The C-terminal sequence of the large hepatitis delta antigen is variable but retains the ability to bind clathrin
© Wang et al; licensee BioMed Central Ltd. 2009
Received: 19 February 2009
Accepted: 16 March 2009
Published: 16 March 2009
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.
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.
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.
Hepatitis delta virus (HDV) is a small defective RNA virus with a negative-stranded genome. It requires for a helper virus, hepatitis B virus (HBV), to supply envelope proteins (HBsAgs) to complete virion assembly and secretion [1–3]. The HDV genome is about 1,700 nucleotides long and is circular in form; it appears to form an unbranched rod-like structure due to a high degree of intra-molecular complementary base-pairing [4, 5]. The genome sequence of HDV is divided into a viroid-like sequence and a protein-coding sequence [1, 6]. It has been hypothesized that HDV resulted from RNA recombination between a viroid sequence and a cellular mRNA coding a DIPA (delta interacting protein) protein [6, 7]. Analysis of HDV sequences from across world has revealed that those from Africa have the highest diversity, which suggests that the first HDV might have arisen in Africa [8, 9]. After the additional isolation of new HDV sequences from Africa, the classification of HDV has been changed from one including genotypes I to III into one involving clades 1 to 8 .
In addition to the isoprenylation signal sequence, a nuclear exporting signal (NES) has also been identified at the C-terminus of LDAg . Within the common 195-amino-acid sequence of SDAg and LDAg, two putative leucine-zipper motifs, an RNA binding motif, and two nuclear localization signals have been identified [21–24]. Both SDAg and LDAg are phosphoprotiens with a different degree of modification . After they have been either phosphorylated by PKC , CKII [18, 26], PKR  or ERK1/2 , they affect HDV replication or are targeted to SC-35 speckles [18, 26–28]. Both acetylation of HDAg at lysine-72 and methylation of SDAg at arginine-13 have also been demonstrated to influence HDV replication [29–31]. Conservation of these post-translational sites of HDAg among all known HDV genotypes suggests that the cellular enzymes responsible for the post-translational modifications of HDAg are at least partial if not all involved in HDV replication.
Co-infection and super-infection of HDV with HBV usually cause more severe liver disease than an HBV single infection . The various HDV genotypes show different geographical distributions and are associated with different disease patterns [7, 33]. HDV genotype I is distributed world-wide and has been linked to a wide spectrum of diseases, ranging from fulminant hepatitis to asymptomatic chronic liver disease. Genotype II is found mainly in Asia, including Japan, Taiwan and Siberia, and seems to give rise to a less severe disease than genotype I. Genotype III is mainly found in the north part of South America and produces a severe form of fulminant hepatitis . The mechanism of HDV pathogenesis would seem to result from complicated interactions between HDV, HBV, and/or host factors and is not completely understood.
Two recent studies have indicated that LDAg rather than SDAg might play a significant role in HDV pathogenesis [34, 35]. One study demonstrated a direct binding of LDAg to Smad-3, which modulates TGF-β signaling to activate plasminogen activator inhibitor-1 expression and c-Jun-induced signal cascades; this would seem to lead to liver cirrhosis . The other study demonstrated that the cytoplasmic form of LDAg binds to the clathrin heavy chain (CHC) and further suggested that this LDAg-CHC interaction is required for HDV assembly. Furthermore, the LDAg-CHC interaction would seem to interfere with the clathrin-mediated endocytosis and exocytosis, which might finally lead to the hepatocytes damage . However, the clathrin-box (199LFPAD203) identified in the LDAg of genotype I is not conserved in the same position in genotype II and III (Fig. 1A). This study was carried out with the aim of verifying whether the clathrin-binding activity is conserved across the three major genotypes of LDAgs.
