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