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

Abstract

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 ␣-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidaseinhibited 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 was also found in the lysosomes. To our knowledge, this is the first report that shows, as a consequence of inhibiting glycosylation processing, certain glycoproteins (i) become dysfunctional and aggregate, yet still depart from the ER, and (ii) have extended rather than shortened half-lives. Taken together, these data suggest that proper intracellular routing of HBV glycoproteins requires ER glucosidase function. It is hypothesized that failure to process N-glycan causes HBV glycoproteins to aggregate and that impaired protein-protein interactions and trafficking are the result of misfolding.

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

“…In the case of the HBV M protein, the most likely effect of NB-DNJ would be that the M protein is severely misfolded. This is supported by the finding of aggregated, hyperglucosylated M in the lysosomes (14). That the M protein is secreted in the presence of tunicamycin and from the Mg Ϫ vector suggest that the degree of misfolding is less in these cases.…”

“…That is, while subviral particles containing mostly the L and S glycoproteins are secreted near normally, as are many host glycoproteins from ␣-glucosidase-inhibited cells (15,19,20), there was a 16-fold reduction the secretion of Mcontaining subviral particles. Preliminary evidence suggest that the subviral particles containing the M protein carry hyperglucosylated glycan structures and are targeted to lysosomal compartments (14). The lack of secretion of Mcontaining subviral particles is thought to be the result of the effect of the ␣-glucosidase inhibitor on the M protein.…”

“…After 1 h the radioactive medium was removed, and the cells were washed with 1ϫ PBS (150 mM NaCl͞10 mM Na 2 HPO 4 ͞1.5 mM K 2 HPO 4 , pH 7.12) and replaced with complete RPMI (10% fetal bovine serum; GIBCO͞BRL) for 24 h. The medium was collected and layered onto a discontinuous sucrose density gradient as described (10). The collected fractions were then analyzed for the presence of S antigen by an antigen capture method as described (14).…”

Inhibition of hepatitis B virus DNA replication by imino sugars without the inhibition of the DNA polymerase: Therapeutic implications

“…[7][8][9][10][11] The ␣-glucosidases (I and II) mediate the first steps in the glycan-processing pathway and allow for downstream glycan-processing events to occur. In addition, the actions of the ␣-glucosidases allow for interactions with the endoplasmic reticulum (ER) chaperones calnexin and calreticulin.…”

“…For example, by disrupting the calnexin-mediated folding of one or more of the HBV glycoproteins, glucosidase inhibitors prevent the formation and secretion of HBV. [7][8][9][10][11]16,17 Abbreviations: HBV, hepatitis B virus; N-butyl-DNJ, N-butyl-deoxynojirimycin; Nnonyl-DNJ, N-nonyl-deoxynojirimycin; N-nonyl-DGJ, N-nonyl-deoxygalactojirimycin; ER, endoplasmic reticulum; MDBK, madin darby bovine kidney cells; HPLC, high performance liquid chromatography; rcDNA, relaxed circular DNA; pgRNA, pregenomic RNA; IC50, concentration required to inhibit 50%.From the …”