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Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy

['Jiacheng Lin', 'Key Laboratory Of Gastrointestinal Cancer', 'Fujian Medical University', 'Ministry Of Education', 'Fuzhou', 'Fujian Key Laboratory Of Tumor Microbiology', 'Department Of Medical Microbiology', 'Limin Yin', 'Xia-Zhen Xu', 'He-Chen Sun']

Date: 2022-02 

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients.

Hepatitis B virus (HBV) infects more than 250 million people worldwide chronically. It is a major pathogen causing liver cirrhosis and hepatocellular carcinoma now. The HBV capsid protein (HBc) plays multiple roles in the viral life cycle, and many antivirals targeting HBc such as Heteroaryldihydropyrimidine compounds (HAPs) are under clinical trial recently. This study aimed to investigate how a HAP compound Bay41-4109 induces the degradation of HBc protein. Bay41-4109 induces aberrant non-capsid polymers, which form in complex with the chaperone-binding E3 ubiquitin ligase protein STUB1 and co-chaperone BAG3 and are transported to the perinuclear compartment. Subsequently, Bay41-4109-induced aberrant non-capsid polymers are removed by p62-mediated macroautophagy and lysosomal degradation. STUB1 overexpression accelerates Bay41-4109-induced degradation of HBc protein, and thus enhances the effect of Bay41-4109 on inhibiting secretion of HBeAg and HBV virions. When Bay41-4109 are enforced during HBV infection, de novo cccDNA formation were also negatively regulated by STUB1 overexpression. Altogether, this study provides novel mechanistic insights into developing more potent and safe HAP-based antiviral treatment.

Funding: The study was supported by the National Natural Science Foundation of China [grant number 81501755], the Excellent Young Talent Program of the Health and Planning Commission of Fujian Province [grant number 2018-ZQN-58], the Joint Funds for the Innovation of Science and Technology in Fujian Province [grant number 2016Y9039], and the Natural Science Foundation of Fujian Province [grant number 2020J01603]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Lin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In the present study, we found that the chaperone-binding E3 ligase STIP1 Homology And U Box-Containing Protein 1 (STUB1) drives Bay41-4109-induced HBc degradation via the macroautophagy-lysosome pathway and that elevation of the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of both HBeAg and HBV virions.

The HBV core protein (HBc) is the structural component of the nucleocapsid packaging of the viral genome and DNA polymerase [ 4 ]. HBc is viewed as a promising target for antiviral drug development because it participates in multiple steps of the viral life cycle, including RNA packaging, DNA synthesis, viral maturation, and recognition of viral envelope proteins [ 5 ]. Drugs known as capsid protein allosteric modulators (CpAMs) have been developed to target HBc. CpAMs can be classified into two types according to their mode of action [ 6 ]. Type II CpAMs, represented by sulfamoylbenzamides (SBAs) such as SBA_R01, prevent the assembly of pgRNA-containing nucleocapsids and thereby lead to the production of defective progeny virions with empty capsids [ 7 – 9 ]. Type I CpAMs [ 10 – 13 ], which are represented by heteroaryldihydropyrimidine (HAP) compounds such as Bay41-4109, can mislead HBc into assembling into “aberrant non-capsid polymers”; some type I CpAMs disrupt assembled nucleocapsids by binding to pre-assembled HBc or assembled capsids at the pocket of the dimer–dimer interface, thereby triggering HBc degradation [ 14 – 18 ]. Given that HAP may also induce aberrant polymers of intracellular HBe, treatment with HAPs disrupts the capacity for HBeAg production [ 17 , 19 ]. Although several HAPs are currently undergoing clinical trials, the molecular basis for the degradation of HAP-induced aberrant non-capsid polymers remains unclear.

Chronic hepatitis B virus (HBV) infection results in a significantly elevated risk of developing liver cirrhosis and hepatocellular carcinoma [ 1 ]. Currently, the approved therapeutic regimens for HBV include pegylated interferon alpha (IFNα) and nucleos(t)ide analogues (NUCs). IFNα induces the non-cytotoxic intracellular suppression of HBV replication; however, the side effects of IFNα limit its extensive use [ 1 , 2 ]. While NUCs can efficiently suppress HBV DNA titers, they cannot eradicate the virus owing to the persistence of covalently closed circular DNA (cccDNA) that remain in the nuclei of hepatocytes [ 3 ]. Thus, new antiviral drugs would be welcomed by the investigators in the field seeking to treat and manage HBV.

