SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication.
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.
Affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.Show MeSH
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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.
Affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.