title: SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Chan-I
authors.initials: CI
authors.lastname: Su
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.|Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Chung-Hsin
authors.initials: CH
authors.lastname: Tseng
authors.affiliation: Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan michlai@gate.sinica.edu.tw epitope@mail.ncku.edu.tw.|Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Chia-Yi
authors.initials: CY
authors.lastname: Yu
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan michlai@gate.sinica.edu.tw epitope@mail.ncku.edu.tw.|Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan.|Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.|Research Center for Emerging Viruses, China Medical University Hospital, Taichung, Taiwan.
authors.forename: Michael M C
authors.initials: MMC
authors.lastname: Lai
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003715:Dengue / Q000235:genetics / Q000378:metabolism / Q000821:virology; D003716:Dengue Virus / Q000502:physiology*; D020868:Gene Silencing; D006801:Humans; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D034622:RNA Interference; D016415:Sequence Alignment; D058207:Sumoylation; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: C582223:NS5 protein, dengue virus; D017361:Viral Nonstructural Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9