N-Linked Glycosylation of Hepatitis B Surface Antigens Is Involved but Not Essential in the Assembly of Hepatitis Delta Virus

Abstract

Hepatitis delta virus (HDV) is a defective virus requiring the hepatitis B virus (HBV) to provide hepatitis B surface antigens as the envelope protein. The hepatitis B surface antigens are posttranslationally modified by N-linked glycosylation, and its significance in HDV assembly was investigated with a cotransfection system using human hepatoma cell line Huh-7. After the N-linked glycosylation of HBsAg was blocked by tunicamycin treatment, the packaging of HDV in the culture system could be suppressed to a level as low as 5-10% of the untreated control. The extent of inhibition correlated with the increased concentrations of tunicamycin. In contrast, the loss of HBsAg glycosylation did not affect the efficiency of assembly of HBV particles. When the N-linked glycosylation site of small HBsAg at amino acid 146 was mutated from asparagine to glutamine, the mutant HBsAg packaged only a modest amount of HDV particles. The quantity and kinetics of formation of HDV particles in culture system were reduced by the depletion of HBsAg glycosylation. Therefore HDV, similar to influenza and vesicular stomatitis viruses, depends on glycosylation of the envelope proteins as a signal for envelope protein maturation and for virion formation.

Molecular interactions between hepatitis B virus and delta virus

“…Furthermore, it has been suggested that a non-glycosylated Ag loop faces the cytoplasm, which possibly masks the cytosolic interaction sites with HDV or hinders appropriate connections between HBsAg and HDV (Figure 1) [11] . Due to deferent propagation responses of HBV and HDV to non-glycosylated Ag loop, it is possible that these viruses apply different mechanisms to interact with HBsAg [21] . Likewise, the lateral S-S interactions between S protein carbohydrates, which play a critical role in virion stability, are suggested to occur differently for HBV and HDV due to their particle sizes [12] .…”

“…In more detailed studies it was shown that N-glycosylation of S-HBsAg, which is mediated by the C-terminal domain of S protein and occurs partially on Asn-146, affects HDV envelopment and secretion [12] . Based on these studies, HDV secretion is delayed or reduced (about ten folds) in the presence of nonglycosylated HBsAg, while HBV and HBsAg formation is not affected [21] . Although a weakened interaction between HBsAg and other components of HDV-RNP complex (rather than L-HDAg) has been suggested for this reduction, based on the luminal positioning of Ag loop in the ER membrane, a direct interaction of this domain and HDV components is unlikely [21,27] .…”

“…Based on these studies, HDV secretion is delayed or reduced (about ten folds) in the presence of nonglycosylated HBsAg, while HBV and HBsAg formation is not affected [21] . Although a weakened interaction between HBsAg and other components of HDV-RNP complex (rather than L-HDAg) has been suggested for this reduction, based on the luminal positioning of Ag loop in the ER membrane, a direct interaction of this domain and HDV components is unlikely [21,27] . Different mechanisms have been suggested to explain the effects of the antigenic domain, especially in its nonglycosylated form, on HDV packaging and secretion.…”

“…Therefore, it is very likely that most of the HDAg binding sites are located on the S domain of HBsAg [20] . While an intact HBsAg is not able to interact with L-HDAg, a denatured form of HBsAg is competent for such interactions suggesting that L-HDAg has no connection to the external domains of HBsAg but rather to the domains inside the particles [21] . The cytoplasmic orientation of the CYLs of the S protein, provides a reasonable condition for these sites to interact with pre-mature HDV virions in the cytosol (Figure 1) [22] .…”

“…Different mechanisms have been suggested to explain the effects of the antigenic domain, especially in its nonglycosylated form, on HDV packaging and secretion. One is a modified maturation and trafficking process for non-glycosylated HBsAg, which in turn will affect the rate of interactions with HDV [21] . The association of HBsAg with calnexin (a molecular chaperon in mutations in the HBV genome of patients co-infected with HDV.…”