Three groups of the C-terminus of LDAg from 43 HDV isolates based on the presence and location of clathrin box
Extra a.a. residues
In this study, we have demonstrated that binding to CHC by the C-terminus of LDAg is conserved across all three major genotypes of HDV, although their amino acid sequences (198LLFPADPPFSPQS210 in genotype I, 198GPSPPQQRLPLLE210 in genotype II, and 198FTPPPPGYYWVPG210 in genotype III) are highly variable in this region (Fig. 1A). Based on the variable sequences, the 43 HDV isolates analyzed can be classified into three groups (Table 1) and the three genotypes (I to III), described earlier in this study, each are represented by one of these groups.
It is reasonable to speculate that the CHC binding by the 13-amino-acid peptide of genotype II LDAg is through a putative clathrin box (LPLLE) that is located between amino acids 206 to 210. It has the highest binding capability to CHC among the three genotypes and this might be a result of a clathrin box at the most proximal end to the C-terminus. Whether such strong binding to CHC reduces genotype II LDAg's interaction with HBsAg and results in poorer packaging by HBsAg as found by Hsu et al.  is not determinable from these results. How the peptide of genotype III LDAg, lacking of a clathrin box, binds to CHC is also unclear. There are two possibilities. Firstly, the proline-rich stretch present in genotype III LDAg, which is similar to that of synaptojanin, might serve as a clathrin binding sequence . Alternatively, cellular factors, such as AP-2, might form a complex with genotype III LDAg, through the 205YYWV208 or 206YWVP209 motif , and CHC. The results show a weaker binding affinity to the CHC(1–107) fragment produced from E. coli than to the full-length of CHC from the HuH-7 cell lysate by the peptide of genotype III LDAg (Fig. 4B vs. Fig. 3B). This suggests that firstly, cell factors are possibly involved in the genotype III LDAg binding to CHC and/or secondly, the fragment of CHC might have a different conformation from the full-length CHC. This change conformation might be less favorable to genotype III LDAg peptide binding. Based on the above results, we believe a further investigation to elucidate the hypotheses is warranted.
It has been demonstrated that the LDAg is required for interacting with HBsAgs to form mature virions or empty particles containing LDAg [16, 42]. Questions on where and how HDV virions and empty particles are produced and mature remain unanswered. It is possible that the interaction between clathrin and the C-terminus of LDAg may assist HDV budding into the lumen of various intracellular membranes, such as ER or Golgi apparatus. Thus the LDAg may act as a late (L) domain similar to the situation during budding of many retroviruses [36, 37]. There are three known classes of L domains for retroviruses, namely, PTAP, PPXY, and YXXL. The 202PPGY205 sequence is present in genotype III LDAg, but is not present in genotype I and II, and this fits well into the second class of L domain. Whether this sequence functions as an L domain for genotype III HDV budding remains to be tested.
The fact that clathrin binding by LDAg is conserved across all genotypes must reflect its biological importance to the HDV life-cycle. We therefore postulate that any sequence at the C-terminus of LDAg that is able to bind to clathrin will be selected during evolution and this explains the great diversity across the various genotypes. Whether LDAg binding to clathrin, which may impair the normal functioning of clathrin, is one of causes of HDV pathogenesis as suggested by Huang et al.  remains to be elucidated, although many clathrin-associated diseases have been reported [43, 44].
Plasmids used in this study
The cDNAs coding LDAg from different HDV genotypes, American strain of genotype I (accession number M28267), Taiwan-3 strain of genotype II (accession number U19598), and Peru-1 strain of genotype III (accession number L22063), were used in this study and have been described previously [33, 45, 46]. In this study, plasmids were divided into two groups: one for transfection into human hepatoma cells, HuH-7 , and the other for transformation into E. coli for protein expression. For transfection studies, the three genotypes of the LDAg coding sequence were constructed into pEGFP-C3 using Bgl II and Eco RI cloning sites as described previously . The plasmids for protein expression were constructed by the insertion of synthetic oligonucleotides (Fig. 1B) downstream of GST in pGEX-4T1 using Eco RI and Sal I sites; this gave pGST-LDIC(198–210), pGST-LDIIC(198–210), and pGST-LDIIIC(198–210), respectively. These clones were first selected checking for the presence of the Eco RV site and then verified by nucleotide sequencing. The cDNA fragment of human clathrin heavy chain containing amino acid 1 to 107 was amplified by RT-PCR and then inserted downstream of hexahistidine-tag sequence of pET-15b to give p6XHis-CHC(1–107); which was used to express 6XHis-CHC(1–107) protein in E. coli. The plasmid was verified by nucleotide sequencing.
