PMC:7464876 / 26152-28610 JSONTXT

{"project":"2_test","denotations":[{"id":"32806600-2214019-20296210","span":{"begin":1369,"end":1371},"obj":"2214019"},{"id":"32806600-12477846-20296211","span":{"begin":1654,"end":1656},"obj":"12477846"}],"text":"3.2. HBV L Recruits Core/Capsid to the Crescent-Shaped ER-Associated Compartments\nTo gain insights into HBV assembly/budding sites, we comparatively inspected the intracellular distribution of wt and mutant HBV replicon constructs on a single cell level. The mutant replicons were either defective in core synthesis (HBV.Cminus), envelope synthesis (HBV.Envminus), L protein expression (HBV.Lminus), reverse transcription (HBV.Polminus) or in nucleocapsid envelopment (HBV.C.K96A). In HBV.Cminus-expressing HuH-7 cells, L appeared in its typical CS structure, implicating that this feature is an intrinsic capacity of L, irrespective of an ongoing replication (Figure 2A). Upon ablation of the synthesis of all three envelope proteins, core yielded a diffuse staining dispersed throughout the cytoplasm with some nuclear labeling (Figure 2B). An almost identical distribution of core was observed, when only the expression of the L envelope protein was prevented (Figure 2C). In the presence of the viral envelope, the core staining pattern thoroughly changed, as it now accumulated in the L-specific CS structure (Figure 2D). A similar overlap and recruitment of core by L was observed when capsid- rather than core-specific antibodies were used for staining (Figure 2E). Even core/capsids defective in reverse transcription, but competent for pgRNA genome packaging [43], were found to extensively colocalize with the characteristic CS structure of L (Figure 2F). Conversely, when the HBV.Cminus replicon was trans-complemented with a core mutant that had been shown to be competent in nucleocapsid formation but defective in envelopment (HBV.C.K96A) [44], no recruitment of core could be detected (Figure 2G). To corroborate whether the spatial overlaps of the core and L-staining pattern relied on true protein interactions, coimmunoprecipitation (CO-IP) studies were performed. To this aim, cell lysates of transfected cells were subjected to a core-specific IP followed by L-specific WB. Thereby, wt core but not the envelopment-defective core.K96A mutant reproducibly brought down L (Figure 2H). Together, the cell imaging and biochemical data indicate that L on its own accumulated in the confined CS structure towards core/capsid particles were actively recruited, likely via a direct interaction between L and core. Since the interaction-defective core.K96A mutant failed to be attracted by L, the CS structure likely mirrors HBV interaction sites."}