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

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral 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 anti-viral 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. Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral 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 anti-viral 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. IFNs 2The abbreviations used are: IFNinterferonISGinterferon-stimulated geneUbubiquitinE3ubiquitin-protein isopeptide ligaseE2ubiquitin carrier proteinWTwild-typeVLPvirus-like particleHAhemagglutininMLFmouse lung fibroblastE1ubiquitin-activating enzyme. are secreted pleiotropic cytokines that mediate diverse biological functions, including induction of the antiviral response and immunomodulatory activities (1Biron C.A. Immunity. 2001; 14: 661-664Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 2Pestka S. Krause C.D. Walter M.R. Immunol. Rev. 2004; 202: 8-32Crossref PubMed Scopus (1334) Google Scholar, 3Sen G.C. Annu. Rev. Microbiol. 2001; 55: 255-281Crossref PubMed Scopus (802) Google Scholar). 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 (4Narasimhan J. Potter J.L. Haas A.L. J. Biol. Chem. 1996; 271: 324-330Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 5Ritchie K.J. Zhang D.-E. Semin. Cell Dev. Biol. 2004; 15: 237-246Crossref PubMed Scopus (120) Google Scholar). Recently, ISG15 was found to function as an antiviral protein against influenza, herpes, Sindbis, and human immunodeficiency virus type 1 (6Lenschow D.J. Giannakopoulos N.V. Gunn L.J. Johnston C. O'Guin A.K. Schmidt R.E. Levine B. Virgin H.W. J. Virol. 2005; 79: 13974-13983Crossref PubMed Scopus (227) Google Scholar, 7Lenschow D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. Garcia-Sastre A. Leib D.A. Pekosz A. Knobeloch K.-P. Horak I. Virgin H.W. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1371-1376Crossref PubMed Scopus (426) Google Scholar, 8Okumura A. Lu G. Pitha-Rowe I. Pitha P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1440-1445Crossref PubMed Scopus (293) Google Scholar), although the molecular mechanism remains largely unknown. interferon interferon-stimulated gene ubiquitin ubiquitin-protein isopeptide ligase ubiquitin carrier protein wild-type virus-like particle hemagglutinin mouse lung fibroblast ubiquitin-activating enzyme. 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 (9Hicke L. Nat. Rev. Mol. Cell Biol. 2001; 2: 195-201Crossref PubMed Scopus (1006) Google Scholar, 10Fang S. Weissman A.M. CMLS Cell. Mol. Life Sci. 2004; 61: 1546-1561Crossref PubMed Google Scholar). The mammalian Nedd4 protein, originally identified as a gene developmentally down-regulated in the early embryonic mouse central nervous system (11Kumar S. Tomooka Y. Noda M. Biochem. Biophys. Res. Commun. 1992; 185: 1155-1161Crossref PubMed Scopus (457) Google Scholar), is a membrane-localized, HECT class Ub-E3 ligase (10Fang S. Weissman A.M. CMLS Cell. Mol. Life Sci. 2004; 61: 1546-1561Crossref PubMed Google Scholar). Nedd4 and Nedd4-like proteins are found in eukaryotes from yeast to mammals and are defined by a similar domain organization (12Flores S.Y. Debonneville C. Staub O. Pfluegers Arch. Eur. J. Physiol. 2003; 446: 334-338Crossref PubMed Scopus (70) Google Scholar, 13Harvey K.F. Kumar S. Trends Cell Biol. 1999; 9: 166-169Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). 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-E3 activity is required for the release of viruses from cells (14Bieniasz P.D. Virology. 