Virology Journal Club 07.03.2013

HepG2 cells infected with the hepatitis B virus This term students taking my virology course at the University of Leicester are doing a series of tutorials involving reading and explaining research papers in virology. This is the sort of exercise which is common in graduate schools and regularly performed by researchers, and is known as a “journal club”. We cannot give students access to the facilities or equipment to study dangerous human viruses at the forefront of research, so gaining an deep understanding of the way into which research is currently being conducted in this area is the closest we can come to allowing them to be “real virologists”. Today, we are looking at the following paper:

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. (2012) eLife, 1: e00049


How do you read a research paper? Start with a quick scan: what’s this all about?

From the Abstract:

  • Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with satellite hepatitis D virus (HDV) have more severe disease.
  • Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding.
  • Near zero distance photo-cross-linking and tandem affinity purification [What is this?] revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver.
  • Silencing [RNA interference] NTCP inhibited HBV and HDV infection, while exogenous NTCP expression made a non-susceptible hepatocarcinoma cell line susceptible to infection.
  • Replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred the ability to support infection.


At this stage it is useful to scan though the figures and explanatory figure legends in the paper to get an overview of the data:


Figure 1. Developing photoreactive peptide ligands and an antibody for identifying pre-S1 binding partner(s) by zero distance cross-linking
A) Monoclonal antibody (mAb) 2D3 binding epitope (grey box) and modified peptides for cross linking. UV cross-linking converts non-covalent interactions between proteins or simply their proximity into covalent bonds.
B) Binding of mutant HDV L proteins to cells (PTH: primary Tupaia hepatocytes).
C) Peptide inhibition of HDV binding.
D) Peptide inhibition of HBV and HDV infection.
E) Epitope mapping of 2D3 antibody.

Figure 2. Identification of pre-S1 binding protein on primary Tupaia hepatocytes with photoreactive peptide Myr-47/WTb
A) Western blot of bound protein.
B) WT but not mutant peptide competes for binding.
C) The abundance of the target protein(s) in PTH cells decreases over time.
D) Cross linked target protein.
E) Purification of target protein(s) for mass spectrometry analysis.
F) Sequence of target protein.

Figure 3. Binding of NTCP to N-terminal peptide of pre-S1 and HDV virions
A) Western blots from 293T cells transfected with hNTCP or tsNTCP expression vectors.
B) Confocal microscopy of 293T cells [human embryonic kidney cell line] transfected with hNTCP or tsNTCP expression vectors.
C) FACS analysis of FITC-labeled pre-S1 peptide binding to hNTCP transiently transfected Huh-7 cells.
D) HDV binding to Huh-7 cells transfected with hNTCP or tsNTCP expression vectors.

Figure 4. HDV and HBV infection of hepatocytes requires NTCP
A) HDV and HBV infection of PTHs is inhibited by tsNTCP siRNA knockdown.
B) HDV and HBV infection of HepaRG is inhibited by NTCP siRNA knockdown.
C) Knockdown of hNTCP in primary human hepatocytes (PHH) hampers HBV infection.

Figure 5. NTCP expression confers Huh-7 susceptibility to HDV infection
A) NTCP mRNA expression levels.
B) Transfected cells stained with 4G5 antibody to HDV delta antigen.
C) Inhibition of HDV infection of Huh-7 cells transfected with hNTCP.
D) HDV RNAs detected by RT-PCR.
E) HCV moi dose response. [Efficiency of infection is low, ~10%]
F) Infection response to hNTCP level.

Figure 6. NTCP expression confers susceptibility to HBV infection [See response to referees comments]
A) Intracellular expression of HBsAg in HBV-infected HepG2-hNTCP stable cells.
B) Secreted HBeAg levels in the supernatants of HBV-infected cells.
C) Dose response of HBV infection.
D) Southern blot analysis of cccDNA
E-F) Kinetic analysis of HBV cccDNA and RNAs in HBV-infected HepG2-hNTCP cells.


