PMC:7464876 / 22098-43693 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7464876","sourcedb":"PMC","sourceid":"7464876","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7464876","text":"3. Results\n\n3.1. HBV L Resides within Markedly Crescent-Shaped ER-Associated Compartments\nUnlike 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).\nTo 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.\n\n3.2. 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.\n\n3.3. HBV Particle Egress Requires the COPII components Sec24A, Sec23B and Sar1\nWe previously showed that HBV spherical SVPs use the cellular COPII machinery for ER export and secretion [29]. Since intracellular trafficking pathways of HBV subviral and viral particles are supposed to differ [2,21,45], a common exploitation of COPII transport carriers is ambiguous. Therefore, we reasoned to investigate the role of COPII factors in the production and release of infectious HBV particles. For RNA interference (RNAi), HuH-7 cells were treated with siRNA duplexes for 48 h prior to transfection with the pHBV* replicon construct. After an additional 72 h, cell lysates and supernatants were harvested. The used siRNAs comprised control duplexes (siCon) and single or siRNA pools targeting Sec24A, Sec24B, Sec23A, Sec23B, Sar1A or Sar1B. Sec24 is the cargo adaptor protein of COPII and associates with Sec23 to form the inner coat layer, while the Sar1 GTPase recruits the Sec23/Sec24 dimer to the ER membrane. To monitor depletion, COPII-specific transcripts were measured by qRT-PCR, which revealed an almost complete knockdown (KD) of each targeted COPII component (Figure 3). Cell lysates were probed for the expression and stability profiles of the viral L envelope and core proteins by specific WBs. As shown in Figure 3, neither knockdown (KD) affected the intracellular steady-state levels of either of the two viral proteins. The formation and release of HBV particles were analyzed by a virion production (VP) assay. For this, intracellular NCs and extracellular virions were immunoprecipitated with capsid- or envelope-specific antibodies, respectively, followed by particle disruption and real-time, multiplex PCR quantification of the number of HBV progeny genomes. Neither RNAi had a substantial effect on the formation of intracellular, replication-competent NCs. However, upon the silencing of Sec24A and Sec23B, the amounts of extracellular virions decreased in a highly significant manner, as compared to siCon-treated cells (Figure 3). Conversely, the loss of the two closely related Sec24B [46] or Sec23A [47] isoforms had no inhibitory effects on virus production and release. This indicates that HBV strictly depends on the specific functions provided by Sec24A and Sec23B in the same manner as HBV SVPs. In case of the KDs of the two Sar1 isoforms, the inhibition of the HBV release was obvious, albeit less pronounced (Figure 3). Since the Sar1A and Sar1B isoforms share 89% sequence identity concomitant with overlapping functions [48], the individual loss of either isoform may be partly compensated by its paralog. Together, these results demonstrate that HBV viral particles, similar to spherical subviral particles, require the COPII machinery for exiting the host cell, involving functions of Sec24A, Sec23B and Sar1. Hence, a COPII-guided export out of the ER appears to be a mandatory step in HBV biology.\n\n3.4. HBV L Colocalizes with ERGIC-53 upon ER Export Block\nGiven that the Sec24A isoform is the essential COPII component linked to the export of both the viral and subviral HBV envelope, we next investigated the fate of the ER-arrested envelopes due to Sec24A KD. To study spherical SVP trafficking, HuH-7 cells were transiently transfected with an expression construct encoding only the S envelope gene (HBV.S) (Figure 4A). Notably, this is an established approach to study exclusively the fate of subviral HBV spheres [29]. Consistent with our previous IF analysis [29] and for recapitulation herein, HBV.S showed a diffuse granular staining pattern typical for viral surface proteins that are synthesized at the ER (Figure 4B). Moreover, it largely colocalized with ERGIC-53-stained structures, implicating that HBV.S is transported out of the ER to the ERGIC in control cells (Figure 4B). Upon Sec24A inactivation, the staining pattern of HBV.S strongly changed, as it now appeared in dot-like structures, reminiscent of ER-arrested aggregates, that no longer coincided with ERGIC-53 signals (Figure 4B). Notably, under these conditions, the distribution of ERGIC-53 also altered. In normal cells, this protein predominantly localizes to the ERGIC compartment, as displayed by the given name ERGIC-53. It is a high mannose-specific lectin that cycles cargoes between the ER and the cis-Golgi through COPII and COPI-dependent pathways (see also Figure 5A) [49,50]. Upon immunostaining, it appeared in juxtanuclear, tubulovesicular clusters in control cells, which shifted to a perinuclear, ER-like structure in Sec24A KD cells (Figure 4B). Hence, the ER export of both HBV.S and ERGIC-53 is apparently impaired in Sec24A-silenced cells.