ISG15 functions as a critical antiviral molecule against influenza virus, with infection inducing both the conjugation of ISG15 to target proteins and production of free ISG15. Here, we report that mice lacking the ISG15 E1 enzyme UbE1L fail to form ISG15 conjugates. Both UbE1L ؊/؊ and ISG15 ؊/؊ mice display increased susceptibility to influenza B virus infection, including non-mouse-adapted strains. Finally, we demonstrate that ISG15 controls influenza B virus infection through its action within radioresistant stromal cells and not bone marrow-derived cells. Thus, the conjugation of ISG15 to target proteins within stromal cells is critical to its activity against influenza virus.One of the earliest host responses to viral infection is the production of type I interferons (IFN-␣ and -) and the subsequent upregulation of IFN-stimulated genes (ISGs). These ISGs generate an antiviral state in nearby cells and also play an important role in shaping the host innate and adaptive immune response (26, 28). We and others have recently identified ISG15 as a critical IFN-induced antiviral molecule. Overexpression of ISG15 by a recombinant Sindbis virus protected IFN-␣ receptor-deficient mice from lethality (13). In a cell culture system, the overexpression of ISG15 also inhibited the release of human immunodeficiency virus virions (20) and decreased alphavirus replication (32). Finally, mice lacking ISG15 are susceptible to several human pathogens, including influenza A and B viruses, herpesviruses, and Sindbis viruses (14). Though it is clear that ISG15 functions as an antiviral molecule, its mechanism and site of action remain poorly understood.ISG15, a 17-kDa ubiquitin-like molecule, contains two ubiquitin domains, including a carboxy-terminal LRLRGG motif, through which it forms conjugates with target proteins (15,19). ISG15 conjugation of target proteins (ISGylation) utilizes an IFN-induced conjugation cascade which includes an E1 (UbE1L/Uba7), an E2 (UbcH8), several E3 ligases (EFP, HHARI, and Herc5), and a deconjugating protease (UBP43/ USP18) (1,11,17,22,31,33,35). Activation of this pathway results in the conjugation of ISG15 to over 100 known target proteins that encompass multiple biological pathways (8,16,30,34). In addition to forming conjugates, free ISG15 also accumulates within cells and is released into the sera of patients following stimulation with IFN (5). Recombinant ISG15 has been reported to function as a cytokine, stimulating IFN-␥ production, NK cell proliferation, neutrophil chemotaxis, and dendritic cell maturation (4,(23)(24)(25). During viral infection in mice, ISG15 exists in three forms: (i) unconjugated within cells, (ii) conjugated to target proteins, and (iii) released into the serum (14). Studies with Sindbis virus suggest that the conjugated form of ISG15 mediates its antiviral activity. The increased lethality seen in ISG15 Ϫ/Ϫ mice can be rescued by a recombinant virus expressing wild-type ISG15 but not mutant ISG15, which cannot form conjugates in vitro (14). In contrast, two recent...
Interferon (IFN)-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that conjugates to target proteins via a C-terminal LRLRGG motif and has antiviral function in vivo. We used structural modeling to predict human ISG15 (hISG15) residues important for interacting with its E1 enzyme, UbE1L. Kinetic analysis revealed that mutation of arginine 153 to alanine (R153A) ablated hISG15-hUbE1L binding and transthiolation of UbcH8. Mutation of other predicted UbE1L-interacting residues had minimal effects on the transfer of ISG15 from UbE1L to UbcH8. The capacity of hISG15 R153A to form protein conjugates in 293T cells was markedly diminished. Mutation of the homologous residue in mouse ISG15 (mISG15), arginine 151, to alanine (R151A) also attenuated protein ISGylation following transfection into 293T cells. We assessed the role of ISG15-UbE1L interactions in control of virus infection by constructing double subgenomic Sindbis viruses that expressed the mISG15 R151A mutant. While expression of mISG15 protected alpha/beta-IFN-receptor-deficient (IFN-␣R ؊/؊ ) mice from lethality following Sindbis virus infection, expression of mISG15 R151A conferred no survival benefit. The R151A mutation also attenuated ISG15's ability to decrease Sindbis virus replication in IFN-␣R ؊/؊ mice or prolong survival of ISG15 ؊/؊ mice. The importance of UbE1L was confirmed by demonstrating that mice lacking this ISG15 E1 enzyme were highly susceptible to Sindbis virus infection. Together, these data support a role for protein conjugation in the antiviral effects of ISG15.
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