Results

STUB1 mediates Bay41-4109-induced HBc degradation HBc is one of the known client proteins of heat shock protein 70 (Hsp70) [20,21]. Some clients of Hsp70 can be degraded by chaperone-binding E3 ubiquitin ligases such as Cullin 5, MDM2, STUB1 and PRKN [22–25]. We hypothesized that one or more of these chaperone-binding ubiquitin E3 ligases may promote HAP-induced HBc degradation. In pursuit of this, we transfected HepAD38 cells [26] (which have a genome containing the 1.1mer HBV genome under the transcriptional control of a tetracycline-responsive promoter (tet-off)) with plasmids for the expression of CUL5, MDM2, STUB1, or PRKN. The overexpression of STUB1, but not the other chaperone-binding E3 ligase, significantly decreased the HBc level in the Bay41-4109-treated cells (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. STUB1 promotes the Bay41-4109-induced degradation of HBc. (A, B) HepAD38 cells were transfected with expression vectors coding for E3 ligases including CUL5, MDM2, STUB1, PRKN, or empty vector (A) or two different siRNAs targeting STUB1 or mock siRNA (B). At 36 hours after transfection, cells were incubated in medium with 1 μM Bay41-4109 or DMSO for 2 d. Cell extracts were then analyzed by western blotting using the indicated antibodies (left panel). The quantification results of three independent immunoblots are shown as relative percentages (HBc/Actin) with mock transfection/transduction samples set to 100% (right panel). The error bars indicate ±SD. Data were analyzed by one-way analysis of variance, followed by Tukey’s comparison test for all groups * indicates p < 0.05. n. s. indicates p > 0.05. (C, D) HepAD38 cells were transduced with LV-STUB1 or LV-control (C) or with LV-sh_STUB1 or mock LV-sh_control (D). At 36 h after transduction, cells were treated with 1 μM Bay41-4109 and 50 μg/ml CHX for the indicated time. The proteins were detected by western blot using the indicated antibodies (left panel). The quantification results of HBc/actin ratio from two independent immunoblots are shown as relative percentages (right panel). The samples of CHX treatment at 0 h were set to 100%. The error bars indicate ±SD.* indicates p < 0.05.**p < 0.01, p was calculated by unpaired two-tailed student’s t-test. https://doi.org/10.1371/journal.ppat.1010204.g001 We next investigated whether STUB1 is required for this observed Bay41-4109-induced reduction of HBc. STUB1 knockdown restored the HBc protein level in Bay41-4109-treated HepAD38 cells (Figs 1B and S1). We also performed a cycloheximide (CHX) chasing experiment in HepAD38 cells with recombinant lentiviruses expressing either STUB1 (LV-STUB1) or shRNA targeting STUB1 (LV-sh_STUB1). Overexpression of STUB1 shortened the half-life of HBc from approximately 6 to 2 h in Bay41-4109-treated cells (Fig 1C). In contrast, the knockdown of STUB1 increased the half-life of HBc from approximately 6 to 22 h (Fig 1D), indicating that STUB1 negatively regulates the stability of HBc in Bay41-4109-treated cells. Of note, manipulation of STUB1 expression only in the cells without Bay41-4109 treatment did not have a significant impact on HBc expression and its degradation (S2 Fig).

