Evidence That N-Linked Glycosylation Is Necessary for Hepatitis B Virus Secretion (original) (raw)

Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: Evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion

Proceedings of the National Academy of Sciences, 1997

The role of N-linked glycosylation and glycan trimming in the function of glycoproteins remains a central question in biology. Hepatitis B virus specifies three glycoproteins (L, M, and S) that are derived from alternate translation of the same ORF. All three glycoproteins contain a common N-glycosylation site in the S domain while M possesses an additional N-glycosylation site at its amino terminus. In the presence of N -butyl-deoxnojirimycin (an inhibitor of α-glucosidase) virions and the M protein are surprisingly retained. Preliminary evidence suggests that the retained M protein is hyperglucosylated and localized to lysosomal vesicles. In contrast, the S and L proteins are secreted, and their glycosylation state is unaffected by the presence of the inhibitor. Site-directed mutagenesis provides evidence that virion secretion requires the glycosylation sequon in the pre-S2 domain of M. This highlights the potential role of the M protein oligosaccharide as a therapeutic target.

Impairment of Hepatitis B Virus Virion Secretion by Single-Amino-Acid Substitutions in the Small Envelope Protein and Rescue by a Novel Glycosylation Site

Journal of Virology, 2010

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 ).

Treatment of hepatitis B virus-infected cells with α-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity

Antiviral Research, 2007

Trimming of the N-glycans attached to the envelope proteins of hepatitis B virus (HBV) is required in different steps of the viral life cycle. Inhibition of the host enzymes ␣-glucosidases, involved in the endoplasmic reticulum (ER)-associated processing of the N-linked glycans, results in misfolding of the HBV envelope proteins, prevention of HBV secretion and accumulation of viral DNA within infected cells. However, the impact of these effects on HBV morphogenesis and infectivity of the viral particles that are still released from cells with inhibited ␣-glucosidase has not been addressed so far. Using N-butyldeoxynojirimycin (NB-DNJ), a competitive inhibitor of the ER ␣-glucosidases, we analyzed the role of these enzymes on HBV assembly and infectivity of the virions released from HepG2.2.2.15 cells. HBV secreted from drug-treated cells contained an envelope with altered composition of the disulfide-linked oligomers and no detectable middle (M) protein. These molecular changes had a significant effect on HBV infectivity, reducing it to 20% compared to controls, for the highest concentrations of NB-DNJ used. Our data show for the first time that an active ␣-glucosidase activity is crucial for production of infectious HBV and provide new insights into the controversial role of the M protein in this process.

Aberrant trafficking of hepatitis B virus glycoproteins in cells in which N-glycan processing is inhibited

Proceedings of the National Academy of Sciences, 1997

The role of N-glycan trimming in glycoprotein fate and function is unclear. We have recently shown that hepatitis B virus (HBV) DNA is not efficiently secreted from cells in which α-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidase-inhibited cells, viral DNA, accompanied by envelope and core proteins, most likely accumulate within lysosomal compartments. Pulse–chase experiments show that although the viral glycoproteins (L, M, and S) are dysfunctional, in the sense that they do not mediate virion egress and are not efficiently secreted from the cell, they all still leave the endoplasmic reticulum (ER). Surprisingly, however, the glycoproteins retained within the cell were not rapidly degraded, appearing as aggregates, enriched for L and M, with intracellular half-lives exceeding 20 h. Moreover, by 24 h after synthesis, a substantial fraction of the detained glycoproteins appeared to return to the ER, although a considerable amount wa...

Hepatitis B Virus MHBs Antigen Is Selectively Sensitive to Glucosidase-Mediated Processing in the Endoplasmic Reticulum

DNA and Cell Biology, 2001

Previous studies have shown that hepatitis B virus (HBV) secretion from HepG 2.2.15 cells is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase under conditions where secretion of cellular glycoproteins are not detectably affected. The 2.2.15 cells are derived from HepG2 and contain intact dimers of the viral genome. They produce and secrete infectious HBV. The secretion of the viral envelope polypeptide, MHBs, was selectively and quantitatively reduced from 2.2.15 cells in which glucosidase was inhibited, whereas the envelope polypeptide, SHBs, was relatively insensitive, being as resistant as were most host glycoproteins. Because 2.2.15 cells express all HBV ORFs, it seemed possible that the sensitivity of MHBs secretion involved its interaction with the viral nucleocapsid or other viral gene products. The work reported here showed that MHBs secretion from HepG2 cells transfected with a plasmid that expresses only the MHBs polypeptide was as sensitive to glucosidase inhibitors as it was from 2.2.15 cells. These data show that the sensitivity of the MHBs polypeptide secretion to glucosidase inhibitors is entirely encrypted within its structural gene. The reasons the MHBs polypeptide, but not SHBs, is so sensitive to glucosidase processing are discussed.