2006; 344: 55-63Crossref PubMed Scopus (304) Google Scholar). In this work, we investigated the role of ISG15 protein in the regulation of Nedd4 Ub-E3 ligase activity. We identified Nedd4 as a substrate of ISG15 modification; however, ISG15 in its free form was sufficient to inhibit the activity of Nedd4 ligase. Further investigation revealed that ISG15 bound Nedd4 specifically and blocked its interaction with Ub-E2 molecules. The ability of ISG15 to interfere with Nedd4-E2 complex formation prevented the Ub transfer from E2 to E3 molecules, thus inhibiting Nedd4 E3 ligase activity. In the presence of ISG15, Nedd4 showed diminished ability to ubiquitinate the Ebola virus matrix protein VP40, which in turn led to a decrease in the release of VP40 VLPs from the cells. Furthermore, we found that the inhibitory potential of ISG15 may not be restricted to Nedd4 alone but rather be a broad inhibition of other Nedd4-like E3 ligases, involved in viral budding and other cellular processes. These data point to a mechanistically novel function of ISG15 during the innate antiviral response through specific inhibition of Nedd4-like Ub-E3 activity. Plasmid Construction—pCAGGS-6×His-mISG15, pCAGGS-HA-UBE1L, pFLAG-CMV2-UbcM8, and pFLAG-CMV2-UbcH6 were described previously (15Kim K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.-E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (188) Google Scholar, 16Zou W. Zhang D.-E. J. Biol. Chem. 2006; 281: 3989-3994Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). pRBG4-His-Myc-Ub (17Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1145) Google Scholar) was from Ron Kopito. pBj-Myc-hNedd4 (WT and cysteine mutant) constructs (18Yasuda J. Nakao M. Kawaoka Y. Shida H. J. Virol. 2003; 77: 9987-9992Crossref PubMed Scopus (160) Google Scholar) were from Hisatoshi Shida. pCXN2-mNedd4.1 (WT and cysteine mutant) constructs (11Kumar S. Tomooka Y. Noda M. Biochem. Biophys. Res. Commun. 1992; 185: 1155-1161Crossref PubMed Scopus (457) Google Scholar) were from Sharad Kumar. HA-tagged mNedd4 constructs were made by PCR-based subcloning of full-length Nedd4 cDNA (WT or cysteine mutant) into the KpnI/XhoI sites of the pcDNA3.1-HA vector (Invitrogen) in-frame with the N-terminal HA tag. pCI-neo-FLAG-hNedd4, -hNEDL1, -hNEDL2, and -hWWP2 were kindly provided by Wesley Sundquist. Ebola Zaire VP40 cDNA in the pCAGGS vector was from Erica Ollmann Saphire. PCR-amplified full-length VP40 cDNA was subcloned into the KpnI/XhoI sites of the pcDNA3.1-HA vector in-frame with the N-terminal HA tag or into the HindIII/ClaI sites of the pCMV2-FLAG vector in-frame with the N-terminal FLAG tag. For retroviral expression, HA-tagged VP40 was subcloned into the BglII/HpaI sites of the pMSCV-PGK-hygro vector. All generated constructs were confirmed by sequencing. Cell Culture and Transfection—HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Mediatech Inc.) with 10% fetal bovine serum (Omega Scientific Inc.) and 2 mm l-glutamine (Invitrogen). Cells were grown on 6-well plates and transfected using PolyFect reagent (Qiagen Inc.) according to the manufacture's instruction. Primary MLFs were prepared as described previously (19Kim K.I. Malakhova O.A. Hoebe K. Yan M. Beutler B. Zhang D.-E. J. Immunol. 2005; 175: 847-854Crossref PubMed Scopus (78) Google Scholar). To create lung fibroblast cell lines, MLFs were immortalized by stable expression of SV40 large T antigen (in pBABE-puro). Stable pools of MLFs expressing HA-VP40 were made after retroviral infection with pMSCV-HA-VP40-PGK-hygro by selection with 500 μg/ml hygromycin (Invitrogen). Antibodies and Reagents—Antibodies against FLAG (Sigma), Ub (Sigma), HA (Covance), Nedd4 (Pharmingen), and Myc (Sigma) were purchased from the indicated manufacturers. Rabbit anti-mouse ISG15 polyclonal antibodies were described previously (20Malakhov M.P. Malakhova O.A. Kim K.I. Ritchie K.J. Zhang D.-E. J. Biol. Chem. 2002; 277: 9976-9981Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Purified Ub, E1, the Ub-conjugating enzyme UbcH6, ISG15, and an ATP energy regeneration system were purchased from Boston Biochem. In Vitro Ubiquitination and Thioester Bond Formation Assays—The in vitro ubiquitination reactions were carried out with reagents purchased from Boston Biochem and Nedd4 protein purified from pcDNA3.1-HA-Nedd4-transfected 293T cells. The in vitro thioester reactions contained different combinations of Ub, E1, UbcH6, Nedd4, and ISG15 and were carried out as described previously (21Anan T. Nagata Y. Koga H. Honda Y. Yabuki N. Miyamoto C. Kuwano A. Matsuda I. Endo F. Saya H. Nakao M. Genes Cells. 