K48-linked polyubiquitination of dengue virus NS1 protein inhibits its interaction with the viral partner NS4B

Virus Res. 2018 Feb 15:246:1-11. doi: 10.1016/j.virusres.2017.12.013. Epub 2017 Dec 30.

Abstract

Dengue virus (DENV) is a member of the Flaviviridae family, which is transmitted to mammalian species through arthropods, and causes dengue fever or severe dengue fever in humans. The DENV genome encodes for multiple nonstructural (NS) proteins including NS1. NS1 plays an essential role in replication by interacting with other viral proteins including NS4B, however how these interactions are regulated during virus infection is not known. By using bioinformatics, mass spectrometry analysis, and co-immunoprecipitation assays, here we show that DENV-NS1 is ubiquitinated on multiples lysine residues during DENV infection, including K189, a lysine residue previously shown to be important for efficient DENV replication. Data from in vitro and cell culture experiments indicate that dengue NS1 undergoes modification with K48-linked polyubiquitin chains, which usually target proteins to the proteasome for degradation. Furthermore, ubiquitinated NS1 was detected in lysates as well as in supernatants of human and mosquito infected cells. Ubiquitin deconjugation of NS1 using the deubiquitinase OTU resulted in increased interaction with the viral protein NS4B suggesting that ubiquitinated NS1 has reduced affinity for NS4B. In support of these data, a K189R mutation on NS1, which abrogates ubiquitination on amino acid residue 189 of NS1, also increased NS1-NS4B interactions. Our work describes a new mechanism of regulation of NS1-NS4B interactions and suggests that ubiquitination of NS1 may affect DENV replication.

Keywords: Dengue virus; Deubiquitinase; K48-linked polyubiquitin; NS1; NS4B; Proteasome; Ubiquitination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line, Tumor
  • Computational Biology
  • Culicidae
  • Dengue Virus / genetics*
  • Dengue Virus / metabolism
  • Gene Expression Regulation, Viral*
  • HEK293 Cells
  • Hepatocytes / drug effects
  • Hepatocytes / virology
  • Humans
  • Lysine / metabolism*
  • Mutation
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Binding
  • Proteolysis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Ubiquitin-Specific Proteases / chemistry
  • Ubiquitin-Specific Proteases / pharmacology
  • Ubiquitination
  • Viral Nonstructural Proteins / genetics*
  • Viral Nonstructural Proteins / metabolism
  • Virus Replication*

Substances

  • NS1 protein, Dengue virus type 2
  • NS4B protein, flavivirus
  • Recombinant Proteins
  • Viral Nonstructural Proteins
  • OTUD1 protein, human
  • Ubiquitin-Specific Proteases
  • Proteasome Endopeptidase Complex
  • Lysine