title: Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site.
abstract: Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.
authors.affiliation: Liver Research Center, Rhode Island Hospital, Warren Alpert School of Medicine, Brown University, Providence, Rhode Island 02906, USA.
authors.forename: Kiyoaki
authors.initials: K
authors.lastname: Ito
authors.forename: Yanli
authors.initials: Y
authors.lastname: Qin
authors.forename: Michael
authors.initials: M
authors.lastname: Guarnieri
authors.forename: Tamako
authors.initials: T
authors.lastname: Garcia
authors.forename: Karen
authors.initials: K
authors.lastname: Kwei
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizokami
authors.forename: Jiming
authors.initials: J
authors.lastname: Zhang
authors.forename: Jisu
authors.initials: J
authors.lastname: Li
authors.forename: Jack R
authors.initials: JR
authors.lastname: Wands
authors.forename: Shuping
authors.initials: S
authors.lastname: Tong
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D019943:Amino Acid Substitution; D015153:Blotting, Western; D004261:DNA Replication; D005838:Genotype; D006031:Glycosylation; D006509:Hepatitis B / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006515:Hepatitis B virus; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011498:Protein Precursors / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*; D014771:Virion / Q000502:physiology*; D014779:Virus Replication
chemical_list: D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen; D000469:Alkaline Phosphatase