Establishment of hepadnaviral infection and viral replication in hepatocytes is critically influenced by e.g. cytokines1 or hormones (glucagon)2. We described previously3 that a natural immune stimulator, the endotoxin (lipopolysaccharide, LPS) of gram-negative bacteria, influences duck hepatitis B virus (DHBV) replication indirectly by activating nonparenchymal liver cells to produce and release soluble mediators. These mediators are polypeptides, most probably cytokines. Preliminary data point to participation of interferon gamma. We now ask the question which step in the DHBV replication cycle was influenced by these mediators.
Liver cells from 2 to 4 week old DHBV infected or uninfected ducks were isolated by a two step collagenase perfusion and differential centrifugation (hepatocytes (HC) 50×g, smaler nonparenchymal cells (NPC) 250×g). NPC were treated for 3h with LPS (Salmonella minnesota; 20ng/ml) on day 1 post plating (p.p.). After extensive washing, LPS-conditioned cell culture medium was collected. On day 2 p.p., hepatocytes were treated with the LPS-conditioned cell culture medium and infected with DHBV (moi 50) or a GFP expressing recombinant DHBV (rDHBV-GFP) (moi 100)4.
First experiments showed a clear decrease of intracellular viral proteins and DNA, as well as progeny virus production, in primary HC which were treated with the LPS-conditioned cell culture medium1. Therefore, we investigated which step of the viral replication cycle was influenced. Using cells with established viral infection, we demonstrated an inhibition of the intracellular DHBV replication cycle. We concluded that very early steps, namly virus uptake and cccDNA formation, are not influenced. An influence on following steps was investigated by infection with rDHBV-GFP. In rDHBV-GFP, the S gene is replaced by the GFP gene. This virus is able to infect HC, but due to the lack of functional polymerase no amplification of nuclear cccDNA occurs and the GFP-reporter is only expressed from the one incoming genome. Treatment with LPS-conditioned cell culture medium significantly reduced the number of GFP expressing cells.
The LPS induced mediators are potent inhibitors of the DHBV replication cycle and therefore of potential therapeutic interest. Our data clearly demonstrate that an early step of the DHBV replication cycle is influenced by the soluble mediators. They block one of the following steps: transcription, RNA export or translation.
1 Schultz U., Koeck J., Schlicht
H. J., Staeheli P., Virology (1995), 212: 641-649.
2 Hild M., Weber O., Schaller H.,
Journal of Virology (1998), 72: 2600-2606.
3 Klöcker U., Urban S.,
Protzer-Knolle U., GfV-Tagung Regensburg 1998.
4 Protzer U., Nassal N., and
Schaller H., manuscript submitted.