The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production

Abstract

Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.

Keywords: HCV; NS5A; SMYD3; TAP-MS; Virus particle assembly.

Fig. 1

Identification and confirmation of SMYD3 as interactor of NS5A. (A) Schematic representation of NS5A-binding proteins identified by TAP-MS with an FC_B score≥4. Node color gradient corresponds to increasing log₂ FC_B scores. Known interactions are depicted by black edges, novel interactions by gray edges. NS5A is highlighted in blue. Two biological replicates were analyzed as technical duplicates. (B) Interaction of overexpressed SMYD3 and NS5A. Myc-NS5A, HA-SMYD3 or catalytic inactive SMYD3 (Y239F) were transiently expressed in HEK 293T cells. 48 h posttransfection, protein complexes were immunoprecipitated and analyzed by Western blot. Representative blots of 3 independent experiments are shown. (C) Interaction of endogenous SMYD3 with TAP-tagged NS5A. Expression of NS5A in HEK FlpIn Strep-HA-NS5A cells was induced by addition of 1 µg/ml doxycyline. After 48 h, NS5A was immunoprecipitated and samples analyzed by Western blot.

Fig. 2

Mapping the binding sites of SMYD3 and NS5A. (A) Schematic representation of full-length SMYD3 and respective deletion mutants. The N-terminal lobe contains the catalytic SET-domain (blue), which is split by the zinc-finger MYND-domain (red). The second lobe consists of regulatory C-terminal domain (purple). Residues spanning the individual domains are indicated. (B) Association of Myc-tagged NS5A with HA-tagged SMYD3 deletion mutants. The indicated plasmids were co-expressed in HEK 293T cells and co-immunoprecipitation experiments were performed as in Fig. 1B. (C) Ratio of co-purified NS5A with the respective SMYD3 mutants. Bars represent the ratio of quantified band signals of the Western blots shown above. (D) Schematic representation of full-length NS5A and respective deletion mutants. NS5A consists of 3 domains (blue, red and purple, respectively) connected by two low-complexity sequences (black). Residues spanning the individual domains are indicated. (E) Association of HA-tagged SMYD3 with Myc-tagged NS5A deletion mutants. The indicated plasmids were co-expressed in HEK 293T cells and co-immunoprecipitation experiments were performed as in (B). (F) Ratio of co-purified SMYD3 with the respective NS5A mutants. Bars represent the ratio of quantified band signals of the Western blots shown above. (B and E) Representative blots of at least 2 similar experiments are shown.

Fig. 3

Interaction of SMYD3 with NS5A in the context of viral replication. (A and C) Huh7.5/HA-SMYD3 cells were electroporated with RNA encoding the JFH1 subgenomic replicon (sgJFH1) or full-length Jc1. 72 h post electroporation, HA-SMYD3 was immunoprecipiated and analyzed by Western blot using anti-HA and anti-NS5A antibodies. (B and D) Enrichment of hyperphosphorylated NS5A (p58) in SMYD3 pull-downs. Western blot bands shown in (A) and (C) corresponding to the p56 and p58 phosphoforms of NS5A were quantified and the p58/P56 ratio calculated. (E) Co-localization of overexpressed HA-SMYD3 (top panel) or endogenous SMYD3 with subgenomic JFH1 NS5A. Huh7.5 cells were electroporated as described above. After 48 h, cells were fixed and proteins stained using SMYD3 (green) and NS5A (red) antibodies. Nuclei were counterstained with DAPI. Images were analyzed by confocal microscopy. Co-localization was quantified using ImageJ and the WCIF ‘Intensity Correlation Analysis’ plugin (upper panel: R_r=0.746; R=0.903; lower panel: R_r=0.620; R=0.781).

Fig. 4

SMYD3 is a negative regulator of HCV infectious particle assembly. (A) Western blot analysis of Huh7-Lunet cells stably expressing wildtype (WT) SMYD3, catalytic inactive (Y239F) SMYD3 or GFP. (B) Effect of SMYD3 overexpression on HCV RNA replication kinetics. Indicated cell lines were electroporated with RNA encoding the subgenomic JFH1 luciferase reporter replicon. Viral replication was measured at the indicated time points by luciferase assay. Luciferase activity is expressed in relative light units (RLU) normalized to the 4 h value to account for different transfection efficiencies. (C) Effect of SMYD3 overexpression on replication of the full-length infectious Renilla reporter virus JcR-2A. Indicated cell lines were electroporated with JcR-2A RNA and harvested after 4 and 48 h. Viral replication was quantified as mentioned above. (D) Effect of SMYD3 overexpression on JcR-2A relative infectivity. Supernatants from (C) were harvested at the indicated time points and used to inoculate naïve Huh7.5 cells in duplicate. 48 h post infection, Renilla activity in reinfected cells was measured as in (B) Viral infectivity is expressed as ratio of re-infection over replication. (E) JcR-2A intra- and extracellular infectivity. Cells pellets and supernatants of cells transfected with JcR-2A RNA were subjected to 3 freeze-thaw cycles 48 h postelectroporation. Viral infectivity in the respective fractions was determined as mentioned above. (F) Effect of SMYD3 overexpression on intra- and extracellular virus titers of the non-reporter virus Jc1. The different Huh7-Lunet cell lines were transfected with Jc1 RNA. 48 h postelectroporation, cell pellets and supernatants were treated by freezing and thawing as described above. Viral infectivity was determined by limiting dilution assay and data are represented as TCID₅₀/ml. (G) Intra- and extracellular amounts of HCV core protein. Core levels in cell lysates and corresponding supernatants were determined by core-specific ELISA 4 and 48 h postelectroporation of Jc1 RNA. Concentrations of core are depicted as pg/ml normalized to intracellular core levels 4 h post electroporation to account for different transfection efficiencies. Bars represent mean values and standard error of the means from 3 (JcR-2A) or 2 (sgJFH1 and Jc1) independent experiments.