Bay41-4109 induces aberrant non-capsid polymers dependent on STUB1 HAPs have been reported to induce the aberrant assembly of viral capsid proteins (as aggregated proteins previously described as “aberrant non-capsid polymers”) [15,17], and the STUB1-based protein quality control system is known to function in degrading misfolded or aggregated proteins [27]. We investigated whether Bay41-4109 induced the formation of these aberrant non-capsid polymers by treating HepAD38 cells with increasing concentrations of Bay41-4109 and performed ultracentrifugation analysis on the cell lysates in iodixanol gradients. In control cells (dimethyl sulfoxide (DMSO)-treated), the amounts of HBc protein peaked in the 8th fraction (Fig 2A and 2B) whereas HBc protein level additionally peaked in the 5th fractions for the cells treated with 1 or 10 μM Bay41-4109. Negative-staining electron microscopy of the 8th fraction from mock-treated cell lysate showed the anticipated icosahedral particles (representing nucleocapsids and/or capsids) (S3 Fig). Fraction 5 from 10 μM Bay41-4109 treated-cell lysates had irregular structures resembling the previously reported aberrant non-capsid polymers. The 5th fraction and the 8th fraction of density gradient samples were also subjected to particle gel assay. When cells were treated with 1 μM or 10 μM Bay41-4109, the bands detected by anti-HBc in the 5th fraction were slightly up-shifted (S4 Fig) as compared to the 8th fraction. This result is similar to the observation of shifted bands of the HAP-induced aberrant polymers in a previous report [28]. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Co-sedimentation and co-localization of STUB1 with Bay41-4109-induced aberrant non-capsid polymers. (A, B) HepAD38 cells were treated with DMSO or increasing concentrations of Bay41-4109 (as indicated) for 36 h. Cytoplasmic lysates from each sample were condensed with a centrifugal filter (100-KDa cutoff) and subjected to iodixanol gradient ultracentrifugation. The levels of the HBc and STUB1 proteins in each fraction were then analyzed with dot blots (A), and were quantified based on two independent experiments by ImageJ (B). (C) HepAD38 cells were treated with 1 μM Bay41-4109 or DMSO as indicated for 48 h. The cells were then immunostained for HBc (green) and stub1 (red). Nuclei were stained with Hochest33342. Areas indicated by white boxes are enlarged. Arrows point to representive co-localized sites. The scale bar is 10 μm. (D) Quantification of the number of HBc puncta co-localized with STUB1 per cell. 100 cells were counted. The error bars indicate ±SD. **p < 0.01, calculated by unpaired two-tailed student’s t-test. https://doi.org/10.1371/journal.ppat.1010204.g002 Given that STUB1 may interact with the aberrant non-capsid polymers, STUB1 was detected in the polymer-containing fractions of 1 μM and 10 μM Bay41-4109-treated cell lysates (Fig 2A and 2B); only a small amount of STUB1 was detected in the 8th fractions from the control cells, probably because the majority of STUB1 was lost in the filtration before gradient centrifugation provided that aberrant polymers were not induced by Bay41-4109. We subsequently immunostained Bay41-4109- or mock-treated HepAD38 cells with the STUB1- and HBc-specific antibodies. 1 μM Bay41-4109 showed scattered HBc puncta, which is likely related to formation of aberrant polymers (Fig 2C) [29]. These HBc puncta were partially merged with STUB1 in Bay41-4109 treated cells (Fig 2C) (average 8 puncta in Bay41-4109-treated cells versus 0.3 in control cells) (Fig 2D). These results together demonstrate that Bay41-4109 induces formation of aberrant non-capsid polymers and that STUB1 associates with these polymers.

STUB1 mediates the lysosomal degradation of Bay41-4109-induced aberrant non-capsid polymers It is well-documented that STUB1 can promote proteasome- and lysosome-mediated protein degradation [30]. Treatment of cells with inhibitors of lysosomal degradation attenuated Bay41-4109-induced reduction in HBc whereas treatment with a proteasome inhibitor did not disrupt the Bay41-4109-induced reduction in HBc (Figs 3A and S5). These results suggest that Bay41-4109 induces degradation of HBc in a lysosome-dependent manner. Immunostaining showed that Bay41-4109 treatment induced co-localization of HBc with LAMP1 (lysosome marker lysosome-associated membrane protein 1); only background co-localization of HBc and LAMP1 was observed in untreated control cells (Fig 3B). We also investigated the potential role of STUB1 in the lysosomal localization of the Bay41-4109-induced aberrant non-capsid polymers. Knockdown of STUB1 in Bay41-4109-treated cells reduced the extent of HBc’s lysosomal localization (Fig 3C and 3D), suggesting that STUB1’s association with aberrant non-capsid polymers somehow mediates trafficking of the polymers to lysosomes. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Bay41-4109 induces STUB1-driven lysosomal degradation of HBc. (A) HepAD38 cells were treated with 1 μM Bay41-4109 or DMSO for 24 h, followed by treatment with 5 μM of the proteasome inhibitor MG132, 0.1 mM of lysosome inhibitor BafA1, or 10 mM of lysosome inhibitor Pep/E64 for another 24 h. Cell extracts were then analyzed by western blotting using the indicated antibodies (upper panel). The quantification results of two independent immunoblots are shown as relative percentages (HBc/actin) with the DMSO-treated sample set to 100% (lower panel). The error bars indicate ±SD. **p < 0.01, *p < 0.05, “n. s.” denotes p > 0.05. p values were calculated by unpaired two-tailed student’s t-test. (B) HepAD38 cells were treated with 1 μM Bay41-4109 or DMSO as indicated for 24 h, followed by treatment with 0.1 mM BafA1 for 12 h. (C) HepAD38 cells transduced with LV-sh_STUB1 or control LV-sh_control were treated with 1 μM Bay41-4109 or DMSO for 24 h. Subsequently, the cells were treated with 0.1 mM BafA1 for 12 h. GFP-positive cells indicate lentivirus transduction (gray). (C, D) The cells were immunostained for HBc (green) and LAMP1 (red). Areas indicated by white boxes are enlarged. Arrows point to typical co-localized sites. Scale bar is 10 μm. (D) Quantification of the number of HBc puncta co-localized with Lamp1 per cell. 100 cells were counted. The error bars indicate ±SD. **p < 0.01, “n. s.” represents p > 0.05, p values were calculated by unpaired two-tailed student’s t-test. https://doi.org/10.1371/journal.ppat.1010204.g003

