Localization of isoprenylated antigen of hepatitis delta virus by anti-farnesyl antibodies.

Hepatitis delta virus (HDV) is a subviral pathogen that requires pre-existing or concurrent infection with hepatitis B virus (HBV). HDV expresses two forms of a single protein, the delta antigen (HDAg), which are identical except for an additional 19 residues at the C terminus of the large form. Within this C-terminal extension a cysteine residue is isoprenylated; this isoprenylation is critical for interaction with HBV envelope proteins to enable virus assembly and release into the medium. Therefore, large HDAg must be recruited to an extracellular compartment. However, immuno-staining with HDAg-specific antibodies has localized the large antigen mainly to the nucleus and supports the notion that large HDAg suppresses virus replication in the nucleus. Since isoprenylation would increase the hydrophobicity of the protein and may favour transport towards specific membranes, the question remains whether the large HDAg detected in the nucleus carries an isoprenyl group. To address this issue, antibodies against the farnesyl modification were generated to allow direct visualization of the antigen by immunofluorescence microscopy. The anti-farnesyl antibodies specifically stained large HDAg expressed in Huh-7 cells, and the signal was largely restricted to the nucleus; the staining pattern could be superimposed on those of cells stained for large HDAg. The large HDAg translocated into the nucleus was therefore isoprenylated. In addition, antibodies specific for the farnesyl modification should be applicable to the study of other similarly isoprenylated proteins.