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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 regulates the stability of NS5. (A and B) BHK-21 cells were transfected with WT or SIM-mutated NS5 for 24 h and then treated with cycloheximide (CHX). Cells were harvested at different time points after CHX treatment as indicated. (A) The NS5 protein expression levels were analyzed by WB using antibodies against HA and actin. (B) The band intensity of NS5 was quantified by ImageJ and normalized to that of actin. The relative quantities (RQ) were calculated and are shown as percentages of the quantities at 0 h. (C) BHK-21 cells were transfected with SIM-mutated NS5 for 24 h, designated hour 0, and then either left untreated or treated with CHX (lanes 2 to 6) together with the indicated inhibitors for another 3 h. Cells were harvested and analyzed by WB with anti-HA and anti-actin antibodies.
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Figure 5: SUMOylation regulates the stability of NS5. (A and B) BHK-21 cells were transfected with WT or SIM-mutated NS5 for 24 h and then treated with cycloheximide (CHX). Cells were harvested at different time points after CHX treatment as indicated. (A) The NS5 protein expression levels were analyzed by WB using antibodies against HA and actin. (B) The band intensity of NS5 was quantified by ImageJ and normalized to that of actin. The relative quantities (RQ) were calculated and are shown as percentages of the quantities at 0 h. (C) BHK-21 cells were transfected with SIM-mutated NS5 for 24 h, designated hour 0, and then either left untreated or treated with CHX (lanes 2 to 6) together with the indicated inhibitors for another 3 h. Cells were harvested and analyzed by WB with anti-HA and anti-actin antibodies.

Mentions: It is noted that the protein expression level of SIM-mutated NS5 was almost the same as that of the WT at 24 h posttransfection but significantly lower than that of the WT at 40 h posttransfection (Fig. 4E, lanes 2, 3, 5, and 6). This finding raised the possibility that SUMOylation stabilizes DENV NS5, thereby enhancing the biological activity of NS5. To check this possibility, A549 cells transfected with WT or SIM-mutated NS5 were treated with cycloheximide (CHX), an inhibitor of protein translation. We found that the amount of WT NS5 protein remained at a high level throughout the 24-h period after CHX treatment, whereas the SIM-mutated NS5 protein degraded rapidly in the same period (Fig. 5A). The quantitative analysis of the kinetics of protein reduction showed that WT NS5 was more stable than the SIM mutant in A549 cells (Fig. 5B). We found that protein expression levels of HA-tagged, SIM-mutated NS5 after CHX treatment was not recovered by either reversible MG132 or irreversible lactacystin, the chemical inhibitors targeting proteasomal machineries (Fig. 5C). Thus, our data suggested that the failure of NS5 to be SUMOylated lowers the stability of NS5 and leads the latter to degradation through proteasome-independent machinery.


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

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

SUMOylation regulates the stability of NS5. (A and B) BHK-21 cells were transfected with WT or SIM-mutated NS5 for 24 h and then treated with cycloheximide (CHX). Cells were harvested at different time points after CHX treatment as indicated. (A) The NS5 protein expression levels were analyzed by WB using antibodies against HA and actin. (B) The band intensity of NS5 was quantified by ImageJ and normalized to that of actin. The relative quantities (RQ) were calculated and are shown as percentages of the quantities at 0 h. (C) BHK-21 cells were transfected with SIM-mutated NS5 for 24 h, designated hour 0, and then either left untreated or treated with CHX (lanes 2 to 6) together with the indicated inhibitors for another 3 h. Cells were harvested and analyzed by WB with anti-HA and anti-actin antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: SUMOylation regulates the stability of NS5. (A and B) BHK-21 cells were transfected with WT or SIM-mutated NS5 for 24 h and then treated with cycloheximide (CHX). Cells were harvested at different time points after CHX treatment as indicated. (A) The NS5 protein expression levels were analyzed by WB using antibodies against HA and actin. (B) The band intensity of NS5 was quantified by ImageJ and normalized to that of actin. The relative quantities (RQ) were calculated and are shown as percentages of the quantities at 0 h. (C) BHK-21 cells were transfected with SIM-mutated NS5 for 24 h, designated hour 0, and then either left untreated or treated with CHX (lanes 2 to 6) together with the indicated inhibitors for another 3 h. Cells were harvested and analyzed by WB with anti-HA and anti-actin antibodies.
Mentions: It is noted that the protein expression level of SIM-mutated NS5 was almost the same as that of the WT at 24 h posttransfection but significantly lower than that of the WT at 40 h posttransfection (Fig. 4E, lanes 2, 3, 5, and 6). This finding raised the possibility that SUMOylation stabilizes DENV NS5, thereby enhancing the biological activity of NS5. To check this possibility, A549 cells transfected with WT or SIM-mutated NS5 were treated with cycloheximide (CHX), an inhibitor of protein translation. We found that the amount of WT NS5 protein remained at a high level throughout the 24-h period after CHX treatment, whereas the SIM-mutated NS5 protein degraded rapidly in the same period (Fig. 5A). The quantitative analysis of the kinetics of protein reduction showed that WT NS5 was more stable than the SIM mutant in A549 cells (Fig. 5B). We found that protein expression levels of HA-tagged, SIM-mutated NS5 after CHX treatment was not recovered by either reversible MG132 or irreversible lactacystin, the chemical inhibitors targeting proteasomal machineries (Fig. 5C). Thus, our data suggested that the failure of NS5 to be SUMOylated lowers the stability of NS5 and leads the latter to degradation through proteasome-independent machinery.

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