ISG15 conjugation system targets the viral NS1 protein in influenza A virus-infected cells.
ISG15 is an IFN-alpha/beta-induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-alpha/beta. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-alpha, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-beta. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a "loss of function" mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.
Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, TX 78712, USA.
Chen
C
Zhao
Tien-Ying
TY
Hsiang
Rei-Lin
RL
Kuo
Robert M
RM
Krug
D000998:Antiviral Agents / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007370:Interferon Type I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D014452:Ubiquitins / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D028901:alpha Karyopherins / Q000378:metabolism
D000998:Antiviral Agents; D016207:Cytokines; C402395:HERC5 protein, human; C085820:INS1 protein, influenza virus; D007370:Interferon Type I; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D014452:Ubiquitins; D017361:Viral Nonstructural Proteins; D028901:alpha Karyopherins; C074034:ISG15 protein, human; D008239:Lysine