Fig 1.
Identification of HBc-interacting proteins in the nucleus of dHepaRG cells.
(A) Schematic view of HBc purification process. Nuclei were purified from differentiated HepaRG-TR cells (dHepaRG-TR) expressing either wt HBc or ST-HBc under the control of a tetracyclin-inducible promoter, lysed and then treated or not with Benzonase. Nuclear extracts were purified on a Streptactin column and protein eluted with desthiobiotin. Input and eluted fractions (E1, E2, and E3) were analyzed by gel electrophoresis followed by silver staining (B) and western blot (C) using an anti-HBc antibody. (D) Venn diagram of proteins significantly associated to HBc common to conditions with and without Benzonase. Proteins in bold correspond to the 11 “founders” RBP common to both conditions (see text, RBMXL is not highlighted because it was considered as a retrogene of RBMX).
Fig 2.
(A) Proteins significantly associated to HBc in the presence of Benzonase were analyzed using the Genemania plugin in Cytoscape (3.7.1). Red lines indicate known physical interactions. Missing nodes are indicated by grey circles. (B) Interaction network of proteins involved in mRNA splicing via spliceosome. Significant proteins, common to the Benzonase-/+ conditions, over-representing the mRNA splicing via spliceosome biological process (“founder proteins” highlighted as orange nodes) were used to initiate the network by querying IntAct database. Red, blue and green nodes denote protein of the computed network that are found in the proteomic hits of both Benzonase-/+ (Benz- or Benz+) conditions. Nodes with a bold border indicate significant proteins (p-value<0.005 and fold change>4) from the proteomics data.
Fig 3.
(A) Relative abundances of the 11”founder” RBPs identified in HBc nuclear complexes submitted or not to Benzonase treatment. The relative abundances of HBc binding partners have been evaluated using the iBAQ metrics [104]. For each replicate, each iBAQ value was normalized by the summed values of the 11 proteins. Error bars represent +/- SD. (B) and (C) Western blot validations in Benzonase treated and streptactin-purified and extracts. Two major isoforms of SRSF10 are visible: the upper at 37KDa and the lower at 20–22 KDa. The band indicated with an asterisk likely corresponds to a band generated by proteolytic cleavage. (D) HBc was immune-precipitated from nuclear extracts purified from liver sections from HBV-infected HuHep mice, using two different anti-HBc antibodies. Eluted proteins were analyzed by western blot using anti HBc and anti-SRSF10 antibodies. The asterisk indicates the positions of IgG heavy chain. (E) Proteins included in the gel band between 35 and 25 KDa were analyzed by MS. The table indicates the list of proteins recovered with the anti-HBc antibody that were also previously found after HBc purification on StrepTactin columns (see Fig 1D). * In the case of MLF, 1 peptide was found in the anti-IgG control IP. The other proteins were found exclusively in the anti-HBc IP.
Fig 4.
Effect of SRSF10 or RBMX KD on HBV replication in PHH.
(A) Outline of the experimental protocol: cells were transfected with siRNA targeting SRSF10 or RBMX or control siRNA (siCTL) and then infected with HBV (MOI of 100 vge/cell). Cells and supernatants were harvested 7 days (D) post-infection (pi) and analyzed to measure intracellular and secreted HBV parameters. (B), (C) and (E) Western bot analysis of NTCP, SRSF10 and RBMX protein levels at D0 of the protocol, respectively. (D) and (F) HBV intracellular and extracellular parameters were measured at D7 pi. Results are expressed as the mean normalized ratio +/- SD, between siSRSF10 or siRBMX and siCTL transfected cells, of 3 independent experiments, each performed in triplicate, with PHH from different donors.
Fig 5.
Effect of 1C8 on an established HBV infection.
(A) Molecular structure of 1C8. (B) Outline of the experimental protocol: HBV-infected dHepaRG cells (C) or PHH (D) were treated three times with Tenofovir (TDF at 10μM), a Core allosteric modulator (CAM at 10μM) or 1C8 (10 μM) starting at D4pi. Intracellular and secreted HBV parameters were quantified 2 days after the last treatment. Results are expressed as the mean normalized ratio +/- SD between non-treated and treated cells of 3 independent experiments, each performed in triplicate.
Fig 6.
Combined effect of sRSF10 KD and 1C8 treatment on HBV-infected dHepaRG cells.
(A) Outline of the experimental procedure: dHepaRG cells were transfected once or twice with siRNA targeting SRSF10, then infected with HBV (MOI of 250 vge/cell), and 7 days later treated three times with 1C8 (10μM). (B) Western blot validation showing SRSF10 depletion. (C) Quantification of intracellular HBV RNAs. Results are expressed as the mean normalized ratio +/- SD between treated and/or siSRSF10 transfected cells and siCTL-transfected cells of 3 independent experiments, each performed in triplicate.
Fig 7.
Analysis of nascent HBV RNAs following SRSF10 KD or 1C8 treatment.
(A) dHepaRG cells were transfected with siRNA against SRSF10 and then infected with HBV (MOI of 250 vge/cell). Edu labelling was performed at D7pi for 2 hours. (B) dHepaRG cells were infected with HBV and then treated three times with 1C8 (40μM) at D7, D9 and D11pi. EU incorporation was performed at D13pi for 2 hours. (C) and (D) Run-on analyses. Intracellular RNA was extracted from transfected/treated cell cells and either directly quantified using HBV primers (Total HBV RNAs) or purified using the Click-iT Nascent RNA Capture kit to quantify newly synthetized RNAs (nascent HBV RNAs). Control was provided by treating cells with Actinomycin D (ActD at 10mg/ml) added to cells 20 min before labeling (see Methods). <LOD: under the limit of detection.