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...
The ubiquitin-like ISG15 protein, as well as its conjugating enzymes, is induced by type I interferons (IFNs). Experiments using ISG15 knockout (ISG15؊/؊ ) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus. However, in contrast to the virus inhibition results for mice, the rates of virus replication in ISG15 ؉/؉ and ISG15 ؊/؊ mouse embryo fibroblasts in tissue culture were similar. Here we focus on human tissue culture cells and on the effect of ISG15 and/or its conjugation on influenza A virus gene expression and replication in such cells. We demonstrate that IFN-induced antiviral activity against influenza A virus in human cells is significantly alleviated by inhibiting ISG15 conjugation using small interfering RNAs directed against ISG15-conjugating enzymes. IFN-induced antiviral activity against influenza A virus protein synthesis was reduced 5-to 20-fold by suppressing ISG15 conjugation. The amounts of the viral proteins that were restored by these siRNA treatments were approximately 40 to 50% of the amounts produced in cells that were not pretreated with IFN. Further, we show that ISG15 conjugation inhibits influenza A virus replication 10-to 20-fold at early times after infection in human cells. These results show that ISG15 conjugation plays a substantial role in the antiviral state induced by IFN in human cells. In contrast, we show that in mouse embryo fibroblasts ISG15 conjugation not only does not affect influenza A virus replication but also does not contribute to the IFN-induced antiviral activity against influenza A virus gene expression.Virus infection activates the synthesis of type I interferons (IFN-␣ and IFN-), which induce the synthesis of a large array of proteins, many of which play crucial roles in the antiviral response (1). One of the most strongly induced proteins is ISG15, a 15-kDa ubiquitin-like protein that becomes conjugated to many cellular proteins (6,8,9,12,18,22,26,30). Three of the human enzymes that catalyze this conjugation, the UbE1L E1 enzyme, the UbcH8 E2 enzyme, and the Herc5 E3 enzyme, are also induced by 10,26,27,29). Although it had been reported that UbcH8 functions in both ISG15 and ubiquitin conjugation (3,10,13,25,28,29), a recent study demonstrated that UbcH8 is unlikely to function in ubiquitin conjugation in vivo for two reasons: K m measurements revealed that the E1 ubiquitin-activating enzyme, unlike UbE1L, exhibits very low affinity for UbcH8, and UbcH8 is poorly, if not at all, expressed in the absence of IFN treatment, indicating that UbcH8 functions only during the IFN response (5). A large number of human proteins that are targets for ISG15 conjugation have been identified (22,26,30). Most of these targets are constitutively expressed proteins that function in diverse cellular pathways, but several of the targets are IFN-␣/--induced antiviral proteins.Because the NS1 protein of influenza B virus (NS1B) was shown to bind ISG15 and inhibit its conjugation to target proteins, it was proposed that ISG15 and/or...
ISG15 is an interferon-induced ubiquitin-like protein that is conjugated to target proteins via the sequential action of three enzymes that are also induced by interferon. Unlike ubiquitin, which is highly conserved, the sequence of ISG15 varies between species. ISG15 conjugation inhibits many viruses, and free (unconjugated) ISG15 can also act as an antiviral protein. Here we focus on the antiviral role of ISG15 conjugation and on countermeasures employed by several viruses. The countermeasure by influenza B virus is unique in that it exhibits species-specificity. Only the antiviral activity of human and non-human primate ISG15s can be blocked, providing one possible explanation for the restriction of influenza B virus to humans.
Retinoic acid inducible gene-I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the immune response against many RNA viruses. Upon RNA ligand binding, RIG-I undergoes a conformational change facilitating its homo-oligomerization and activation that results in its translocation from the cytosol to intracellular membranes to bind its signaling adaptor protein, mitochondrial antiviral-signaling protein (MAVS). Here we show that RIG-I activation is regulated by reversible acetylation. Acetyl-mimetic mutants of RIG-I do not form virus-induced homo-oligomers, revealing that acetyl-lysine residues of the RIG-I repressor domain prevent assembly to active homo-oligomers. During acute infection, deacetylation of RIG-I promotes its oligomerization upon ligand binding. We identify histone deacetylase 6 (HDAC6) as the deacetylase that promotes RIG-I activation and innate antiviral immunity to recognize and restrict RNA virus infection.
We demonstrate that phosphorylation of the NS1 protein of a human influenza A virus occurs not only at the threonine (T) at position 215 but also at serines (Ss), specifically at positions 42 and 48. By generating recombinant influenza A/Udorn/72 (Ud) viruses that encode mutant NS1 proteins, we determined the roles of these phosphorylations in virus replication. At position 215 only a T-to-A substitution attenuated replication, whereas other substitutions (T to E to mimic constitutive phosphorylation, T to N, and T to P, the amino acid in avian influenza A virus NS1 proteins) had no effect. We conclude that attenuation resulting from the T-to-A substitution at position 215 is attributable to a deleterious structural change in the NS1 protein that is not caused by other amino acid substitutions and that phosphorylation of T215 does not affect virus replication. At position 48 neither an S-to-A substitution nor an S-to-D substitution that mimics constitutive phosphorylation affected virus replication. In contrast, at position 42, an S-to-D, but not an S-to-A, substitution caused attenuation. The S-to-D substitution eliminates detectable double-stranded RNA binding by the NS1 protein, accounting for attenuation of virus replication. We show that protein kinase C ␣ (PKC␣) catalyzes S42 phosphorylation. Consequently, the only phosphorylation of the NS1 protein of this human influenza A virus that regulates its replication is S42 phosphorylation catalyzed by PKC␣. In contrast, phosphorylation of Ts or Ss in the NS1 protein of the 2009 H1N1 pandemic virus was not detected, indicating that NS1 phosphorylation probably does not play any role in the replication of this virus. Influenza A viruses cause a highly contagious respiratory disease in humans, resulting in annual epidemics and periodic worldwide pandemics. The smallest of the eight negative-sense viral gene segments encodes the NS1 protein, a nonstructural protein that plays multiple crucial roles in virus replication (5). Most NS1 proteins are 230 to 237 amino acids long, although shorter forms have also been found. The NS1 protein comprises two functional domains: the N-terminal (amino acids 1 to 73) RNA-binding domain (RBD) and the C-terminal (amino acids 74 to 230/237) effector domain (ED), which binds several cellular proteins.The NS1 protein undergoes three posttranslational modifications: phosphorylation by cellular kinases (4, 12, 13), ISG15 modification by the interferon (IFN)-induced ISG15 conjugation system (17, 21), and modification by SUMO (11,20). It has been reported that these modifications affect the NS1 protein and the phenotype of the virus (4, 17, 21). Phosphorylation of threonines (Ts) of the NS1 protein was observed many years ago (12, 13). It was reported that T at position 215 (T215) of the NS1 protein of the human H3N2 influenza A/Udorn/72 virus (Ud) is phosphorylated and that this phosphorylation is important for virus replication (4). The basis for the latter conclusion was that a recombinant Ud virrus encoding an NS1 protein with a T-to...
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