The C-terminal sequence of the large hepatitis delta antigen is variable but retains the ability to bind clathrin

BACKGROUND: Hepatitis delta virus (HDV) is a defected RNA virus and requires its encoded large antigen (LDAg) to interact with helper viral proteins (HBsAgs) during assembly. Recently, a study demonstrated a direct binding of the LDAg C-terminus from genotype I HDV to the clathrin heavy chain (CHC), which suggests that this interaction might facilitate HDV assembly. If LDAg binding to clathrin is essential to HDV life cycle, a clathrin box sequence at the C-terminus of LDAg should be conserved across all HDV. However, the C-terminal sequence of LDAg is variable among 43 HDV isolates. RESULTS: Based on the presence and location of clathrin box at the C-terminus of LDAg from 43 isolates of HDV, we classified them into three groups. Group 1 (13 isolates) and 2 (26 isolates) contain a clathrin box located at amino acids 199–203 and 206–210, respectively, as found in genotype I and genotype II. Group 3 (4 isolates) contains no clathrin box as found in genotype III. CHC binding by three different LDAg (genotype I to III) was then tested by in vivo and in vitro experiments. Transfection of plasmids which encode fusion proteins of EGFP and full-length of LDAg from three genotypes into HuH-7 cells, a human heptoma cell line, was performed. GFP-pull down assays showed that a full-length of CHC was co-precipitated by EGFP-LDI, -LDII and -LDIII but not by EGFP. Further in vitro studies showed a full-length or fragment (amino acids 1 to 107) of CHC can be pull-down by 13-amino-acid peptides of LDAg from three genotypes of HDV. CONCLUSION: Both in vivo and in vitro studies showed that CHC can bind to various sequences of LDAg from the three major genotypes of HDV. We therefore suggest that the clathrin-LDAg interaction is essential to the HDV life-cycle and that sequences binding to clathrin are evolutionarily selected, but nonetheless show the diversity across different HDV genotypes.