Listen Now Download as mp3 from the show Elife Episode 1: Multicellular life, potato blight and Hepatitis B
The liver virus hepatitis B is thought to have infected up to a third of the world’s population – 2 billion people, half a billion of them, chronically. Hep B also has a partner in crime called hepatitis D which is the subviral particle that borrows the coat proteins of hepatitis B and quite literally rides on its coat tails. And although there's an excellent vaccine that can prevent infection with both of these agents, scientists still didn’t know how the virus was recognising and entering liver cells. And this is important because if we know how it gets in, we can potentially design drugs to block the process, helping people who are already infected. Wenhui Li from the National Institute of Biological Sciences in Beijing has made just that discovery.
Wenhui - We want to find the functional receptor for hepatitis B and hepatitis D virus, which infected a third of the world population, and how it enters our hepatocytes, which is the target cells of that, has been unknown for several decades. We just want to identify the receptor for this virus.
Chris - So, although we know that hepatitis B and its relative hepatitis D get in to liver cells, until now, no one knew exactly how they were doing that.
Wenhui - Yes. So, we’ve identified very specific bile acid transporter which could function as a functional receptor for this virus.
Chris - So, this is just a chemical which is present on the cells in the liver which the virus is docking onto and using to get in.
Wenhui - Yes. It’s a protein mainly expressed in the liver, the protein name is sodium taurocholate cotransporting polypeptide. In short, NTCP and is a multiple transmembrane protein and predominantly expressed in liver. It’s in the sinusoidal membrane that is that blood side of the hepatocyte and then it functions as a bile acid transporter and is critical for the enterohepatic circulation of bile acid.
Chris - Can you explain to me how the technique worked? How did you home in on that protein in the membrane in order to prove that this was the receptor that hepatitis B and hepatitis D were using?
Wenhui - Yeah. So, we combined biochemistry and biological approaches. We developed a unique and very highly efficient approach for tandem purification of complex membrane proteins. We utilised a viral protein ligand in which we incorporate three tags of photoreactive or nonnatural amino acids in the receptor binding sites epitope and adjacent to the receptor binding site for antibody recognition and a biotin tail. So, the photo-leucine allowed zero distance cross-linking of the peptide ligand and then expand the receptor upon UV irradiation.
Chris - So, what you've done is to insert some fake – for want of a better word – amino acids into the virus protein which when you shine ultraviolet light on them will form bonds to anything nearby them. You've also got a tag on there so that you can grab it with an antibody or biotin. And you incubate this modified virus protein with cell surface membrane and then zap them with a light to make the proteins stick on irreversibly to anything it’s bonded to at the moment, at that moment in time and then you purify it. Is that right?
Wenhui - Right, yeah. That's exactly what we did and that we then use the MS that is mass spectrometry to analyse the target protein.
Chris - So, by basically asking, what is this molecule, you come up with a chemical formula for the protein. But how do you work out what that is in the cell because there must be lots of proteins in the cells that are going to have a similar chemical formula?
Wenhui - Yes. We took several approaches to prove that is the molecule that we want. First of all, we used small interfering RNAs to knockdown this molecule in the cells. We checked whether the infection of HBV and HDV is affected by this gene silencing. And then we express this NTCP molecules into the Hu stem cells, which normally does not express this protein, and we evaluate whether the viral infection could be enhanced by transfecting this molecule.
Chris - That's quite neat. So effectively, you turn a cell which is uninfectable into a cell that becomes infectable as soon as you express this molecule in it. Thus, proving that this is necessary and sufficient for hepatitis B and hepatitis D viruses to get into these cells.
Wenhui - Correct.
Chris - So, now that you know this target, is it the only target on cells? In other words, if you take cells in a dish and as you have done, put this receptor molecule into them, do you get the same sort turnover or growth of the cells or efficiency of infection of the cells that you would in a human who is challenged with hepatitis B?
Wenhui - That's a very important question. We think that its molecule is a predominant one. If not, the only of the receptor. However, if you transfected this receptor into cells, it can support viral infection but not as efficient as in vivo. There's many reasons for this and detailed mechanism is still unknown.
Chris - Now that you know what the receptor is though, what do you think the implications of your discovery are?
Wenhui - This discovery advances our understanding of HBV and HDV infection and I think it also may raise a possibility to develop new therapeutic approaches for these viral infections. There are some drugs available against this NTCP, but we don't know yet if they can block the viral infection.