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SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication.

Su CI, Tseng CH, Yu CY, Lai MM - J. Virol. (2016)

Bottom Line: By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation.SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling.Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

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SUMOylation of DENV NS5 is required for the replication of DENV replicon and for the antagonism of IFN signaling. (A) Endogenous Ubc9 protein expression level in Huh7 cells stably expressing shRNA targeting control (Ctrl) or Ubc9 (Ubc9) were analyzed by WB. (B) Huh7 cells stably expressing shRNA targeting Ctrl or Ubc9 were transfected with DENV replicon RNA harboring WT or SIM-mutated NS5 as indicated. The cell lysates were harvested and analyzed by luciferase assay after 24 h of transfection. The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test. (C) A549 cells were cotransfected with Vip-Luc (0.3 μg) and pRL-TK (0.1 μg) plus LacZ control, WT, or SIM-mutated DENV NS5 (0.5 μg) for 16 h. Subsequently, transfected cells were treated with IFN (500 U) or left untreated (0 U) for 8 h. The cells were harvested and analyzed by dual-luciferase assay. Data are expressed as means and SD (n = 3 per group) and were compared by two-tailed Student's t test. (D) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 16 h, followed by another 8-h treatment of IFN (500 U). The cells were collected and analyzed by WB with the indicated antibodies. (E) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 24 or 40 h and then analyzed by WB using the indicated antibodies.
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Figure 4: SUMOylation of DENV NS5 is required for the replication of DENV replicon and for the antagonism of IFN signaling. (A) Endogenous Ubc9 protein expression level in Huh7 cells stably expressing shRNA targeting control (Ctrl) or Ubc9 (Ubc9) were analyzed by WB. (B) Huh7 cells stably expressing shRNA targeting Ctrl or Ubc9 were transfected with DENV replicon RNA harboring WT or SIM-mutated NS5 as indicated. The cell lysates were harvested and analyzed by luciferase assay after 24 h of transfection. The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test. (C) A549 cells were cotransfected with Vip-Luc (0.3 μg) and pRL-TK (0.1 μg) plus LacZ control, WT, or SIM-mutated DENV NS5 (0.5 μg) for 16 h. Subsequently, transfected cells were treated with IFN (500 U) or left untreated (0 U) for 8 h. The cells were harvested and analyzed by dual-luciferase assay. Data are expressed as means and SD (n = 3 per group) and were compared by two-tailed Student's t test. (D) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 16 h, followed by another 8-h treatment of IFN (500 U). The cells were collected and analyzed by WB with the indicated antibodies. (E) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 24 or 40 h and then analyzed by WB using the indicated antibodies.

Mentions: Since the SIM motif is required for NS5 SUMOylation, we next determined its potential biological roles in DENV infection. Huh7 cells stably expressing the control shRNA (Ctrl) or Ubc9-targeting shRNA (Fig. 4A) were transfected with in vitro-transcribed RNA derived from WT or a SIM-mutated DENV subgenomic replicon containing a luciferase reporter gene, which reflects the viral RNA replication level. By measuring the luciferase activity, we found that the replication level of WT replicon was consistently and significantly lower in the cells stably expressing Ubc9-targeting shRNA than that of control shRNA (Fig. 4B), suggesting that Ubc9 activity is required for DENV RNA replication. In contrast, with the SIM-mutated replicon, the luciferase activity was further reduced in both the control and Ubc9 knockdown cells. Collectively, these results suggested that Ubc9-mediated SUMOylation is required for DENV RNA replication, and this posttranslational modification relies on the presence of the putative SIM motif of DENV NS5.


SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication.

Su CI, Tseng CH, Yu CY, Lai MM - J. Virol. (2016)

SUMOylation of DENV NS5 is required for the replication of DENV replicon and for the antagonism of IFN signaling. (A) Endogenous Ubc9 protein expression level in Huh7 cells stably expressing shRNA targeting control (Ctrl) or Ubc9 (Ubc9) were analyzed by WB. (B) Huh7 cells stably expressing shRNA targeting Ctrl or Ubc9 were transfected with DENV replicon RNA harboring WT or SIM-mutated NS5 as indicated. The cell lysates were harvested and analyzed by luciferase assay after 24 h of transfection. The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test. (C) A549 cells were cotransfected with Vip-Luc (0.3 μg) and pRL-TK (0.1 μg) plus LacZ control, WT, or SIM-mutated DENV NS5 (0.5 μg) for 16 h. Subsequently, transfected cells were treated with IFN (500 U) or left untreated (0 U) for 8 h. The cells were harvested and analyzed by dual-luciferase assay. Data are expressed as means and SD (n = 3 per group) and were compared by two-tailed Student's t test. (D) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 16 h, followed by another 8-h treatment of IFN (500 U). The cells were collected and analyzed by WB with the indicated antibodies. (E) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 24 or 40 h and then analyzed by WB using the indicated antibodies.
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Figure 4: SUMOylation of DENV NS5 is required for the replication of DENV replicon and for the antagonism of IFN signaling. (A) Endogenous Ubc9 protein expression level in Huh7 cells stably expressing shRNA targeting control (Ctrl) or Ubc9 (Ubc9) were analyzed by WB. (B) Huh7 cells stably expressing shRNA targeting Ctrl or Ubc9 were transfected with DENV replicon RNA harboring WT or SIM-mutated NS5 as indicated. The cell lysates were harvested and analyzed by luciferase assay after 24 h of transfection. The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test. (C) A549 cells were cotransfected with Vip-Luc (0.3 μg) and pRL-TK (0.1 μg) plus LacZ control, WT, or SIM-mutated DENV NS5 (0.5 μg) for 16 h. Subsequently, transfected cells were treated with IFN (500 U) or left untreated (0 U) for 8 h. The cells were harvested and analyzed by dual-luciferase assay. Data are expressed as means and SD (n = 3 per group) and were compared by two-tailed Student's t test. (D) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 16 h, followed by another 8-h treatment of IFN (500 U). The cells were collected and analyzed by WB with the indicated antibodies. (E) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 24 or 40 h and then analyzed by WB using the indicated antibodies.
Mentions: Since the SIM motif is required for NS5 SUMOylation, we next determined its potential biological roles in DENV infection. Huh7 cells stably expressing the control shRNA (Ctrl) or Ubc9-targeting shRNA (Fig. 4A) were transfected with in vitro-transcribed RNA derived from WT or a SIM-mutated DENV subgenomic replicon containing a luciferase reporter gene, which reflects the viral RNA replication level. By measuring the luciferase activity, we found that the replication level of WT replicon was consistently and significantly lower in the cells stably expressing Ubc9-targeting shRNA than that of control shRNA (Fig. 4B), suggesting that Ubc9 activity is required for DENV RNA replication. In contrast, with the SIM-mutated replicon, the luciferase activity was further reduced in both the control and Ubc9 knockdown cells. Collectively, these results suggested that Ubc9-mediated SUMOylation is required for DENV RNA replication, and this posttranslational modification relies on the presence of the putative SIM motif of DENV NS5.

Bottom Line: By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation.SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling.Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Show MeSH
Related in: MedlinePlus