STUB1 drives the p62-mediated macroautophagy of Bay41-4109-induced aberrant non-capsid polymers STUB1 is known to promote the lysosomal degradation of proteins via chaperone-mediated autophagy and p62-mediated macroautophagy [27]. Large cargoes such as protein aggregates and cell organelles are delivered to the lysosomes by p62-mediated macroautophagy [29]. Given the large size of the Bay41-4109-induced aberrant polymers, we incubated Bay41-4109-treated HepAD38 cells with the macroautophagy inhibitor 3-methyladenine (3-MA) and found that 3-MA reversed the Bay41-4109-induced reduction of HBc levels (Fig 4A). Additionally, treatment with 3-MA dramatically decreased the number of HBc-LAMP1 co-localized puncta (from 11 ± 4 to 4 ± 2 puncta per cell) (Fig 4B). Thus, Bay41-4109-induced aberrant non-capsid polymers are trafficked to lysosomes for degradation via macroautophagy. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Aberrant non-capsid polymers induced by Bay41-4109 are degraded via STUB1-mediated macroautophagy. (A) HepAD38 cells were treated with 1 μM Bay41 or DMSO for 1 d, followed by treatment with 10 mM 3-MA or DMSO as indicated for 24 h. Proteins were detected by western blot using antibodies recognizing the indicated proteins (left panel). (B) HepAD38 cells were treated with 1 μM Bay41-4109 or DMSO for 24 h followed by treatment with 0.1 mM BafA1 and 10 mM 3-MA (or BafA1 alone) for 12 h. The cells were immunostained for HBc (green) and LAMP1 (red) as indicated. Nuclei were stained with Hochest33342. Areas indicated by white boxes are enlarged. Arrows point to typical co-localized sites. Scale bar is 10 μm. (C) HepAD38 cells were transduced with LV-sh_STUB1 or LV-sh_control, cultured in the the presence or absence of Dox, and treated with 1 μM Bay41-4109 or DMSO for 24 h. (D) HepAD38 cells were transduced with LV-sh_STUB1 or LV-sh_control, treated with 1 μM Bay41-4109 or DMSO for 1 d, and then treated with 0.1 mM BafA1 for 12 h. Cell lysates were immunoprecipitated with HBc antibodies or mock antibodies. Proteins were detected by western blot using antibodies recognizing the indicated proteins. (E) Huh7 cells expressing HBc or lysine-free HBc mutant (HBc_K7R/K96R) were treated with 1 μM Bay41-4109 or DMSO. The proteins were detected by western blot using the indicated antibodies. (F) HepAD38 cells were transfected with STUB1, STUB1_H260Q, or empty vector. At 24 h after transfection, the cells were incubated with or without 1 μM Bay41-4109 for 2 d. The proteins were detected by western blot using the indicated antibodies. (A, B, C, E, F) The right panels show the quantification results of three independent immunoblots as relative percentages (HBc/actin) (A, E, F) or (LC3-I/LC3-II) (C) with the control sample set to 100%. The error bars indicate ±SD. **p < 0.01, p value was calculated by unpaired two-tailed student’s t-test. https://doi.org/10.1371/journal.ppat.1010204.g004 We further examined the macroautophagy process (including involvement of STUB1) by measuring the LC3-II to LC3-I ratio [31] in lysates of HepAD38 cells. Bay41-4109 promoted the conversion of cytosolic LC3-I to the lipidated, autophagosome-associated LC3-II in the cells expressing HBc, but not in the HBc non-expressed cells (Fig 4C), indicating the aberrant polymer dependent activation of autophagy. Knockdown of STUB1 restored the LC3-II/LC3-I ratio to the level observed in untreated control cells (Fig 4C). These findings support that STUB1 is required for the stimulation of macroautophagy observed in cells with Bay41-4109-induced aberrant polymers. P62 is a macroautophagy receptor that directs substrate proteins to autophagosome by simultaneously interacting with a substrate protein and with LC3 [31]. We investigated whether Bay41-4109-induced aberrant polymers interact with p62, and assessed whether the interaction may be facilitated by STUB1. Co-immunoprecipitation analysis showed that p62 was detected in the anti-HBc precipitates from Bay41-4109-treated HepAD38 cells but not in the precipitates from DMSO control cells. Of note, owing to the rapid degradation of HBc, the lysosomal inhibitor Baf A1 was included in all samples in these experiments (Fig 4D). No p62 was detected among the anti-HBc precipitates from STUB1 knockdown and Bay41-4109-treated cells (Fig 4D), indicating that STUB1 facilitates the interaction between p62 and Bay41-4109-induced aberrant polymers. P62-mediated macroautophagy selectively recognizes ubiquitinated substrate proteins although it is known that some non-ubiquitinated proteins can also be degraded via macroautophagy [32]. Our in vivo ubiquitination assays showed no obvious ubiquitination of HBc in HepAD38 cells treated with Bay41-4109, even when STUB1 was overexpressed (S6 Fig). Consistently, we found that the level of HBc_K7R/K96R (an HBc mutant missing 2 potential ubiquitin modification sites) [33], was decreased upon treatment of cells with Bay41-4109 (Fig 4E). Moreover, the STUB1 mutant variant STUB1H260Q, which lacks E3 ubiquitin ligase activity [23], exhibited the same ability to reduce the HBc protein level as STUB1wild-type (Fig 4F). These results demonstrate that STUB1 drives autophagosome formation for P62-mediated macroautophagy of Bay41-4109-induced aberrant non-capsid polymers. Importantly, this process is independent of STUB1’s E3 ubiquitin ligase activity while our data suggest that STUB1 somehow promotes the interaction between p62 and the aberrant polymers.

