ISG15 Inhibits Nedd4 Ubiquitin E3 Activity and Enhances the Innate Antiviral Response*

Abstract

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in antiviral responses. The interferon-inducible ubiquitin-like protein ISG15 is expressed in cells in response to a variety of stress conditions like viral or bacterial infection and is present in its free form or is conjugated to cellular proteins. In addition, protein ubiquitination plays a regulatory role in the immune system. Many viruses modulate the ubiquitin (Ub) pathway to alter cellular signaling and the antiviral response. Ubiquitination of retroviral group-specific antigen precursors and matrix proteins of the Ebola, vesicular stomatitis, and rabies viruses by Nedd4 family HECT domain E3 ligases is an important step in facilitating viral release. We found that Nedd4 is negatively regulated by ISG15. Free ISG15 specifically bound to Nedd4 and blocked its interaction with Ub-E2 molecules, thus preventing further Ub transfer from E2 to E3. Furthermore, overexpression of ISG15 diminished the ability of Nedd4 to ubiquitinate viral matrix proteins and led to a decrease in the release of Ebola VP40 virus-like particles from the cells. These results point to a mechanistically novel function of ISG15 in the enhancement of the innate antiviral response through specific inhibition of Nedd4 Ub-E3 activity. To our knowledge, this is the first example of a Ub-like protein with the ability to interfere with Ub-E2 and E3 interaction to inhibit protein ubiquitination. IFNs2 are secreted pleiotropic cytokines that mediate diverse biological functions, including induction of the antiviral response and immunomodulatory activities (1-3). IFNs activate the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway, which leads to the induction of hundreds of ISGs. Along with other ISGs, IFN stimulation leads to the up-regulation of ISG15, a Ub-like protein that is present in cells either in the free form or conjugated to various cellular substrates (4, 5). Recently, ISG15 was found to function as an antiviral protein against influenza, herpes, Sindbis, and human immunodeficiency virus type 1 (6 -8), although the molecular mechanism remains largely unknown.Ubiquitination and Ub-mediated proteolysis play a regulatory role in many biological systems. In general, polyubiquitination targets proteins to degradation by the 26 S proteasome, whereas monoubiquitination signals for internalization and vesicle sorting (9, 10). The mammalian Nedd4 protein, originally identified as a gene developmentally down-regulated in the early embryonic mouse central nervous system (11), is a membrane-localized, HECT class Ub-E3 ligase (10). Nedd4 and Nedd4-like proteins are found in eukaryotes from yeast to mammals and are defined by a similar domain organization (12, 13). All contain a catalytic HECT domain at the C terminus and an N-terminal region involved in substrate recognition that includes a C2 domain and a series of WW domains. The WW domains of Nedd4 bind to the viral PPXY motifs, and its Ub-...

ISG15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis C virus production in vitro: implications for chronic infection and response to treatment

“…All three forms could potentially affect HCV viral production. In other systems, the free form of ISG15 has been shown to inhibit the release of Ebola virus-like particles by interfering with the activity of Nedd4 (Malakhova & Zhang, 2008;Okumura et al, 2008). ISGylation is critical to the effect of ISG15 on Sindbis virus, and ISGylation is targeted by the human influenza virus NS1 protein (Yuan & Krug, 2001;Lenschow et al, 2007).…”

Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation

“…4C. Protein ISGylation may interfere with the UPS function in multiple arms, such as the competition of E1/E2/E3s for ubiquitylation (50) and the competition of ubiquitylation sites on substrates (51). One particular effect may be through ISGylation of ubiquitylated proteins, which disrupts the formation of long ubiquitin chains (52).…”

Expression, regulation and function of the ISGylation system in prostate cancer

“…So far, the biological function of ISGylation has only been elucidated for some cellular target proteins. These include the functional inhibition of enzymes Takeuchi et al, 2006;Zou et al, 2007;Malakhova and Zhang, 2008) and the enhancement of the cap structure-binding activity of the translational suppressor, 4EHP (Okumura et al, 2007). Recent studies have shown that ISGylation negatively regulates the ubiquitin-proteasome pathway by direct interference with polyubiquitination (Desai et al, 2006).…”