title: ISG15 Arg151 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of Sindbis virus.
abstract: 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-alphabetaR(-/-)) 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-alphabetaR(-/-) 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.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Nadia V
authors.initials: NV
authors.lastname: Giannakopoulos
authors.forename: Elena
authors.initials: E
authors.lastname: Arutyunova
authors.forename: Caroline
authors.initials: C
authors.lastname: Lai
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Lenschow
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Haas
authors.forename: Herbert Whiting
authors.initials: HW
authors.lastname: Virgin
mesh_terms: D018354:Alphavirus Infections / Q000276:immunology*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012845:Sindbis Virus / Q000276:immunology*; D016019:Survival Analysis; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: D016207:Cytokines; C489550:G1p2 protein, mouse; D014452:Ubiquitins; C074034:ISG15 protein, human; C471622:UBE2L6 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C534879:UBA7 protein, human; D044764:Ubiquitin-Activating Enzymes