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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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Silencing SUMO E2 enzyme Ubc9 represses DENV replication. (A and B) A549 cells were transfected with the indicated siRNAs for 48 h and then infected with DENV serotype 2 (MOI of 0.1) for another 48 h. Intracellular protein expression levels and infectious DENV titer in the culture supernatant were measured by Western blotting (WB) (A) and by plaque forming assay (B), respectively. Data are expressed as means and standard deviations (SD) (n = 3 per group) and were compared to those for siCtrl by two-tailed Student's t test. (C and D) A stable BHK-21 cell line harboring DENV2 replicon were transfected with the indicated siRNAs for 48 h. WB (C) and luciferase assay (D) were carried out to analyze the indicated protein expression levels and the replication levels of DENV2 replicon. (E and F) A549 cells stably expressing shRNA targeting control LacZ or Ubc9 were infected with DENV (MOI, 0.1 and 10) for 24 h. Cell lysates were analyzed by WB with the indicated antibodies (E), and culture supernatants were harvested for virus titration by performing plaque assays (F). The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test.
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Figure 1: Silencing SUMO E2 enzyme Ubc9 represses DENV replication. (A and B) A549 cells were transfected with the indicated siRNAs for 48 h and then infected with DENV serotype 2 (MOI of 0.1) for another 48 h. Intracellular protein expression levels and infectious DENV titer in the culture supernatant were measured by Western blotting (WB) (A) and by plaque forming assay (B), respectively. Data are expressed as means and standard deviations (SD) (n = 3 per group) and were compared to those for siCtrl by two-tailed Student's t test. (C and D) A stable BHK-21 cell line harboring DENV2 replicon were transfected with the indicated siRNAs for 48 h. WB (C) and luciferase assay (D) were carried out to analyze the indicated protein expression levels and the replication levels of DENV2 replicon. (E and F) A549 cells stably expressing shRNA targeting control LacZ or Ubc9 were infected with DENV (MOI, 0.1 and 10) for 24 h. Cell lysates were analyzed by WB with the indicated antibodies (E), and culture supernatants were harvested for virus titration by performing plaque assays (F). The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test.

Mentions: Recent evidence indicated that the cellular SUMO conjugase Ubc9, a key component for protein SUMOylation, interacts with several dengue viral proteins (32, 34, 38). To determine whether the SUMO modification pathway is involved in DENV replication, we suppressed the SUMOylation system by knocking down Ubc9 expression through RNA interference (RNAi) silencing approaches. In A549 cells transfected with Ubc9-specific siRNA, the expression of Ubc9 was substantially suppressed; correspondingly, the production of viral nonstructural proteins (represented by NS3 and NS5) also was substantially decreased after DENV infection (Fig. 1A). For comparison, the transfection of the DENV-specific siRNA into DENV-infected cells showed an almost complete loss of viral replication. Consistent with this, culture media derived from DENV-infected siUbc9 cells also showed a lower viral titer than that from the control siRNA-transfected cells (Fig. 1B). To further understand the mechanism of the involvement of the cellular SUMO modification system in the DENV replication processes, we used a stable cell line, BHK21-DENV2-SGR, containing a replication-competent DENV subgenomic RNA replicon, which expresses viral nonstructural proteins and core protein. A firefly luciferase reporter gene was fused in frame with the DENV replicon to monitor the viral RNA translation and replication statuses (36). We found that both viral protein expression (Fig. 1C) and replicon luciferase activity (Fig. 1D) were inhibited by silencing endogenous Ubc9 expression in BHK21-DENV2-SGR cells. The human A549 cells harboring shRNA that stably knocked down endogenous Ubc9 displayed similar characteristics upon DENV infection. We found that both the expression of viral proteins (Fig. 1E) and the production of viral progeny (Fig. 1F) were slightly reduced in shUbc9 cells upon DENV infection at either a low or high multiplicity of infection (MOI). Despite the incomplete knockdown of Ubc9 in A549 cells (Fig. 1E), these results consistently indicated that the cellular SUMO modification system is involved in the DENV life cycle.


