Type I IFNs induce the expression of IFN-stimulated gene 15 (ISG15)and its conjugation to cellular targets. ISGylation is a multistep process involving IFN-inducible Ube1L, UbcH8, and a yet-to-be identified E3 ligase. Here we report the identification of an IFNinduced HECT-type E3 protein ligase, HERC5͞Ceb1, which mediates ISGylation. We also defined a number of proteins modified by ISG15 after IFN triggering or HERC5 overexpression. A reduction in endogenous HERC5 by small interfering RNA inhibition blocks the IFN-induced ISG15 conjugation. Conversely, HERC5 coexpression with Ube1L and UbcH8 induces the ISG15 conjugation in vivo independent of IFN stimulation. A targeted substitution of Cys-994 to Ala in the HECT domain of HERC5 completely abrogates its E3 protein ligase activity. Therefore, this study demonstrates that HERC5͞Ceb1 is involved in the conjugation of ISG15 to cellular proteins.ISG15 ͉ Ceb1 ͉ innate immunity ͉ antiviral proteins T ype I IFNs (IFN-␣͞␤) play an essential role in both innate antiviral and adaptive immune responses and are rapidly produced in response to microbial infection (1-3). They exert signals through the activation of the Janus kinase-signal transducer and activator of transcription pathway that mediates rapid induction of IFN-stimulated genes (ISGs) (4, 5). ISG15 is one of the most strongly induced genes after IFN treatment (6, 7) and is also significantly induced by viral infection (8, 9) and LPS treatment (10, 11). The ISG15 protein starts with two ubiquitin-related domains that have Ϸ27% sequence identity to ubiquitin and terminate in a conserved 152 LRLRGG 157 ubiquitin C-terminal motif. This study suggests that ISG15 could act in a similar way to ubiquitin and other ubiquitin-like proteins such as SUMO by forming an isopeptide bond with cellular proteins (6, 12, 13). The crystal structure of ISG15 revealed that ISG15 consists of two domains with ubiquitinlike folds joined by a linker sequence (14).Conjugation of ISG15 to cellular proteins occurs in a parallel but distinct mechanism to that of ubiquitin (15-17). The E1 enzyme for ISG15, Ube1L, is a single-subunit enzyme and is identified in vitro by its ability to catalyze the formation of a thioester bond between ISG15 and Ube1L (17,18). The Ube1L protein is highly similar to the E1 enzyme for ubiquitin at the protein level. However, this protein does not form a conjugate with ubiquitin, indicating that Ube1L is an E1 enzyme for the ISG15 conjugation system (ISGylation). Influenza B virus blocks protein ISGylation by inhibiting the activation step through the interaction of the NS1B viral protein with ISG15 (18). This finding was the first suggestion that ISGylation might be important for protecting cells from viral infection.Two groups recently found that a member of the ubiquitin E2-conjugating enzyme family, UbcH8, is involved in the ISGylation (19,20). Like ISG15 and Ube1L, the expression of UbcH8 is also induced by IFN (21). The suppression of UbcH8 protein expression by RNA interference is shown to dramatically inhib...

Supplemental Digital Content is available in the text.