Antibodies and affinity beads
The antibodies used in this study were purchased from different commercial companies. Anti-GFP was obtained from Clontech (California, USA) and Chemicon (California, USA). Anti-GST and anti-6XHis were bought from LTK BioLaboratories (Taoyuan, Taiwan). Anti-clathrin heavy chain was purchased from BD (California, USA). Anti-actin was obtained from Novus Biologicals (Colorado, USA). The secondary antibody conjugated with horseradish peroxidase was bought from Chemicon. Glutathione beads and nickel beads were purchased from GE (New Jersey, USA) and Qiagen (California, USA), respectively.
Cell culture and plasmid transfection
HuH-7 is a fully-differentiated human hepatoma cell line  and was cultured at 37°C under 5% CO2 using Dulbecco's modified Eagle's medium supplement with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and 1% non-essential amino acid. Cells at 60% confluence in a 10 cm Petri dish were transfected with 10 μg of plasmid by the calcium phosphate/DNA precipitation method or by adding lipofectamin 2000 (Invitrogen; California, USA). The transfection rate of each experiment was determined by the GFP expression under a fluorescence microscope.
Protein expression in E. coli and purification
E. coli strain BL21 (DE3) was used to express GST and GST fusion proteins as well as 6XHis-CHC(1–107). Bacteria were grown in LB medium supplement with 100 μg/ml ampicillin while vigorously shaken at 37°C until an O.D. 600 nm of 0.6 was reached. Production of protein was induced by the addition of 1 mM IPTG to the medium for 1 to 3 h. The resulting fusion proteins were further purified by one step binding to either glutathione affinity beads or nickel beads as appropriate and then analyzed by SDS-PAGE and Western blotting.
Immunoprecipitation, GST-protein pull down and Westernblotting
At 24 h post-transfection with pEGFP-C3, pEGFP-LDI, pEGFP-LDII or pEGFP-LDIII into HuH-7 cells, the cells were treated with TNF-α (30 ng/ml) for 2 h . Cells were lysed and immunoprecipitatd by anti-GFP. The precipitated proteins were fractionated by SDS-PAGE and electrotransferred onto PVDF membranes. The membrane was then incubated with anti-clathrin, anti-GFP, and anti-actin antibodies individually. After incubation with the secondary antibody conjugated with horseradish peroxidase, the blots were developed by enhanced chemiluminescence using a commercial kit (Pierce; Illinois, USA). The intensity of the protein bands was quantified by the program Image J software (NIH, Maryland, USA). In order to compare the pull-down efficiency of the different genotype LDAgs, the amount of CHC was normalization against actin and the amount pulled down by genotype I EGFP-LD was designated as 1. Three independent results were averaged and compared. Glutathione Sepharose 4B beads containing various GST fusion proteins were incubated with HuH-7 cell lysates or E. coli produced 6XHis-CHC(1–107) for 16 h at 4°C. After three washes with 0.05% Tween-20 in PBS (phosphate buffered saline), the proteins bound to the beads were analyzed and quantified by Western blot and Image J software as described above.
hepatitis B virus
surface antigen of HBV
hepatitis delta virus
hepatitis delta antigen
large delta antigen
small delta antigen
clathrin heavy chain
enhanced green fluorescence protein
We thank Dr. Jim-Tong Horng (Chang Gung University) for helpful suggestions on the study and Dr. Ralph Kirby (National Yang Ming University) for English editing of the manuscript. This work was supported by grants from NHRI (EX9521BI, EX9621BI, and EX9721BI) to S.J.L.
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