\nIn contrast, the HBV L protein, synthesized in HBV-replicating cells, could not be traced within the ERGIC, as no colocalization between L and ERGIC-53 was detectable in the control cells (Figure 4C). In further difference to spherical SVPs, the naturally occurring structure of L was preserved in Sec24A KD cells without any signs of aggregation. Quite unexpectedly, in this setting, ERGIC-53 intensively colocalized with L, implicating that the lectin is seemingly entrapped by the L-specific CS structure if arrested in the ER. Similar staining experiments were done in cells depleted for the related Sec24B isoform. However, this intervention did not affect the typical juxtanuclear distribution of ERGIC-53 and, consequently, did not result in ERGIC-53/L colocalization (Figure 4C). We infer from these results that ERGIC-53 may be a preferred cargo of Sec24A rather than of Sec24B. If arrested in the ER, ERGIC-53 tightly associated with the HBV L envelope protein either by chance or for action.\n\n3.5. HBV Particle Egress Requires ERGIC-53, while HBV Spherical Subviral Particle Egress Does Not\nTo distinguish between these possibilities, we probed for a functional role of ERGIC-53 in HBV propagation. HuH-7 cells were treated with control RNA duplexes or a single ERGIC-53-specific siRNA prior to transfection with the HBV replicon (pHBV*). The analysis of ERGIC-53-specific transcript and protein levels revealed an almost complete KD without any signs of cell cytotoxicity, as evidenced by anti-tubulin immunoblotting of the same cell lysates (Figure 5B). The loss of ERGIC-53 did not affect the L or core protein expression, nor the N-glycosylation of L (Figure 5B). Importantly, the virus production assay revealed that the silencing of ERGIC-53 significantly reduced the amounts of extracellular virions without altering the intracellular nucleocapsid assembly (Figure 5B). Hence, these results indicate that the lectin plays an essential role in HBV envelopment and transport out of the cell.\nGiven the important role of Sec24A in guiding ERGIC-53 ER export and its involvement in HBV subviral and viral envelope trafficking, we next investigated whether ERGIC-53 may likewise contribute to HBV spherical SVP secretion. For this, we employed an SVP release assay and transiently transfected HuH-7 cells with an expression construct encoding only the S envelope gene (HBV.S) with a C-terminally fused HA-tag and examined the intra- and extracellular HBV.S levels by HA-specific WB. For RNAi, cells were treated with control or ERGIC-53-specific RNA duplexes (72 h) prior to transfection with HBV.S (48 h). To control the accuracy of the SVP release assay, cells were depleted for Sec24A, an intervention known to block SVP secretion [29]. Lysates and supernatants were harvested, and SVPs present in the supernatants were collected by ultracentrifugation through sucrose cushions. Immunoblotting of lysates with anti-ERGIC-53 and anti-Sec24A antibodies demonstrated efficient depletions without any signs of codepletions (Figure 5C). Consistent with our previous results [29], Sec24A silencing strongly inhibited the secretion of HBV.S as compared to the control cells and concomitantly increased the intracellular S.HA levels (Figure 5C). In contrast, the KD of ERGIC-53 neither interfered with the synthesis of the nonglycosylated and glycosylated HBV.S forms nor with their secretion (Figure 5C). Together, these data indicate that the export routes for HBV viral and spherical subviral particles dramatically differ in their requirements for ERGIC-53.\n\n3.6. Blocking N146-linked Glycosylation Inhibits HBV Egress without Affecting Envelope/Core Interactions\nSince ERGIC-53 acts as a high mannose-specific lectin, we next focused on the HBV-specific N-glycans. A number of studies have shown that HBV release requires sugar moieties linked to the N-glycan acceptor N146 of the envelope proteins [11,12,13,14,15,16]. To ascertain the importance of the N146-linked glycans in our experimental settings, we disabled N-glycosylation by creating an asparagine-to-aspartic acid substitution in the HBV envelope expression vector (Env.N146D). This vector was then used for trans-complementation of the envelope-defective replicon HBV.Envminus, followed by WB and VP assays. Consistent with the literature [11,12,13,14,15], the Env.N146D mutant was exclusively synthesized in the nonglycosylated p39 form and, importantly, inhibited the HBV virion release (Figure 6A). Previous studies have attributed the virion release defect of Env.N146D to possible structural alterations of L that might prevent its interaction with the viral capsid [13]. To address this point, we performed CO-IP and CO-IF experiments of HBV-replicating cells expressing either the wt Env or mutant Env.N146D proteins. Neither assay rendered any evidence for a disturbed L/core interaction, as core efficiently coprecipitated Env.N146D (Figure 6B) and thoroughly colocalized with L.N146D in the characteristic CS structure (Figure 6C). We thus infer that the N146-linked N-glycan plays a pivotal role in HBV egress apart from envelope/core interactions.