The Large Surface Protein of Hepatitis B Virus Is Retained in the Yeast Endoplasmic Reticulum and Provokes Its Unique Enlargement

Abstract

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.

Cloning, Expression, and Purification of Histidine-Tagged preS Domains of Hepatitis B Virus

“…However, the detailed study of all these properties has been complicated due to the difficulty in isolating these proteins. Both the L and M proteins are present in small amounts in HBV (23), and the recombinant L particles are poorly expressed in eucaryotic cells because the protein is retained by the preS1 region in the cell endoplasmic reticulum (34). An alternative approach is the synthesis of the preS domains in the absence of the S polypeptide since half of the preS sequences have been shown to be exposed in the virions (7,8) and there are several indications suggesting that the preS exists as an independent domain (or domains) retaining its native structure and functions in the absence of the S protein (24).…”

Hepatocyte-Specific Binding of L/S-HBV Particles Expressed in Insect Cells

“…To generate an expression vector for the small surface antigen (pVL-N-941 -S), a PCR fragment of HBV ayw genome (bp 157-834) was inserted into a derivative of pVL-941 (Wippler et a/., 1994) under control of the polyhedrin promoter. Since the large surface antigen is known to be retained in the endoplasmatic reticulum (ER) in yeast (Biemans et al, 1991) and insect cells (Lanford et al, 1989), we constructed several variants of the L gene. In the vector pRS-L-Ala2 a known myristylation site (Prange et al, 1991;Towler et al, 1988) was removed by replacing the glycine2 by an alanine.…”

Enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinantSaccharomyces cerevisiae at high cell concentration

“…Evidently, the 24 kDa protein is the core N-glycosylated IL-1␤ ⅐ rhG-CSF and hence is located in the ER. The retention of secretory proteins in the ER has been also discussed in detail in the secretion of both native and foreign proteins (Lodish et al, 1983;Shuster, 1991;Biemans et al, 1991). The effect of key genetic/cultivation factors related to the transit from the ER to the Golgi is still under investigation.…”