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 Mr 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.
ChangC., EndersG., SprengelR., PetersN., VarmusH. E., GanemD.1987; Expression of the precore region of an avian hepatitis B virus is not required for viral replication. Journal of Virology 61:3322–3325
ChenH.-S., KewM. C., HornbuckleW. E., TennantB. C., CoteP. J., GerinJ. L., PurcellR. H., MillerR. H.1992; The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host. Journal of Virology 66:5682–5684
GalibertF., ChenT. N., MandartE.1982; Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence. Journal of Virology 41:51–65
GarciaP. D., OuJ.-H., RutterW. J., WalterP.1988; Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm. Journal of Cell Biology 106:1093–1104
HantzO., PichoudC., VitvitskiL., TrepoC.1983; Use of the cross-reactivity with hepatitis B virus antigens and antibodies for the demonstration of a woodchuck hepatitis virus ‘e’ antigen-antibody system. Journal of Virological Methods 7:45–55
Jean-JeanO., LevreroM., WillH., PerricaudetM., RossignolJ.-M.1989a; Expression mechanism of the hepatitis B virus (HBV) C gene and biosynthesis of HBeAg antigen. Virology 170:99–106
Jean-JeanO., SalhiS., CarlierD., ElieC., de RecondoA.-M., RossignolJ.-M.1989b; Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site. Journal of Virology 63:5497–5500
MclachlanA., MilichD. R., RaneyA. K., RiggsM. G., HughesJ. L., SorgeJ., ChisariF. V.1987; Expression of hepatitis B virus surface and core antigens: influence of pre-S and precore sequences. Journal of Virology 61:683–692
MilichD. R., JonesJ. E., HughesJ. L., PriceJ., RaneyA. K., MclachlanA.1990; Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero?. Proceedings of the National Academy of Sciences, U.S.A 87:6599–6603
NakabayashiH., TaketaK., MiyanoK., YamaneT., SatoJ.1982; Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Research 42:3858–3863
NassalM., GalleP. R., SchallerH.1989; Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease. Journal of Virology 63:2598–2604
OuJ.-H., LaubO., RutterW. J.1986; Hepatitis B virus gene function: the precore region targets the core-antigen to cellular membranes and causes the secretion of the e antigen. Proceedings of the National Academy of Sciences, U.S.A 83:1578–1582
RoossinckM. J., JameelS., LoukinS. H., SiddiquiA.1986; Expression of hepatitis B viral core region in mammalian cells. Molecular and Cellular Biology 6:1393–1400
SawhneyR. S., HeringT. M., SandellL. J.1991; Biosynthesis of small proteoglycan II (decorin) by chondrocytes and evidence for procore protein. Journal of Biological Chemistry 266:9231–9240
SchlichtH. J.1991; Biosynthesis of the secretory core protein of duck hepatitis B virus: intracellular transport, proteolytic processing, and membrane expression of the precore protein. Journal of Virology 65:3489–3495
SchlichtH. J., SalfeldJ., SchallerH.1987; The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation. Journal of Virology 61:3701–3709
StandringD. N., OuJ.-H., MasiarzF. R., RutterW. J.1988; A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e antigens in Xenopus oocytes. Proceedings of the National Academy of Sciences, U.S.A 85:8405–8409
TakahashiK., MachidaA., FunatsuG., NomuraM., UsudaS., AoyagiS., TachibanaK., MiyamotoH., ImaiM., NakamuraT., MiyakawaY., MayumiM.1983; Immunochemical structure of hepatitis B e antigen in the serum. Journal of Immunology 130:2903–2907
TongS., DiotC., GriponP., LiJ., VitvitskiL., TrepoC., Guguen-GuillouzoC.1991; In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region. Virology 181:733–737
WangJ., LeeA. S., OuJ.-H.1991; Proteolytic conversion of hepatitis B virus e antigen precursor to end product occurs in a postendoplasmic reticulum compartment. Journal of Virology 65:5080–5083