Clathrin is involved in the endocytosis and exocytosis of cellular proteins and 
the process of virus infection. We have previously demonstrated that large 
hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the 
detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis 
delta virus (HDV) are not clear. In this study, we found that clathrin heavy 
chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a 
single amino acid substitution at the clathrin box retained nuclear export 
activity but failed to interact with CHC and to assemble into virus-like 
particles. Downregulation of CHC function by a dominant-negative mutant or by 
short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion 
of hepatitis B virus subviral particles. In addition, the coexistence of a 
cell-permeable peptide derived from the C terminus of HDAg-L significantly 
interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and 
CHC were found to colocalize with the trans-Golgi network and were highly 
enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which 
assembles less efficiently than genotype I HDV does, has a putative clathrin box 
in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of 
the various HDV genotypes correlates well with the CHC-binding activity of their 
HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin 
box and the nuclear export signal at the C terminus of HDAg-L are potential new 
molecular targets for HDV therapy.