ISG15 conjugation system targets the viral NS1 protein in influenza A virus–infected cells

Abstract

ISG15 is an IFN-α/β-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-α/β. 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-α, 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-β. 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.SG15 is a ubiquitin-like molecule that is highly induced by IFN α/β (1). It is conjugated to more than 100 cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β: E1 (Ube1L) (2), E2 (UbcH8) (3, 4), and E3 (Herc5) (5, 6). The vast majority of IFN-induced ISG15 conjugation is mediated by a single E3 enzyme, Herc5, in contrast to the ubiquitin system that uses a large number of E3 enzymes to accomplish target selectivity (7).ISG15 and/or its conjugation play important roles in innate immunity against several viruses. The first clue to the antiviral property of ISG15 conjugation was the finding that the NS1 protein of influenza B virus binds ISG15 and blocks its conjugation, suggesting that ISG15 and/or its conjugation is inhibitory to the replication of influenza B virus (2). Subsequently, the antiinfluenza activity of ISG15 and/or its conjugation was established by the demonstration that ISG15 knockout (ISG15 −/− ) mice are more susceptible to both influenza A and B virus infection (8). Experiments with Ube1L −/− mice established that ISG15 conjugation rather than free ISG15 inhibits influenza B virus replication (9). Further, we established that ISG15 conjugation plays a large role in the IFN-induced antiviral state against influenza A virus in human tissue culture cells (10). Thus, siRNA-silencing of ISG15 conjugation enzymes inhibited IFN-induced ISG15 conjugation and parti...

Activation of Double-stranded RNA-activated Protein Kinase (PKR) by Interferon-stimulated Gene 15 (ISG15) Modification Down-regulates Protein Translation

“…This is also true for modification by other ubiquitin like proteins, such as SUMO. It has been reported that these minor ISGylated fractions have important physiological roles, such as tumor-suppressive effect through down-regulation of ⌬Np63␣ (46), inhibition of influenza A virus replication (47), sustained IRF3 activation (48), and prevention of JNK cascade (49). These reports support the significant physiological role of ISGylated PKR on the protein translation although ISGylated PKR is minor fraction.…”

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

“…The first was SUMOylation, the conjugation of the small ubiquitin-like modifier (SUMO) to NS1 (29). The second was ISGylation, the conjugation of IFNstimulated gene 15 (ISG15) to NS1 (30). The initial report identifying NS1 as an ISGylated protein indicated that ISGylation prevented the interaction of NS1 with importin-␣, a karyopherin essential for its nuclear import (30).…”

“…The second was ISGylation, the conjugation of IFNstimulated gene 15 (ISG15) to NS1 (30). The initial report identifying NS1 as an ISGylated protein indicated that ISGylation prevented the interaction of NS1 with importin-␣, a karyopherin essential for its nuclear import (30). A subsequent report described three additional roles for NS1 ISGylation: disrupting NS1 dimerization, preventing its interaction with PKR, and exhibiting an overall negative effect on NS1's ability to neutralize the IFN system (31).…”

Structural basis for the sequence-specific recognition of human ISG15 by the NS1 protein of influenza B virus

“…IFN-induced ISG15 conjugation plays an important antiviral role against several viruses, including influenza A and B viruses, Sindbis virus, herpes simplex virus type 1, murine gammaherpes virus, Japanese encephalitis virus, and vaccinia virus (8)(9)(10)(11)(12)(13). A major mechanism by which ISG15 conjugation inhibits one of these viruses, influenza A virus, has been identified (10,14).…”

“…A major mechanism by which ISG15 conjugation inhibits one of these viruses, influenza A virus, has been identified (10,14). Specifically, the ISG15 conjugation system targets a viral protein, the NS1 protein (NS1A), which is responsible for countering key components of the host antiviral system.…”