The hepatitis B virus (HBV) e antigen (HBeAg) is a 15 kDa soluble antigen derived from a precursor protein (precore protein) by two processing events, cleavage of the N-terminal signal peptide and cleavage of the Cterminal 34 amino acids. So far, the role of the Cterminal sequences in secretion has not been analysed in full. In this study deletion of the last 60 amino acids was found to abrogate HBeAg secretion whereas deletions of the last 10, 25 or 39 amino acids decreased its secretion rate. These data demonstrate that C-terminal precore protein sequences are crucial for HBe secretion and determine its secretion rate.
In hepatitis B virus (HBV) the precore gene encodes a protein from which derives P22, the precursor of the mature secreted hepatitis B virus e antigen (HBeAg). Circumstantial evidences suggest that HBeAg and/or its precursor P22 are important for establishing persistent infection. Although P22 is essentially present in the secretory pathway, a substantial fraction has been found in the cytosol. In order to get new insights into the biological function of P22, we looked for cellular proteins which could strongly associate with this protein. Using immunoprecipitation studies on human cell extracts, we found that a non-secreted cellular protein of about 32 kDa (P32) bound with a high specificity to P22. P32 associated neither with HBeAg nor with the viral core protein P21 which exhibits the same amino acids sequence as P22 but is N-terminally shorter by 10 residues. We also demonstrated that this interaction depended on the presence of the P22 C-terminal domain. Our data argues for a potential biological function of P22.
The biosynthesis of the secretory core gene product of the woodchuck hepatitis virus (WHV) was studied in human cells. We have shown that the WHV e antigen was a N-glycosylated (most likely a diglycosylated) protein, with an apparent M r of 24K. To demonstrate that the WHV precore protein was correctly processed in human cells, we engineered chimeric proteins in which signal peptides or arginine-rich domains of WHV and hepatitis B virus (HBV) precore proteins were exchanged. Our results showed that both the signal peptide and the arginine-rich region of WHV precore protein were cleaved off during the secretion pathway, as previously reported for precore protein of human HBV and duck HBV. These observations demonstrate that the maturation process of the e antigen is conserved in hepadnaviruses. In addition, on the basis of inhibition experiments, we suggest that the cleavage of the carboxy terminus of the WHV precore protein occurred in a post-endoplasmic reticulum compartment, most likely beyond the medial Golgi, and that this cleavage was catalysed by an aspartyl protease.
The Hepatitis B virus encodes the secreted e antigen (HBe) whose function in the viral life cycle is unknown. HBe derives from a 25-kDa precursor that is directed to the secretory pathway. After cleavage of the signal sequence, the resulting 22-kDa protein (P22) is processed in a post-endoplasmic reticulum compartment to mature HBe by removal of the 34-amino acid C-terminal domain. The efficiency of HBe secretion is specifically decreased in cells grown in the presence of tunicamycin, an inhibitor of N-glycosylation. Inasmuch as HBe precursor is not N-glycosylated, our data suggest that a cellular tunicamycin-sensitive protein increases the intracellular transport through the HBe secretory pathway. The study of the secretion of HBe derived from C-terminal-truncated precursors demonstrates that the tunicamycin-sensitive secretion absolutely requires a part of the C-terminal region that is removed to form mature HBe, indicating that the cellular tunicamycinsensitive protein increases the efficiency of the intracellular transport of P22. We have also shown that the Escherichia coli -galactosidase can be secreted when fused to the HBe precursor signal sequence and that the P22 C-terminal domain renders the secretion of this reporter protein also tunicamycin-sensitive.
The C gene products of all mammalian hepadnaviruses contain a region with sequence similarities to the catalytic center of the aspartyl proteases. This region could have the capacity to cleave precore proteins, leading to the synthesis of e antigen. By site-directed mutagenesis on a plasmid containing the hepatitis B virus C gene, we have replaced either the Asp residue of the putative aspartyl protease catalytic center or an Asp residue located 3 amino acids upstream. Transient expression of the mutated hepatitis B virus C gene in human and mouse cells showed that none of these mutations prevented the secretion of an accurately processed HBe antigen. Thus, we demonstrated that the aspartyl protease responsible for e antigen precursor processing is not C gene encoded but is more likely to be a cellular enzyme. From these results, we suggest a model for the mechanism of e antigen synthesis.
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