1998; 3: 751-763Crossref PubMed Scopus (88) Google Scholar). Immunoprecipitation and Western Blot Analyses—For immunoprecipitation, cells were lysed in 1× Tris-buffered saline (50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 1% Nonidet P-40) by sonication. Immunocomplexes from 0.5–1 mg of total cell lysates were precipitated with a mixture of protein A- and protein G-agarose (Amersham Biosciences). Immunoprecipitates were washed three times with the same buffer, boiled in SDS-PAGE loading buffer, and subjected to SDS-PAGE followed by Western blotting. Western blotting was performed as described previously (22Malakhova O.A. Kim K.I. Luo J.K. Zou W. Kumar K.G. Fuchs S.Y. Shuai K. Zhang D.-E. EMBO J. 2006; 25: 2358-2367Crossref PubMed Scopus (334) Google Scholar). Nickel-nitrilotriacetic acid (Qiagen Inc.) purification of His6-tagged proteins was performed under native conditions according to the instructions suggested by the manufacturer. Preparation of VLPs—Preparation of VLPs was as described previously (23Timmins J. Scianimanico S. Schoehn G. Weissenhorn W. Virology. 2001; 283: 1-6Crossref PubMed Scopus (161) Google Scholar). Nedd4 Ub-E3 Ligase Activity Is Inhibited in the Presence of ISGylation—We have identified Nedd4 Ub-E3 ligase in yeast two-hybrid screening for Ubp43-interacting proteins. Because Ubp43 was found to be a bona fide de-ISGylating enzyme (20Malakhov M.P. Malakhova O.A. Kim K.I. Ritchie K.J. Zhang D.-E. J. Biol. Chem. 2002; 277: 9976-9981Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar), we also confirmed that Nedd4 is a target of protein modification by ISG15 that is reversed by Ubp43 (data not shown). To elucidate the effect of ISGylation on Nedd4 function, we first examined whether ISGylation affects the Ub-E3 activity of Nedd4. Transfection of 293T cells with WT Nedd4 in combination with Ub led to a general induction of ubiquitination in the cells (Fig. 1A, upper left panel, lanes 1 and 2) as well as automonoubiquitination of Nedd4 itself (middle left panel, lanes 1 and 2). As expected, upon introduction of ISG15 and E1 + E2 enzymes responsible for ISGylation in 293T cells in combination with Nedd4, the ISGylated form of Nedd4 was readily detectable (Fig. 1A, middle right panel, lane 3). When both Ub and the ISGylation system were coexpressed with Nedd4 in 293T cells, we observed a significant decrease in Nedd4-mediated cellular ubiquitination (Fig. 1A, upper left panel, lanes 2 and 4) as well as a decrease in Nedd4 autoubiquitination (middle left and right panels, lanes 2 and 4). Both the total level of ISGylation and ISGylation of Nedd4 remained unaffected by coexpression of Ub (Fig. 1A, upper and middle right panels, lanes 3 and 4), and the total Nedd4 level was comparable between samples (∼18% variation). These results suggested that ISGylation may have a negative effect on Nedd4 activity. ISG15 Suppresses Nedd4 Interaction with Ub-E2 Enzymes and Prevents Ub Transfer to the Active Site of Nedd4—Because Nedd4 E3 activity may be influenced by multiple factors in vivo, we next investigated whether ISG15 can directly inhibit the activity of this enzyme in a cell-free system. We performed in vitro ubiquitination reactions containing purified Ub, E1 (UBE1), E2 (UbcH6), and Nedd4 in the absence or presence of purified ISG15 protein (Fig. 1B). As expected, no proteins were in the absence of Nedd4 (Fig. lanes 1 and 2). The of Nedd4 in autoubiquitination of Nedd4 itself (Fig. upper panel, lane as well as of other protein panel, lane that are to be the of the energy regeneration system. the presence of ISG15 protein in the enzyme the ubiquitination (Fig. lane 4), that free ISG15 protein is of directly with the transfer of Ub between E1 and Nedd4 E3 To further we in vitro thioester formation between Ub and Nedd4 in the absence of ISG15. As expected, Nedd4 was with Ub, E1, and the formation of a complex between Nedd4 and Ub was observed (Fig. upper panels, lane 3). The of free ISG15 the formation of the thioester (Fig. upper panels, lane 4), that ISG15 the Ub transfer from E2 to thus inhibiting Nedd4 E3 activity. E2 including UbcH6, and were to transfer Ub to the Nedd4 cysteine (21Anan T. Nagata Y. Koga H. Honda Y. Yabuki N. Miyamoto C. Kuwano A. Matsuda I. Endo F. Saya H. Nakao M. Genes Cells. 