B. Background on HDV Antigens:HDV encodes two proteins of its own: a large and small hepatitis delta antigen (HdAg-S and HdAg-L) on a single coding sequence. The small antigen is expressed earlier in infection and mediates replication. The HdAg-L is expressed later in infection, and is involved in mediating assembly by interacting with Hepatitis B antigen and in inhibiting viral replication. HdAg-L can also form "empty" virus like particles (VLPs) without HDV RNA or HBAg. HDAg-L is expressed when cellular processes (cellular enzyme ADAR1) mutate the UAG stop codon on HDAg-S to a UGG tryptophan codon, adding a 19 amino acid carboxyl terminal extension which includes a CXXQ farnesylation segment which is involved in assembly and inhibition. While the functions of this added segment in the HDAg-L are known, the mechanisms by which they work have not been fully discovered.

O'Malley, B. and Lazinski, D. 2005. Roles of Carboxyl-Terminal and Farnesylated Residues in the Functions of the Large Hepatitis Delta Antigen. J. Virology. 79(2):1142-1153.Pubmed

The nature of the structure of the HdAg-L contributes to its main functions. Genotypes of HdAg are typically classified into three categories, 1, 2, and 3. The three genotypes have shown differences in disease phenotype as well. By examining the efficiency of different genotypes of HdAg-L of forming virus like particles (VLPs), O<92>Malley and Lazinski examined how the amino acid sequence particularly of the unique portion of the HDAg-L contributes to its particular functions, assembly and inhibition of replication. From this they drew several conclusions:

• certain specific positions in the HDAg-L carboxyl terminus interact with the HBAg
• the presence of HDAg-S can stimulate farnesylation of HDAg-L (and in nature, HDAg-L is never expressed without HDAg-S), perhaps by stabilizing HDAg-L in its optimal conformation
• inhibition of HDV replication derives from association with membranes
• myristoylation of the amino terminus (amino acids 1 through 195 that is part of the HDAg-S as well) will allow association with membranes (and thus inhibition of replication) and will allow HDAg-S to interact with HBAg and form VLPs
• however, farnesylation at the carboxyl terminus allows HDAg-L to perform these functions (membrane association and thus inhibition and association with HBAg to form VLPs) far more efficiently (roughly ten-fold more efficient)
• association with HBAg is brought about both by association with membranes and by induction of conformational change in the region that interacts with HBAg
• There is no direct association between efficiency of assembly and disease virulence (as previously thought)

Wang YH, Chang SC, Huang C, Li YP, Lee CH, Chang MF. 2005. Novel nuclear export signal-interacting protein, NESI, critical for the assembly of hepatitis delta virus. J of Virology. 79(13):8113-20.

Previous studies by Wang et al demonstrated that a Nuclear Export Signal (NES) on HDAg-L is involved in HDV assembly. Wang et al further explored the potential proteins interacting with the NES(HDAg-L) site. A product of an mRNA found mainly in liver tissue was discovered to interact with the site, and was named NESI (NES(HDAg-L)-interacting protein). NESI is 469 amino acids long and includes an actin binding site and a nuclear localization signal. It interacts with HDAg-L and is involved in HDV assembly.