STUB1 mediates the transportation of Bay41-4109-induced aberrant polymers to the perinuclear region A previous study reported that STUB1-bound, aggregated proteins can accumulate in the perinuclear region before being delivered to autophagosomes [27]. We next studied whether a similar process may occur with Bay41-4109-induced aberrant non-capsid polymers. Interestingly, immunostaining with anti-HBc antibody showed that co-treatment of Bay41-4109 with the macroautophagy inhibitor 3-MA led to obvious perinuclear accumulation of HBc puncta whereas no such perinuclear accumulation was observed in cells treated with Bay41-4109 alone. Furthermore, the number of perinuclear puncta was significantly decreased upon STUB1 knockdown (Fig 5A and 5B). These results collectively support that the Bay41-4109-induced aberrant non-capsid polymers are transported to the perinuclear region and that STUB1 is essential for this perinuclear translocation. Moreover, these findings suggest that Bay41-4109-induced aberrant non-capsid polymers are transported from the perinuclear region to lysosomes in a macroautophagy-dependent manner. PPT PowerPoint slide

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TIFF original image Download: Fig 5. STUB1 mediates transport of Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region. (A) HepAD38 cells were treated with 1 μM Bay41-4109 alone, Bay41-4109 and 10 mM 3-MA, or DMSO for 2 d. For STUB1 silencing, cells were transfected with si_STUB1 or si_control at 36 h before treatment with both Bay41-4109 and 10 mM 3-MA. Cells were immunostained for HBc (green) as indicated. Nuclei were stained with Hochest33342. The “enlargement” column shows enlarged images of the areas indicated by white boxes in the “merge” column. Scale bars are 30 μm in the “merge” column and 3 μm in the “enlargement” column. (B) Numbers of perinuclear HBc puncta (diameter exceeds 3 μM in any direction) per cell. 100 cells were counted. The error bars indicate ±SD. **p < 0.01, p value was calculated by unpaired two-tailed student’s t-test. (C) HepAD38 cells were treated with 1 μM Bay41-4109 and 10 mM 3-MA for 2 d. Cells were immunostained for HBc (green) and STUB1 (red) as indicated. Nuclei were stained with Hochest33342. Areas indicated by white boxes are enlarged. Arrows point to typical co-localized sites. Scale bar is 10 μm. (D) HepAD38 cells transduced with LV-sh_STUB1 or LV-sh_control were treated with 1 μM Bay41 or DMSO for 24 h, and all the cells were treated with 0.1 mM BafA1 for 12 h. Cell lysates were immunoprecipitated with HBc antibodies or mock antibodies. Proteins were detected by western blot using antibodies recognizing the respective proteins. (E) HepAD38 cells were transfected with two different siRNAs targeting BAG3 or mock siRNA. At 36 hours after transfection, cells were incubated in medium with 1 μM Bay41-4109 or DMSO for 2 d. Cell extracts were then analyzed by western blotting using the indicated antibodies (left panel). The quantification results of three independent immunoblots are shown as relative percentages (HBc/Actin) with mock transfection/transduction samples set to 100% (right panel). (F) HepAD38 cells were transfected with si_BAG3 or si_control. 36 h after transfection, HepAD38 cells were treated with 1 μM Bay41-4109 and 10 mM 3-MA, or DMSO for 2 d. Cells were immunostained for HBc (green) as indicated. Nuclei were stained with Hochest33342. The “enlargement” column shows enlarged images of the areas indicated by white boxes in the “merge” column. Scale bars are 30 μm in the “merge” column and 3 μm in the “enlargement” column. https://doi.org/10.1371/journal.ppat.1010204.g005 We immunostained HepAD38 cells treated with Bay41-4109 and 3-MA against STUB1 and HBc and observed co-localization of STUB1 and HBc in the perinuclear space (Fig 5C). Considering previous studies reporting that the BAG3 protein mediates the transport of STUB1- and Hsp70-bound aggregate proteins to the perinuclear compartment and