1998; 3: 751-763Crossref PubMed Scopus (88) Google Scholar). the interaction between Nedd4 and specific cellular E2 enzymes is for Nedd4-mediated ubiquitination. To the mechanism by which ISG15 affects Nedd4 E3 we examined whether ISG15 the of Nedd4 with 293T cells were with combinations of and ISG15, followed by with antibodies (Fig. and of the with (middle and Both (Fig. middle right panel, lane 2) and ISG15 right panel, lane were specifically with Nedd4, that proteins in Nedd4 was coexpressed with in the presence of ISG15, a significant decrease in complex formation was but complex formation remained unaffected (Fig. middle and right panels, lane 4). The of Nedd4, UbcH6, and ISG15 were comparable in transfected cells as in results were also or Ub-E2 enzymes were used in similar (data not that of the E2 enzymes can bind Nedd4 in the presence of ISG15. These data that expression of ISG15 the interaction between Nedd4 and Ub-E2 molecules. ISG15 Nedd4-mediated Ubiquitination of Ebola VP40 and from the matrix protein of both Ebola and a at its VP40 can mediate its release from mammalian cells, and the PPXY is important for The budding of VP40 VLPs was to be regulated by Nedd4 through the interaction with the viral PPXY (18Yasuda J. Nakao M. Kawaoka Y. Shida H. J. Virol. 2003; 77: 9987-9992Crossref PubMed Scopus (160) Google Scholar, G. J. Virol. 2001; PubMed Scopus (176) Google Scholar). the of our we investigated the role of ISG15 in Nedd4-mediated of the Ebola 293T cells were transfected with VP40 protein alone or with different combinations of ISG15, and either WT or human Nedd4. The level of VP40 ubiquitination and release from the cells were As in total protein Nedd4 protein and ISG15 this effect (Fig. left To the level of we carried out to followed by with antibodies to In the presence of Ub, VP40 was in 293T cells (Fig. upper right panel, lane and the ubiquitination level of VP40 was upon overexpression of WT Nedd4, but not its 2 and 3). the introduction of ISG15 led to a significant decrease in VP40 ubiquitination (Fig. upper right panel, lane 4) and also significant on VP40 ubiquitination by WT Nedd4 The expression level of VP40 remained between different (Fig. middle right The ubiquitination level of VP40 well with the of VLPs into the medium as in right The ability of Nedd4 to Ebola budding was by coexpression of ISG15, the of Nedd4 E3 activity. Because not Nedd4 but other Nedd4-like proteins with similar were in the of viral we that the antiviral potential of ISG15 may not be restricted to Nedd4, but rather be a broad cellular to the To the of ISG15, we performed with other Nedd4-like E3 enzymes that were previously to ubiquitinate viral matrix proteins and to be involved in the of viral of 293T cells with HA-tagged VP40 protein alone or with different combinations of ISG15 and human Nedd4, or we the level of VP40 expression and release from the cells As in the level of VP40 not between the As previously (14Bieniasz P.D. Virology. 2006; 344: 55-63Crossref PubMed Scopus (304) Google Scholar), overexpression of various Nedd4-like proteins VP40 release into the however, the ability of E3 ligases to Ebola budding was by coexpression of ISG15 (Fig. the of Nedd4-like E3 activity involved in the of Ebola virus release. These results were also confirmed in MLFs (Fig. upper cells transfected with HA-VP40 showed an in VP40 release into the medium (Fig. with WT cells, although both pools of VP40 protein (middle the results the role of ISG15 in the of viral budding by inhibition of Nedd4-like Ub-E3 ligase to the of viral release from the cells. In the of a we identified Nedd4 Ub-E3 ligase as a Ubp43-interacting Because Ubp43 is an enzyme (20Malakhov M.P. Malakhova O.A. Kim K.I. Ritchie K.J. Zhang D.