Effect of glycosylation on the conformational epitopes of the glycoprotein of vesicular stomatitis virus (New Jersey serotype)

Virology, 1991

The conformational epitopes reactive with neutralizing monoclonal antibodies (MAbs) appear to be clustered at the middle third of the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) and are flanked by two N-linked carbohydrate chains (W. Keil and R. R. Wagner, Virology 170, 392-407, 1989). We report here studies on the effect of glycosylation on the reactivity of VSV-NJ G protein with MAbs directed to conformational epitopes. When VSV-NJ was grown in the presence of tunicamycin, the unglycosylated G protein derived from released virions or immunoprecipitated from pulse-labeled cells was not significantly affected in its reactivity with MAbs directed to epitope IV mapped toward the amino-terminus, nor to the centrally located conformational epitopes VI, VIII, and IX. However, there was a 5-to 15-fold decrease in the reactivity with MAb of epitopes VI, VIII, and IX on unglycosylated G protein either isolated from a ribosome-enriched membrane fraction or immunoprecipitated from whole VSV-infected cells labeled for 15 hr in the presence of tunicamycin. In sharp contrast, epitope V and to a somewhat lesser extent epitope VII exhibited decreased reactivity with their respective MAbs when unglycosylated G protein was isolated from released viral particles or from pulse-labeled cells infected with VSV-NJ in the presence of tunicamycin. Enzymatic removal of preformed carbohydrate chains with N-glycanase had little or no effect on the MAb-reactivity of epitopes V and VII, indicating that the carbohydrate chains per se do not influence the antigenic specificity of VSV-NJ G protein. These data suggest that the formation of N-linked carbohydrate chains influences the structure of the VSV-NJ G protein in such a way that epitopes V and VII are shielded from reactivity with their specific MAbs from an early stage of G-protein processing and to a much lesser extent epitopes VI, VIII, and IX at late stages of intracellular processing. These results are compatible with, but do not prove, the hypothesis that N-linked glycosylation plays a key role in promoting the formation and the stability of the disulfide bonds that determine the epitope-specific conformational integrity of the VSV-NJ glycoprotein. o ISSI Academic press, IX.

Role of N Glycosylation of Hepatitis B Virus Envelope Proteins in Morphogenesis and Infectivity of Hepatitis Delta Virus

Journal of Virology, 2003

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.

Tunicamycin resistant glycosylation of a coronavirus glycoprotein: Demonstration of a novel type of viral glycoprotein

Virology, 1981

Tunicamycin has different effects on the glycosylation of the two envelope glycoproteins of mouse hepatitis virus (MHV), a coronavirus. Unlike envelope glycoproteins of other viruses, the transmembrane glycoprotein El is glycosylated normally in the presence of tunicamycin. This suggests that glycosylation of El does not involve transfer of core oligosaccharides from dolichol pyrophosphate intermediates to asparagine residues, but may occur by O-linked glycosylation of serine or threonine residues. Synthesis of the peplomeric glycoprotein E2 is not readily detectable in the presence of tunicamycin. Inhibition of N-linked glycosylation of E2 by tunicamycin either prevents synthesis or facilitates degradation of the protein moiety of E2. Radiolabeling with carbohydrate precursors and borate gel electrophoresis of glycopeptides show that different oligosaccharide side chains are attached to El and E2. The two coronavirus envelope glycoproteins thus appear to be glycosylated by different mechanisms. In tunicamycin-treated cells, noninfectious virions lacking peplomers are formed at intracytoplasmic membranes and released from the cells. These virions contain normal amounts of nucleocapsid protein and glycosylated El, but lack E2. Thus the transmembrane glycoprotein El is the only viral glycoprotein required for the formation of the viral envelope or for virus maturation and release. The peplomeric glycoprotein E2 appears to be required for attachment to virus receptors on the plasma membrane. The coronavirus envelope envelope glycoprotein El appears to be a novel type of viral glycoprotein which is post-translationally glycosylated by a tunicamycin-resistant process that yields oligosaccharide side chains different from those of N-linked glycoproteins. These findings suggest that El may be particularly useful as a model for studying the biosynthesis, glycosylation, and intracellular transport of O-linked glycoproteins.

The structural proteins of infectious pancreatic virus are not glycosylated

Journal of virology, 1996

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.