facilitates the interaction of aggregated proteins with p62[27,29], we next tested whether BAG3, Hsp70 and STUB1 may interact and potentially form a complex with Bay41-4109-induced aberrant non-capsid polymers. Immunoprecipitation using anti-HBc showed that BAG3 and STUB1 were detected in precipitates from Bay41-4109 and Baf A1-treated HepAD38 cells, but not those from DMSO and Baf A1-treated control cells (Fig 5D). No BAG3 was co-immunoprecipited with HBc in STUB1 knockdown cells (Fig 5D). To validate the physiological role of BAG3 in processing Bay41-4109-induced aberrant polymers, we transfected cells with BAG3-targeting siRNA and treated cells with Bay41-4109 or DMSO. Silencing BAG3 restored Bay41-4109-induced degradation and perinuclear accumulation (upon 3-MA co-treatment) of HBc protein (Fig 5E and 5F). Thus, STUB1 facilitates recruitment of BAG3 to the aberrant non-capsid polymers. Hsp70 binds to the complex of STUB1, BAG3 and Bay41-4109-induced aberrant polymers (Fig 5D). This host chaperone is also required for the degradation of Bay41-4109-induced aberrant polymers as knockdown of Hsp70 abrogated such degradation (S7 Fig). Since Hsp70 binding to unfolded or aggregated proteins can be promoted by a cationic rhodacyanine MKT-077 [34], we assumed that MKT-077 would enhance the degradation of Bay41-4109-induced aberrant polymers. Indeed, MKT-077 further decreased HBc level in the cells exposed to Bay41-4109 (S7 Fig), indicating that enhanced activity of Hsp70 can facilitate Bay41-4109-induced degradation of HBc.

STUB1 protects cells against toxicity from Bay41-4109-induced aberrant non-capsid polymers Many studies have demonstrated that protein aggregates can cause cytotoxicity if they are not removed rapidly [29,35,36]. We investigated whether blocking the degradation of the aberrant non-capsid polymers by knocking down STUB1 could increase cytotoxity. To this end, we knocked down STUB1 by shRNA and treated cells with 1 μM or 3 μM Bay41-4109. These concentrations are 10–35 times lower than the previously reported 50% cytotoxic concentration (CC 50 ) of Bay41-4109 [37], but can still induce formation of aberrant non-capsid polymers [32]. STUB1 knockdown significantly decreased the viability of Bay41-4109-treated HepAD38 cells. Furthermore, STUB1 knockdown had no impact on the viability of HepAD38 cells when HBc protein expression was turned off by Dox (Fig 6). These results support that STUB1 reduces the cytotoxicity caused by Bay41-4109-induced aberrant non-capsid polymers, likely by promoting their degradation. PPT PowerPoint slide

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TIFF original image Download: Fig 6. STUB1 protects cells from cytotoxity of Bay41-4109-induced aberrant non-capsid polymers. HepAD38 cells were cultured in the presence (right panel) or absence (left panel) of Dox. Cells were transduced with LV-sh_STUB1 or LV-sh_control. 36 h later, cells were treated with the indicated concentrations of Bay41-4109 for 6 d. A CCK-8 assay was used to quantify cell viability. The error bars indicate ±SD. **p < 0.01, p value was calculated by unpaired two-tailed Student’s t-test. https://doi.org/10.1371/journal.ppat.1010204.g006

STUB1 enhanced the inhibitory effect of Bay41-4109 on the production of HBeAg and virions from HepAD38 cells We reasoned that STUB1-mediated degradation of the aberrant non-capsid polymers upon Bay41-4109 treatment would likely reduce the extent of intracellular capsids, which function in both HBV DNA replication and virion secretion [4]. We therefore assessed whether STUB1 facilitates Bay41-4109’s inhibition with respect to the intracellular capsid and extracellular virion levels. Immunoblot analysis of intracellular capsids that were separated using native agarose electrophorese showed that STUB1 overexpression in Bay41-4109-treated cells reduced the level of intracellular viral capsids by approximately 5 times compared to empty vector control cells (Fig 7B). PPT PowerPoint slide