Analysis Checklist

What did the authors want to find out or prove? Why? (Introduction)
HBV-related liver diseases remain a major public health problem, causing approximately 1 million deaths per year. Progress in HBV research has been impeded by the lack of understanding of HBV entry process by which the virus specifically infects human liver cells. HDV is a small satellite RNA virus of HBV carrying all three HBV envelope proteins and can only propagate when coexisting with HBV. The pre-S1 domain of the L protein is a key determinant for entry of both HBV and HDV and was believed to mediate viral interaction with the cellular receptor(s) on hepatocytes. An N-terminal myristoylated peptide corresponding to amino acids 2–48 of the pre-S1 domain of the L protein has been shown to block both HBV and HDV infection of hepatocytes. By using a synthetic modified peptide originating from the native aa 2–48 lipopeptide (Myr-47/WT) as a probe the authors set out to identify the HBV/HDV receptor.

What exactly did they do? (Methods)
HBV infects only primary hepatocytes in humans, chimpanzees and a primate-like animal called the treeshrew (Tupaia belangeri), but it does not infect other animals such as monkeys, rats, mice or rabbits. So far, no transformed or immortalized cell lines can be infected with HBV. HBV research is limited by the availability of primary hepatocytes from permissive hosts. The authors used primary hepatocytes from Tupaia and human liver and a number of cell lines to investigate HBV/HDV infection.
Infection of cells was measured by antibody staining and real time RT-PCR analysis of HDV genome and antigenome RNAs. HBV covalently closed circular DNA (cccDNA) was measured by Southern blot analysis and qPCR.
Expression vectors expression Tupaia and human PTH were made from cDNA libraries.
Photo-cross-linking of peptide ligand and tandem purification of the target molecule(s) was carried out. Cross-linked target proteins were identified by mass spectrometry and bioinformatic data bases.
Gene knockdowns of NTCP in cultured cells was performed by siRNA interference.

What were their results? (Results)
Photo-cross-linking of a synthetic peptide derived from the native pre-S1 peptide with certain residues replaced by nonnatural amino acids (L-photo-leucine, L-2-amino-4,4-azi-pentanoic acid) identified NTCP as a specific binding protein of pre-S1 (partial sequence obtained by mass spectrometry (MS) analysis).
This was confirmed by cloning and expressing human and Tupaia NTCP and showing that NTCP expression was required for HBV and HDV infection, and conversely that HBV/HDV infection is reduced by siRNA knockdown of NTCP.
NTCP expression made non-susceptible hepatocarcinoma cells (Huh-7 and HepG2) permissive for HDV and HBV infection.
Residues 157 to 165 of hNTCP are critical for pre-S1 binding and virus infection.

What do these results mean? (Discussion)
The liver bile acid transporter, NTCP, specifically interacts with a key region in the pre-S1 domain of the HBV envelope L protein, and functions as a major receptor of HBV/HDV.
Why this protein? NTCP is a multiple transmembrane glycoprotein presumed to span the cellular membrane up to 10 times with small extracellular loops and is mainly expressed in the liver consistent with the liver tropism of HBV and HDV.
NTCP is functionally conserved in mammalians, but protein sequences of NTCP vary among species, which is likely to contribute to the narrow species tropism of HBV infection.

What else could the authors have done? What should they do next? What are the strengths and weaknesses of this paper? Why does this research matter? (Synthesis)
Identification of NTCP as a functional receptor for HBV and HDV advances our understanding of their entry into host cells and may lead to new prevention and treatment strategies against these viruses and related diseases.

Interestingly, eLife is one of the new generation of scientific journals that publish referees comments on the submitted manuscript and the authors responses to these, a stage in scientific research is normally kept secret. In this case, there was an interesting discussion around the fact that the efficiency of infection is low (5-10%). Does this indicate that NTCP is not the only HBV receptor in vivo, that other soluble factors in the blood or other co-receptor/entry components boost the efficiency in vivo, or that the microenvironment and architecture of hepatocytes in the liver affects the infection process?

In any case, this paper is a major technical tour de force and a major advance in understanding HBV infection.


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