-E. J. Biol. Chem. 2002; 277: 9976-9981Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar), we further whether Nedd4 is a target of protein modification by ISG15. We found that Nedd4 was modified by ISG15 in various cell after IFN To elucidate the effect of ISGylation on Nedd4 function, we first examined whether ISGylation affects the Ub-E3 activity of Nedd4. We found that coexpression of the ISGylation system with Ub and Nedd4 ubiquitination in general as well as the autoubiquitination of Nedd4, that ISGylation may negatively regulate Nedd4 ligase activity. the level of Nedd4 ISGylation in not with the of of Nedd4 activity in the presence of ISG15. to the ability of free ISG15 protein to inhibit the enzyme activity of Nedd4. In this we have that ISG15 in its free form is sufficient to inhibit Nedd4 in both in vitro ubiquitination and in Because cell a level of free ISG15 expression upon IFN stimulation or viral results the observed negative effect of ISG15 on Nedd4 activity in Further investigation into the molecular mechanism of inhibition of Nedd4 activity revealed that ISG15 by Nedd4 interaction with Ub-conjugating E2 enzymes the Ub transfer to the of Nedd4, thus preventing further ubiquitination of Nedd4 the viral Ebola VP40 protein, which on Nedd4 and Nedd4-like enzyme activity for its release from cells, we that overexpression of ISG15 of VLPs from cells. We also observed a significant decrease in Nedd4-mediated ubiquitination of VP40 protein in the presence of ISG15 that well with the decrease in release into the a similar effect of ISG15 was observed with three other Nedd4 E3 family and that ISG15 a broad of substrates multiple of the Nedd4-like protein which elucidate its inhibitory These data were further confirmed in that showed VP40 the role of ISG15 in the negative of Nedd4-mediated viral Furthermore, ISG15 to be a J. S. J. Immunol. 1996; Google Scholar), may be and secreted from cells and may Nedd4 family Ub ligase activity in cells that have to be to To the role of ISG15 as an antiviral protein examined by in a of data the of ISG15 to inhibit both and including human immunodeficiency virus type 1 A. Lu G. Pitha-Rowe I. Pitha P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1440-1445Crossref PubMed Scopus (293) Google Scholar), and viruses D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. Garcia-Sastre A. Leib D.A. Pekosz A. Knobeloch K.-P. Horak I. Virgin H.W. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1371-1376Crossref PubMed Scopus (426) Google Scholar, EMBO J. 2001; PubMed Scopus Google Scholar), the virus type 1 and and virus (6Lenschow D.J. Giannakopoulos N.V. Gunn L.J. Johnston C. O'Guin A.K. Schmidt R.E. Levine B. Virgin H.W. J. Virol. 2005; 79: 13974-13983Crossref PubMed Scopus (227) Google Scholar, 7Lenschow D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. Garcia-Sastre A. Leib D.A. Pekosz A. Knobeloch K.-P. Horak I. Virgin H.W. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1371-1376Crossref PubMed Scopus (426) Google Scholar). In of our further the of ISG15 antiviral activity. out the of ISG15 to cellular or viral target proteins for antiviral activity. although not a role of ISGylation in the of viral that ISG15 in its free form is sufficient to mediate its antiviral effect by the of Nedd4 activity. our of the potential of ISG15 in the inhibition of cellular E3 viral Because not Nedd4 but other Nedd4-like in the of and showed enzyme activity in the presence of ISG15, the antiviral potential of ISG15 may be a broad cellular to the with other E3 and viral ISG15 We Sharad Ron Hisatoshi Wesley and Erica Ollmann for Knobeloch and for ISG15 of the Zhang for and for the and for the

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.