Ebola virus budding is mediated by the VP40 matrix protein. VP40 can bud from mammalian cells independent of other viral proteins, and efficient release of VP40 virus-like particles (VLPs) requires interactions with host proteins such as tsg101 and Nedd4, an E3 ubiquitin ligase. Ubiquitin itself is thought to be exploited by Ebola virus to facilitate efficient virus egress. Disruption of VP40 function and thus virus budding remains an attractive target for the development of novel antiviral therapies. Here, we investigate the effect of ISG15 protein on the release of Ebola VP40 VLPs. ISG15 is an IFN-inducible, ubiquitinlike protein expressed after bacterial or viral infection. Our results show that expression of free ISG15, or the ISGylation system (UbE1L and UbcH8), inhibits budding of Ebola virus VP40 VLPs. Addressing the molecular mechanism of this inhibition, we show that ISG15 interacts with Nedd4 ubiquitin ligase and inhibits ubiquitination of VP40. Furthermore, the L-domain deletion mutant of VP40 (⌬PT/PY), which does not interact with Nedd4, was insensitive to ISG15-mediated inhibition of VLP release. These data provide evidence of antiviral activity of ISG15 against Ebola virus and suggest a mechanism of action involving disruption of Nedd4 function and subsequent ubiquitination of VP40.Ebola virus ͉ interferon ͉ innate immunity ͉ ubiquitin T he IFN pathway activates hundreds of cellular IFNstimulated genes (ISGs), some of which have direct antiviral activity (1-5). For example, ISG15 was one of the first recognized ISG proteins whose expression was up-regulated not only by IFN but also by viral infection, LPS treatment, and retinoic acid (6-8). ISG15 has high homology to ubiquitin and can be detected in cells in both free and conjugated forms (9, 10). ISG15 conjugation (ISGylation) to proteins uses cascades of enzymatic reactions similar to those used in protein ubiquitination pathways (11). Some of these enzymes, like ubiquitin E1-like protein (UBE1L) (12, 13), are unique for ISG15, whereas two E2 enzymes, UbcH8 and UbcH6, are also used in the ubiquitination pathway (14)(15)(16) The observation that ISG15 conjugation targets many components of the antiviral signaling pathway suggests that ISG15 may play a role in the innate antiviral response (12,17). To this effect, it was shown that the NS1 protein of the Influenza B virus inhibits ISGylation (12), and ISG15 expression decreased Sindbis virus replication and provided protection against lethal infection (12, 18). Also, ISG15-null mice showed an increase susceptibility to both DNA-and RNA-containing viruses in vivo, including influenza, herpes simplex, and Sindbis viruses (19). Recently, inhibition of HIV-1 virion release in IFN-treated cells was shown to be mediated by ISG15 (20). Several reports have also linked induction and expression of ISG15 to inhibition of important viral pathogens of fish (21,22). Together, these studies suggest that ISG15 has a potent and broad-based antiviral effect; however, the mechanism of action of ISG15 remains to be d...