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TIFF original image Download: Fig 7. STUB1 inhibits secretion of virions and HBeAg from Bay41-4109-treated HepAD38 cells, and reduces intracellular p17 level in p17-transfected cells. (A, B) HepAD38 cells transduced with LV-STUB1 or LV-control were treated with 1 μM Bay41-4109 or DMSO for 2 d. Capsids precipitated from cell lysates were subjected to agarose gel electrophoresis and western blotting using a rabbit polyclonal anti-HBc antibody (A). Capsid levels of two independent immunoblots were quantified by ImageJ (B). (C, D) HepAD38 cells transduced with LV-STUB1 or LV-control were treated with the indicated concentrations of Bay41-4109 for 6 d. Media were refreshed every 2 d. The number of HBV DNA copies (C) and HBeAg level (D) in media were quantified. The error bars indicate ±SD. **p < 0.01, calculated by unpaired two-tailed student’s t-test. (E, F) HepAD38 cells were transfected with pcDNA3.1-STUB1 or control plasmid, and pcDNA3.1-p25ΔCTD or pcDNA3.1-p25. At 1 d after transfection, cells were incubated in medium with 1 μM Bay41-4109 or DMSO for 2 d. Cell extracts were then analyzed by western blotting using antibodies recognizing myc tag or actin (E). The quantification results of three independent immunoblots are shown as relative percentages (p17/Actin or precore/Actin) with mock-treated samples set to 100% (F). https://doi.org/10.1371/journal.ppat.1010204.g007 Consistently, quantification of HBV DNA derived from extracellular virions demonstrated that overexpression of STUB1 decreased the effective concentration at 50% (EC50) of Bay41-4109 from approximately 42.5 to 23 nM (Fig 7C). No potentiating effects from STUB1 overexpression were observed when HepAD38 cells were treated with a type II CpAM (SBA_R01) (S8 Fig), nor did we observe STUB1-driven transport of HBc to lysosomes in SBA-R01-treated cells (S8 Fig). This is consistent with the known effects of Type II CpAMs for inducing the formation of empty capsids (i.e., unrelated to the known effects of type I HAPs for inducing formation of aberrant non-capsid polymers). Moreover, HAP compounds are known to directly reduce HBeAg production because HAP may binds to intracellular HBeAg dimers and induce aberrant polymers [12,17,19]. As STUB1 mediates the degradation of HAP-induced aberrant non-capsid polymers, it is plausible that STUB1 may promote HAP-mediated inhibition of HBeAg secretion. Recent findings demonstrated that in addition to HBeAg, HBc protein can also be secreted by HBV-infected hepatocyte [38–40]. HBc shares 149–159 aa with HBeAg. To distinguish HBeAg from extracellular HBc protein, we used a specific antibody, recognizing the 5th to10th amino acids on the N-terminal residues of HBeAg that has been proven to have no cross-reactivity with HBcAg, to detect HBeAg in cell culture media [41]. The overexpression of STUB1 in Bay41-4109-treated HepAD38 cells (by LV-STUB1 transduction) significantly lowered the level of HBeAg in culture supernatants (Fig 7D). In contrast, the extracellular level of HBsAg—which is not involved in HAP-induced aberrant non-capsid polymers—was not affected by STUB1 overexpression in Bay41-4109-treated cells (S9 Fig). It has been demonstrated that HAP reduces the level of intracellular HBe (p17) [19] or precore protein (p22), i.e. the precursor of HBe [17]. We next evaluated whether STUB1 affects the level of intracellular precore protein or HBe protein. We constructed precore and p17 expression plasmids by inserting the signal peptide-included and c-terminal Myc-tagged p25 ORF sequence or p25ΔCTD into the pcDNA3.1, respectively. As previsously reported, transfection of signal peptide-included p25 and p25ΔCTD leads to expression of the signal peptide-deleted precore protein p22 and p17, respectively [38,42]. p17 level was significantly reduced by Bay41-4109 treatment and further lowered upon STUB1 overexpression (Fig 7E and 7F); the level of precore protein was slightly reduced by Bay41-4109 treatment, but was not further affected by STUB1 overexpression (Fig 7E and 7F). These results supported that Bay41-4109 reduces the level of intracellular p17 by a STUB1-mediated degradation, which may lead to a lower level of secreted HBeAg.

