Cytokeratins 8 and 18 are responsible for distribution and secretion of hepatitis B virus surface proteins

Chronic Hepatitis B (HBV) induces liver cirrhosis and liver cancer (HCC). The pathologic phenotype is based on host autoimmune reactions against the virus and by direct cytotoxic effects of viral components, e.g. the accumulation of the large surface protein of HBV (LHBs) in the endoplasmic reticulum (ER) of hepatocytes, which leads to ER-stress. If the cells cannot normalize the state by activating of protecting mechanisms they finally undergo apoptosis or transform into tumor cells. In this work we could show that cytokeratins are responsible for intracellular distribution and also secretion of HBV surface proteins (HBsAg).

The hepatitis B surface DNA sequences of small (SHBs) and large (LHBs) of the three HBV surface proteins and a construct expressing all three (L, M and S) HBV surface proteins together with HBV X-protein (LHBs/HBx) were cloned separately into lentiviral vectors and infectious lentiviruses transduced stably the human hepatoma cell line Huh7 and the untransformed mouse fibroblast cell line NIH3T3. AML-12 cells (murine hepatic cell line) expressing SHBs or LHBs/HBx were generated by transfection.

The LHBs and also the LHBs/HBx protein expression causes intracellular aggregate-formation of LHBs protein within NIH3T3, but a scattered distribution pattern of LHBs appeared in Huh7 cells by immunofluorescence staining. AML-12 cells showed either large aggregates and a fine LHBs distribution. In contrast a scattered distribution of SHBs protein was seen with SHBs expression in all three cell lines.

Further studies revealed that the breakdown of cytokeratin filament network by phosphatase inhibitors e.g. okadaic acid resulted in a perinuclear co-collapse of distributed LHBs and cytokeratin 8/18 (CK8/18) into one focus in Huh7 and AML-12 cells. However SHBs pattern in all three cell lines was not significantly affected.

Confocal microscopy and proximity ligation assays of co-stained LHBs and CK8/18 in AML-12 and Huh7 cells expressing LHBs/HBx confirmed a colocalization of LHBs with CK8/18. Moreover CK18 transfection in NIH3T3 cells expressing LHBs could prevented the formation of large LHBs aggregates and led to a finely distributed LHBs pattern and strong colocalization with CK18.

We also analysed the effect of CK8/18 on secretion of HBsAg with Huh7 and AML-12 cells transiently transfected with a LHBs/HBx construct under the HBV promoter ensuring a natural expression ratio of surface proteins. CK8/18 breakdown led to an okadaic acid concentration dependent increase of HBsAg secretion into the cell culture medium.

Cytokeratins 8 and 18 are responsible for distribution and secretion of HBV surface proteins. These new findings could be relevant for new therapeutic options in the field of HBV therapy.