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Visualisation of the early steps of human hepatitis B virus infection: a colourful approach

Final Activity Report Summary - HBVENTRYVISUALISED (Visualisation of the early steps of human hepatitis B virus infection: a colourful approach)

Due to restrictions in cell culture systems, the study of the early infection events as well as productive infection of the hepatocyte by the hepatitis B virus (HBV) has been limited. Recent progress has been made using the HepaRG cell line (Gripon et al., PNAS, 2002). In order to study HBV binding and uptake, we utilised HepaRG cells, as well as cutting edge techniques such as incorporation of fluorescent dyes into HBV particles and quantitative, real-time PCR. We found that after four hours of incubation at 37 degrees Celsius, only around 5 % of input virus was associated with HepaRG cells, representing a maximum of 500 genome equivalents per cell. Furthermore, these genomes were distributed in specific cells, the target cells for the viruses were differentiated cells on the edge of island. In total, only 5 % of differentiated cells were positive for HBV after four hours incubation. The binding of HBV to HepaRG cells was trypsin and glycine-resistant, suggesting that binding was perhaps to a receptor of non-proteinaceous origin. However, binding could be eliminated by pre-incubation of HBV with anti-HBV antibodies, but not with an irrelevant antibody. Interestingly, pre-incubation of HepaRG cells with a highly active preS1 peptide, although totally inhibiting infection, did not inhibit binding to or uptake into differentiated cells. Furthermore, mutants that we have previously shown to be deficient in infection (Engelke et al., Hepatology, 2006) were not deficient in binding, suggesting a two-step process of HBV infection. In support of this hypothesis, HBV was found to bind to a number of cell lines, both non-human and non-liver in origin.

The study of productive infection, that is infection between days 4 and 12, including the study of distribution of HBV-infected cells as well as the rate of infection and dynamics of HBV antigen expression at a cellular level requires visualisation of infected cells. We examined these aspects of HBV infection of HepaRG cells using fluorescence microscopy. We discovered that only a small percentage (10 %) of differentiated cells were infected, and displayed a striking distribution within differentiated cells. During a time course of 12 days, the number of infected cells increased only incrementally, which was the result of a highly restricted dispersion to neighbouring cells. Treatment with an inhibitory peptide post-infection indicated that this dispersion takes place to some degree via a cell-to-cell route. EGTA treatment of cells directly prior to infection leads to an increase in infection, hinting at an initial physical restriction of infection.

In order to investigate whether this restriction is related to the induction of polarity during differentiation, markers for hepatocyte differentiation were studied. HepaRG cells were found to display an unusual polarisation, which was restricted to cells within a differentiated island. Such cells were resistant to infection. Taken together, these data seem to indicate an initial physical restriction of infection, followed by a biochemical restriction to unpolarised cells.