Abstract
BackgroundThe addition of N-linked glycans to proteins is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. Here, we report on an exception to this rule occurring on the hepatitis B virus (HBV) large L envelope protein that is a subject to co-plus posttranslational N-glycosylation.ResultsBy using an improved detection system, we identified so far unrecognized, novel isoforms of L. Based on mutational analyses, the use of N-glycosylation inhibitors, and pulse-chase studies, we showed that these isoforms are due to posttranslational N-glycan addition to the asparagines 4 and 112 within the preS domain of L. While an inhibition of N-glycosylation and glycan trimming profoundly blocked virus assembly and release, the posttranslational N-glycosylation of L itself was found to be dispensable for HBV morphogenesis.ConclusionThese data together with previous results implicate that the N-glycosylation requirements of virion release are due to functional inhibition of cell glycoproteins engaged by HBV.
Highlights
The addition of N-linked glycans to proteins is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum
We found that mutational inactivation of the L-specific glycosylation sites did not affect virus maturation, whereas inhibitors of N-glycosylation and glycan trimming blocked virus production, indicating that proper N-glycosylation and processing of host cell components are involved in hepatitis B virus (HBV) multiplication
When microsomal fractions of COS-7 cells transiently expressing the wild-type L protein were analyzed by Western blotting with the L-specific antibody MA18/7, L appeared in its characteristic doublet of a 39-kDa nonglycosylated (p39) and a 42-kDa single-glycosylated species due to partial cotranslational N-glycosylation in its S domain as expected (Fig. 2A)
Summary
The addition of N-linked glycans to proteins is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. We report on an exception to this rule occurring on the hepatitis B virus (HBV) large L envelope protein that is a subject to co-plus posttranslational N-glycosylation. N-linked glycans are added to proteins en bloc in the lumen of the endoplasmic reticulum (ER) as presynthesized oligosaccharides. As the OST-complex is associated with the translocon, N-glycosylation normally occurs cotranslationally when the nascent protein chain grows into the ER lumen [1,2]. In the present study we show that the large envelope protein of the hepatitis B virus (HBV) is modified by posttranslational N-glycosylation. The M protein is modified by cotranslational N-glycosylation (page number not for citation purposes)
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