3.1. HBV L Resides within Markedly Crescent-Shaped ER-Associated Compartments
Unlike those of HBV subviral envelope particles (SVPs), intracellular trafficking pathways of the viral envelope are poorly understood. Unlike those of HBV spherical subviral envelope particles (SVPs) formed by S alone, intracellular trafficking pathways of the L are poorly understood. To study HBV envelope transport through the cell secretory system, we used a transient replication system by transfecting HuH-7 cells with a replication-competent HBV replicon plasmid and performed IF analysis. For organelle-specific reporters, cells were cotransfected with either pYFP.ER, pYFP.GRASP65 or pDsRed.Golgi that encode autofluorescent marker proteins specific for the ER, cis Golgi or medial/trans Golgi, respectively. Three days post-transfection, cells were fixed with PFA, permeabilized and stained with antibodies against the L envelope protein. To locate the ER/Golgi intermediate compartment (ERGIC), cells were co-stained with antibodies against ERGIC-53, an approved marker of this organelle [42]. As shown in Figure 1A, L reproducibly appeared in a confined, crescent-shaped structure in the perinuclear region, partly emerging with dissections. For short, this structure will be termed CS herein. Worth mentioning, a similar pattern emerged when S domain-specific antibodies were used for staining (data not shown), indicating that the CS structure is specific for the viral envelope rather than for L. As would be expected, a similar pattern emerged when S domain-specific antibodies were used for staining (Figure 1A). To assess the impact of the L protein on the formation of the CS structure, comparative studies were performed with a mutant HBV.Lminus replicon construct that is defective in L protein synthesis [33]. In the absence of L, the S domain-specific antibody rendered a diffuse granular staining pattern of the M and S envelope proteins (Figure 1A), indicating that the CS structure is specific for L. As would be expected for a viral transmembrane envelope protein, the L-specific staining pattern clearly colocalized with the reticular structures labeled by the ER marker YFP.ER (Figure 1B). However, unlike many other viral envelope proteins synthesized within and transported through the secretory system, the L-specific CS structure neither colocalized with ERGIC- nor Golgi-specific areas (Figure 1B).
To investigate whether HBV might traverse post-ER compartments or not, we analyzed the nature of the N-glycans that are added to the asparagine acceptor site N146 of about half of the L molecules during their integration into the ER membrane. During glycoprotein transport through the secretory pathway, Golgi-residing enzymes process glycoproteins and modify N-linked mannose-rich oligosaccharides into complex glycans that are resistant to treatment with Endoglycosidase H (EndoH). By implication, an EndoH sensitivity of glycoproteins indicates the presence of high-mannose glycans and the absence of glycoprotein modification by Golgi-residing enzymes and vice versa. We therefore conducted EndoH digestions of intra- and extracellular HBV particles. As shown in Figure 1C, L is naturally synthesized in nonglycosylated p39 and single-glycosylated gp42 forms due to its partial N-glycosylation. Cell-associated L was entirely sensitive to EndoH, which converted the gp42 form to the p39 form. By contrast, extracellular L resisted EndoH digestion concomitant with an increased molecular weight of its gp42 form by about one kDa as a consequence of N-glycan processing. Accordingly, the viral envelope must have traversed the Golgi apparatus en route to the exterior. To account for the intracellular EndoH sensitivity of L together with its missing colocalization with ERGIC- and Golgi-specific markers, L might be blocked in a pre-ERGIC compartment for much of its time in the cell to match the rate-limiting step in viral particle assembly. Once budded, the viral particles appeared to be rapidly released out of the cell.