(I) Dendritic cells take up viral antigens but do not support the early steps of Hepatitis B Virus infection (II) Generation and characterization of Hepatitis B virus surface proteins fused to GFP and RFP
(I)Dendritic cells (DC), being key players in antigen presentation and initiation of virus-specific T-cell responses, have been reported to exhibit functional impairment in HBV carriers. Possible explanations for this phenomenon are infection of DCs with HBV or alteration of DC function by HBV. Therefore it was analysed whether DCs support the different steps of HBV infection: uptake, delivery of the HBV genome to the nucleus, antigen expression, and progeny virus release. When HBV genomes were artificially introduced into monocyte-derived DCs (moDC) by adenoviral vectors, low-level expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) but no HBV replication was detected. When subjecting moDCs to recombinant HBV expressing Renilla luciferase either under a non�liver-specific promoter or under a HBV promoter, no luciferase activity was detected. After incubation with wild-type HBV, intracellular HBV rcDNA was detected in a low percentage of cells, but nuclear cccDNA was not formed. This indicates that either uncoating or nucleocytoplasmic transport were blocked. To verify the observation in the in vivo situation, myeloid (mDC) and plasmacytoid DCs (pDC) were isolated from blood of high viremic HBV carriers, and analysed by quantitative polymerase chain reaction (PCR) and electron microscopy. Although circulating DCs had in vivo been exposed to more than 104 HBV virions per cell, HBV genomic DNA was hardly detected, and no nuclear cccDNA was detected at all. By using electron microscopy, subviral particles were found in endocytic vesicles, but virions were undetectable, as were viral capsids in the cytoplasm. Quantitative PCR analysis of B cells, monocytes and an enriched T-cell fraction of chronic HBV carriers showed uptake of HBV particles in low amounts, but no establishment of an infection. In conclusion, circulating DCs may take up HBV antigens, but neither support nucleocytoplasmic transport nor replication of HBV. It can be excluded that HBV infects DCs with a frequency sufficient to explain the functional impairment of the virus-specific T-cell response in chronic HBV carriers. One can hypothesise that the contact of DCs with HBV antigens, which are present in sera of infected persons in high amounts, influences DC and T-cell function. (II)Fluorescent labelling of viral proteins and viruses, or virus-like particles, has been shown to provide a powerful tool for investigating unexplored aspects in viral life cycles. A commonly used technique is the genetic incorporation of fluorophores into virus proteins. The insertion of GFP into the Gag protein of HIV for instance allowed studying the trafficking of this protein, as well as production of infectious virions (Müller et al., 2004). In case of adeno-associated virus, Lux et al. (2005) were able to directly monitor the cytosolic and nuclear trafficking with GFP-tagged virus particles. Since many aspects of the HBV life cycle still remain unclear, the visualisation of viral (VP) and subviral (SVP) HBV particles is a promising approach to elucidate different steps of virus entry and egress. GFP or RFP fused HBV S, M and L surface proteins were constructed and analysed for their fluorescence and stability properties. To ensure that the fusion did not alter the characteristic properties of the HBV surface proteins, they were compared to the parental proteins with regard to their localisation in cellular compartments and the ability to form subviral and viral particles. The GFP fused proteins did not co-localise with the parental proteins within the cell, suggesting that they were unstable. The RFP fusion proteins clearly co-localised with the parental proteins. But although they behaved similar to the parental proteins with regard to their distribution in the ER and golgi compartment, neither of them contributed to the formation of SVP or VP. Thus, GFP and RFP fusion to HBV surface proteins is not suitable to generate fluorescent HBV particles. The main impediment is most probably misfolding of the fusion proteins. The bulky GFP and RFP domains seem to have topological and sterical effects, affecting stability, correct integration into the ER membrane or assembly and secretion of properly formed particles. Labelling with small fluorescent molecules can minimise or overcome these problems and therefore perhaps provides a better strategy