\n\n3.7. HBV L Interacts with ERGIC-53 in a N146-Glycan-Dependent Manner\nAccordingly, we next investigated whether there might be physical links between the viral N-glycan and cellular ERGIC-53. As above, HuH-7 cells were depleted for Sec24A in order to arrest ERGIC-53 within the ER, following a retransfection with the wt replicon construct or the envelope-defective HBV.Envminus replicon trans-complemented with Env.N146D. In control cells, neither the wt L nor the L.N146D mutant exhibited any colocalization with the tubulovesicular clusters stained with anti-ERGIC-53 (Figure 7). Upon depletion of Sec24A, the ERGIC-53-specific labeling pattern changed and again appeared in ER-like reticular areas. Importantly, this pattern strongly coincided with the CS structure of wt L (Figure 7A) but poorly with the CS structure of L.N146D (Figure 7B). To corroborate these findings, CO-IP analyses were performed. Since ERGIC-53/cargo interactions are known to be transient and short-lived [42,50], we here used the ectopic expression of a Myc-tagged ERGIC-53 construct together with the wt or mutant HBV replicons. As shown in Figure 7C, wt L, but not L.N146D, efficiently coprecipitated ERGIC-53. Together, these results point to a productive interaction between L and ERGIC-53 that is primary mediated by the N146-linked glycan.\n\n3.8. HBV Envelopment of Nucleocapsids Occurs prior to the Actions of ERGIC-53 and Sec24A\nTo narrow down the roles of ERGIC-53 and Sec24A in the HBV´s life cycle, colocalization studies were performed in RNAi-treated HBV-replicating cells. Aside, cells were treated with BFA, an established fungal compound that inhibits protein trafficking and secretion in mammalian cells via the collapsing of ER exit sites and, hence, of ER export [51]. The double-staining with anti-L and anti-core antibodies showed that neither the loss of ERGIC-53 nor of Sec24A hindered the recruitment of core to the typical CS structure of L (Figure 8A), indicating that both host factors are dispensable for L/core contact formation. Similar results were obtained in BFA-treated cells (Figure 8A), implicating that core is recruited by L prior to ER exit. To test whether ERGIC-53 and Sec24A might play a role in HBV assembly/budding or trafficking, we modified the VP assay in such that we screened for virions in cell extracts. In order to prevent the destruction of the viral envelope during lysis, cells were mechanistically disrupted. Extracts were next subjected to an L-specific IP in the absence of detergents, followed by particle disruption and PCR quantification of the number of HBV progeny genomes (Figure 8B). To assure the IP efficiency under these conditions, extracts precipitated in the same manner were analyzed by L-specific immunoblotting, which demonstrated effective and comparable precipitations of L (Figure 8C). Noteworthy, the pull-downed L pool may comprise both viral particles and subviral filamentous SVPs. Even so, filaments lack nucleocapsids and viral genomes and are not detectable by PCR analysis. In respect thereof, the PCR data, shown in Figure 8B, demonstrated that the depletion of either ERGIC-53 or Sec24A led to a significant increase of intracellular virions as compared to the control cells. This indicates a blockage of viral transport more than viral assembly.\n\n3.9. HBV Recruits the ESCRT-specific Vps4 ATPase to the Crescent-Shaped ER-Associated Compartments\nSeveral studies have shown that HBV budding and egress require functions of the ESCRT complex, a membrane scission machinery operating at several cellular membranes [20,21,22,23,24]. To examine whether the ER-related, core-recruiting CS structure of L might have a link to ESCRT, we inspected the intracellular distribution of the Vps4 ATPase, the terminal ESCRT subunit [25], in HBV-replicating and control HuH-7 cells. To this aim, cells were transfected with an EGFP-tagged version of Vps4A together with either empty plasmid DNA or the HBV replicon and processed for IF analysis. In control cells, Vps4A was distributed throughout the cytoplasm, along with some nuclear localization (Figure 9A). Conversely, in HBV-positive cells, the distribution of Vps4A changed in such that it now additionally appeared in the CS structure reminiscent for HBV (Figure 9A). The apparent recruitment of Vps4A to the L/core complex suggested an ESCRT intervention occurring at the ER-related CS structure. To further investigate the spatial and temporal interconnections between HBV and the ESCRT cascade, we reasoned to study the N-glycan pattern of L in order to learn whether the HBV budding driving activity of ESCRT occurred before or after its acquisition of EndoH resistance. For ESCRT inactivation in HBV-replicating HuH-7 cells, we silenced the expression of EAP20, one subunit of the heterotetrameric ESCRT-II complex. Consistent with our previous report [22], this led to a substantial decline of extracellular virion amounts (Figure 9B). In parallel, cellular lysates were mock-treated or digested with EndoH and assayed by L-specific WB. Upon ESCRT-II inactivation, cell-associated L displayed the same EndoH sensitivity as observed in siCon-treated cells (Figure 9B), implicating that EAP20 is acting prior to L arrival at the cis/medial Golgi 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