription of five RNAs of 3.5 (precore and pregenomic RNA), 2.4, 2.1 and 0.7 kb that, respectively, encode the HBeAg, Core protein (HBc), viral polymerase, three surface glycoproteins (S, M and L; all defining the HBsAg), and X protein (HBx).…”

Section: Introductionmentioning

confidence: 99%

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

et al. 2020

View full text Add to dashboard Cite

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

show abstract

“…Indeed, this step relies on the cellular DNA repair machinery, and cell culture conditions may largely influence this process. Cell-based genetic approaches, using siRNA or Crispr/Cas9, are difficult to implement because of the importance of the DNA repair proteins in cell survival and in vitro biochemical systems have been used to mimic rcDNA to cccDNA [ 26 , 27 ]. We propose to use these different culture conditions as a complementary approach to confirm the involvement of cellular factors in this process [ 26 ] and possibly discover additional ones.…”

Section: Discussionmentioning

confidence: 99%

Fast Differentiation of HepaRG Cells Allowing Hepatitis B and Delta Virus Infections

Michelet

Salvetti

Durantel

2020

Cells

View full text Add to dashboard Cite

HepaRG cells are liver bipotent progenitors acquiring hepatocytes features when differentiated in the presence of dimethylsulfoxide (DMSO). Differentiated HepaRG (dHepaRG) are considered the best surrogate model to primary human hepatocytes (PHH) and are susceptible to several hepatotropic viruses, including Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV) infection. Despite these advantages, HepaRG cells are not widely used for the study of these two viruses because of their long differentiation process and their rather low and variable infection rates. Here, we tested the use of a cocktail of five chemicals (5C) combined or not with DMSO to accelerate the cells’ differentiation process. We found that NTCP-mediated HDV entry and replication are similar in HepaRG cells cultivated for only 1 week with 5C and DMSO or differentiated with the regular 4-week protocol. However, even though the NTCP-mediated HBV entry process seemed similar, cccDNA and subsequent HBV replication markers were lower in HepaRG cells cultivated for 1 week with 5C and DMSO compared to the regular differentiation protocol. In conclusion, we set up a new procedure allowing fast differentiation and efficient HDV-infection of HepaRG cells and identified differential culture conditions that may allow to decipher the mechanism behind the establishment of the HBV minichromosome.

show abstract

Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation

Cited by 86 publications

References 35 publications

Synchronized 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

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

Fast Differentiation of HepaRG Cells Allowing Hepatitis B and Delta Virus Infections

Contact Info

Product

Resources

About