EVIDENCE THAT N-LINKED GLYCOSYLATION IS NECESSARY FOR HEPATITIS-B VIRUS SECRETION

Human hepatitis B virus (HBV) envelopes contain three distinct glycopr oteins called L, M, and S HBsAg. Each is posttranstationally modified to contain N-linked oligosaccharides. N-linked oligosaccharides, after attachment to a polypeptide backbone, are processed by enzymes within the endoplasmic reticulum (ER). There is uncertainty about what role, if any, these N glycans and their modification in the ER play in the function of the HBV envelope proteins. By treating hepatoblastoma cult ures which secrete HBV(HepG 2.2.15 cells) with inhibitors of different steps of the glycosylation and glycan modifying pathway, we provide e vidence that glycosylation and the first step in the processing pathwa y are necessary for virion, but not subviral particle, secretion. That is, using a highly sensitive immunoprecipitation/polymerase chain rea ction system, enveloped HBV could not be detected in the medium of Hep G2.2.15 cells incubated with tunicamycin. However, HBV subviral partic le secretion was not prevented by tunicamycin. Moreover, inhibitors of alpha-glucosidase I (the first step in the glycan processing pathway) also prevented virion secretion. Inhibitors of mannose trimming (a la ter step) and glycolipid synthesis, did not prevent virion secretion, defining the limits of the glycosylation requirements in secretion. Th ese results demonstrate a requirement for N-glycosylation and glucosid ase processing in the secretion of virions and further distinguish bet ween the requirements for virion and subviral particle secretion. (C) 1995 Academic Press, Inc.