STUB1 overexpression enhances the inhibitory effects of Bay41-4109 against secretion of HBV virions and HBeAg, and cccDNA formation in HBV-infected HepG2-NTCP cells We next examined the influence of STUB1 on the inhibitory effect of Bay41-4109 in the context of HBV infection. We initially transduced HepG2-NTCP cells with LV-STUB1 or LV-control and infected these cells with HBV virions 6 days later (day 0). These cells were treated with Bay41-4109 over a 6-day time course, and the supernatant was collected every other day for the quantification of secrected HBeAg and HBV DNA levels (Fig 8A). By 6 days after HBV infection, STUB1 overexpression in Bay41-4109-treated cells lead to a 50% decrease in extracellular HBeAg and a 60% decrease in extracellular HBV DNA (Fig 8B and 8C). Furthermore, we did not observed a decrease in the viability of LV-STUB1-transduced cells in HBV infection assay, so the reduction of HBeAg and virions were not related to the cytotoxicity caused by long-term overexpression of STUB1 (S10 Fig). Immunostaining against HBc at 6 d after HBV infection showed that more than 85% cells were infected by HBV (Fig 8D). Treatment with Bay41-4109 reduced the fluorescence signal intensity of the HBc protein, and induced HBc puncta. STUB1 overexpression in Bay41-4109-treated cells further decreased Bay41-4109-induced puncta of HBc protein (Fig 8D and 8E); consistently, STUB1 overexpression reduce the total HBc protein level in Bay41-4109-treated, HBV-infected cells as detected by immunoblot (S11 Fig). When STUB1 was knocked down by LV-sh_STUB1, the fluorescence intensity of Bay41-4109-induced HBc puncta increased (S12 Fig). Altogether, these findings are consistent with our earlier results from the HepAD38 cells and support that STUB1 can promote Bay41-4109’s anti-viral efficacy. PPT PowerPoint slide

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TIFF original image Download: Fig 8. The effect of STUB1 overexpression in Bay41-4109-treated HBV-infected HepG2-NTCP cells. (A) Schematic diagram of the experimental procedure for HepG2-NTCP cells in Fig 8B–8D. (B, C, D) HepG2-NTCP cells transduced with LV-STUB1 or LV-control were infected with HBV at MOI of 500 genome equivalents in the presence of 2% DMSO. Bay41-4109 (1 μM) or DMSO was added during HBV infection. The HBeAg level (B) and HBV DNA copies (C) in media were quantified at 2, 4, and 6 d after HBV infection. The error bars indicate ±SD.*p < 0.05.**p < 0.01, p value was calculated by unpaired two-tailed student’s t-test. (D, E) Cells were immunostained for HBc (green) as indicated. Nuclei were stained with Hochest33342 (D) The fluorescence signal intensity was quantified by ImageJ (E). *p < 0.05, p value was calculated by unpaired two-tailed student’s t-test. (F) Schematic diagram of the experimental procedure of adding Bay41-4109 during (co-infection) or post HBV infection (post-infection) in LV-control- or LV-STUB1-transduced HepG2-NTCP cells. (G) Cells were treated with Bay41-4109 during (co-infection) or post HBV infection as descripted in Fig 8G. cccDNA were isolated by hirt extraction, and their levels were quantified by qPCR. https://doi.org/10.1371/journal.ppat.1010204.g008 It has been demonstrated that HAP compound acts on capsid of incoming virus particles and inhibits the infectivity in de novo HBV infection and cccDNA synthesis [16,28]. After establishment of cccDNA, HAP can also mislead progeny nucleocapsid assembly. It is very interesting to know whether STUB1 enhances this early effect of HAP on the procedure before de novo cccDNA synthesis. We transduced HepG2-NTCP cells with LV-STUB1 or LV-control. 6 days later, we treated the cells with Bay41-4109 at the same time as HBV infection, and quantified cccDNA level at day 3 post HBV infection (Fig 8F). Interestingly, qPCR results demonstrated that STUB1 overexpression further decreased cccDNA levels in Bay41-4109-treated cells (Fig 8G). It is known that cccDNA level reaches the maximum at 2 day post HBV infection in HepG2-NTCP cells [16]. It is interesting to unveal whether Bay41-4109 could reduce cccDNA level after cccDNA formation has been established, and whether STUB1 can enhance Bay41-4109-induced cccDNA degradation, if any. We treated HBV-infected, LV-STUB1- or LV-control-transduced HepG2-NTCP cells with Bay41-4109 at 2 days after HBV infection, and harvest cells at day 5 post HBV infection (Fig 8F). Intriguingly, cccDNA level was not affected when Bay41-4109 was enforced after HBV infection (Fig 8G). STUB1 overexpression caused no reduction of cccDNA level. Altogether, Bay41-4109 and STUB1 overexpression reduced early de novo synthesis of cccDNA, but not induced cccDNA level reduction after HBV infection has established.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010204

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