Small ubiquitin-like modifier (SUMO) participates in a reversible 
posttranslational modification process (SUMOylation) that regulates a wide 
variety of cellular processes and plays important roles for numerous viruses 
during infection. However, the roles of viral protein SUMOylation in dengue 
virus (DENV) infection have not been elucidated. In this study, we found that 
the SUMOylation pathway was involved in the DENV life cycle, since DENV 
replication was reduced by silencing the cellular gene Ubc9, which encodes the 
sole E2-conjugating enzyme required for SUMOylation. By in vivo and in vitro 
SUMOylation assays, the DENV NS5 protein was identified as an authentic 
SUMO-targeted protein. 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. A 
DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe 
defect in viral RNA replication, supporting the notion that NS5 SUMOylation is 
required for DENV replication. SUMOylation-defective mutants also failed to 
suppress the induction of STAT2-mediated host antiviral interferon signaling. 
Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 
protein, which could account for most of the biological functions of SUMOylated 
NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is 
one of the mechanisms regulating DENV replication.
IMPORTANCE: SUMOylation is a common posttranslational modification that 
regulates cellular protein functions but has not been reported in the proteins 
of dengue virus. Here, we found that the replicase of DENV, nonstructural 
protein 5 (NS5), can be SUMOylated. It is well known that providing 
RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN 
signaling are a "double indemnity" of NS5 to support DENV replication. Without 
SUMOylation, NS5 fails to maintain its protein stability, which consequently 
disrupts its function in viral RNA replication and innate immunity antagonism. 
DENV threatens billions of people worldwide, but no licensed vaccine or specific 
therapeutics are currently available. Thus, our findings suggest that rather 
than specifically targeting NS5 enzyme activity, NS5 protein stability is a 
novel drug target on the growing list of anti-DENV strategies.