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

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

Silencing SUMO E2 enzyme Ubc9 represses DENV replication. (A and B) A549 cells were transfected with the indicated siRNAs for 48 h and then infected with DENV serotype 2 (MOI of 0.1) for another 48 h. Intracellular protein expression levels and infectious DENV titer in the culture supernatant were measured by Western blotting (WB) (A) and by plaque forming assay (B), respectively. Data are expressed as means and standard deviations (SD) (n = 3 per group) and were compared to those for siCtrl by two-tailed Student's t test. (C and D) A stable BHK-21 cell line harboring DENV2 replicon were transfected with the indicated siRNAs for 48 h. WB (C) and luciferase assay (D) were carried out to analyze the indicated protein expression levels and the replication levels of DENV2 replicon. (E and F) A549 cells stably expressing shRNA targeting control LacZ or Ubc9 were infected with DENV (MOI, 0.1 and 10) for 24 h. Cell lysates were analyzed by WB with the indicated antibodies (E), and culture supernatants were harvested for virus titration by performing plaque assays (F). The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test.
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Figure 1: Silencing SUMO E2 enzyme Ubc9 represses DENV replication. (A and B) A549 cells were transfected with the indicated siRNAs for 48 h and then infected with DENV serotype 2 (MOI of 0.1) for another 48 h. Intracellular protein expression levels and infectious DENV titer in the culture supernatant were measured by Western blotting (WB) (A) and by plaque forming assay (B), respectively. Data are expressed as means and standard deviations (SD) (n = 3 per group) and were compared to those for siCtrl by two-tailed Student's t test. (C and D) A stable BHK-21 cell line harboring DENV2 replicon were transfected with the indicated siRNAs for 48 h. WB (C) and luciferase assay (D) were carried out to analyze the indicated protein expression levels and the replication levels of DENV2 replicon. (E and F) A549 cells stably expressing shRNA targeting control LacZ or Ubc9 were infected with DENV (MOI, 0.1 and 10) for 24 h. Cell lysates were analyzed by WB with the indicated antibodies (E), and culture supernatants were harvested for virus titration by performing plaque assays (F). The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test.
Mentions: Recent evidence indicated that the cellular SUMO conjugase Ubc9, a key component for protein SUMOylation, interacts with several dengue viral proteins (32, 34, 38). To determine whether the SUMO modification pathway is involved in DENV replication, we suppressed the SUMOylation system by knocking down Ubc9 expression through RNA interference (RNAi) silencing approaches. In A549 cells transfected with Ubc9-specific siRNA, the expression of Ubc9 was substantially suppressed; correspondingly, the production of viral nonstructural proteins (represented by NS3 and NS5) also was substantially decreased after DENV infection (Fig. 1A). For comparison, the transfection of the DENV-specific siRNA into DENV-infected cells showed an almost complete loss of viral replication. Consistent with this, culture media derived from DENV-infected siUbc9 cells also showed a lower viral titer than that from the control siRNA-transfected cells (Fig. 1B). To further understand the mechanism of the involvement of the cellular SUMO modification system in the DENV replication processes, we used a stable cell line, BHK21-DENV2-SGR, containing a replication-competent DENV subgenomic RNA replicon, which expresses viral nonstructural proteins and core protein. A firefly luciferase reporter gene was fused in frame with the DENV replicon to monitor the viral RNA translation and replication statuses (36). We found that both viral protein expression (Fig. 1C) and replicon luciferase activity (Fig. 1D) were inhibited by silencing endogenous Ubc9 expression in BHK21-DENV2-SGR cells. The human A549 cells harboring shRNA that stably knocked down endogenous Ubc9 displayed similar characteristics upon DENV infection. We found that both the expression of viral proteins (Fig. 1E) and the production of viral progeny (Fig. 1F) were slightly reduced in shUbc9 cells upon DENV infection at either a low or high multiplicity of infection (MOI). Despite the incomplete knockdown of Ubc9 in A549 cells (Fig. 1E), these results consistently indicated that the cellular SUMO modification system is involved in the DENV life cycle.

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