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).