L’érythropoïèse constitue un processus continu et ordonné au cours duquel des progéniteursérythroides commis prolifèrent et se différencient en globules rouges. Au cours d’un criblage visant à identifierdes gènes dont l’expression est régulée au cours de la différenciation terminale, nous avons identifié ISG15comme un gène dont l’expression est induite dans les stades tardifs de la différenciation érythroïde. ISG15appartient à la famille des protéines apparentées à l’ubiquitine et est conjuguée directement à des résidus Lysinede protéines cibles. La séquence des évènements moléculaires qui président à la modification de résidus Lysinedes protéines par ISG15 (ISGylation) est très semblable à ceux mis en oeuvre dans l’ubiquitination des protéines.Nous avons montré que l’expression d’ISG15 et des enzymes liées au processus d’ISGylation Ube1L, UbcM8 etHerc6 est induite au cours de l’érythropoièse in vivo et aussi dans des érythroblastes en culture induits à sedifférencier en réponse à l’Epo. Grâce à ce système de culture in vitro, nous avons montré que l’induction de cesgènes est majoritairement indépendante de la voie de signalisation IFN, et partiellement dépendante de la voie designalisation Epo. La comparaison de l’érythropoïèse dans des souris contrôles et des souris dans lesquelles legène ISG15 a été inactivé (ISG15-/-), montre une diminution du nombre de progéniteurs BFU-E et CFU-E dansla moelle osseuse avec une augmentation des BFU-E/CFU-E dans la rate des animaux ISG15-/-. Nous avonsaussi montré que les érythroblastes ISG15-/- sont inhibés dans leur différenciation terminale, à la fois in vivo et invitro. A l’inverse, la surexpression d’ISG15 dans les érythroblastes se révèle faciliter la différenciationérythroide. Au niveau moléculaire, nous avons montré que des acteurs importants de la différenciation érythroidepeuvent être ISGylés, comme par exemple STAT5, Globine, PLCγ et ERK2. En ce qui concerne plusprécisément STAT5, nous avons montré que son ISGylation est induite au cours de la différenciation et avonscherché à identifier les conséquences de son ISGylation. Dans le contexte d’une protéine de fusion, STAT5ISGylée se lie mieux à son site de liaison à l’ADN, une propriété associée avec une régulation positive de TfR etBCL-XL, deux gènes précédemment décrits comme étant régulés par STAT5 dans les érythroblastes. L’ensemblede ces résultats établit un nouveau rôle pour ISG15 au cours de la différenciation érythroide, en plus de sesfonctions anti-virales bien caractérisées / Erythropoiesis is an orderly continuous process during which committed erythroid progenitorcells proliferate and differentiate into mature red blood cells. In a search for genes that are deregulated duringthis differentiation process, we have identified ISG15 as being induced during the late stages of erythroiddifferentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins bythe enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we haveshown that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6are induced during erythroid differentiation. Moreover, using in vitro differentiating erythroblasts, we haveshown that the induction of these genes is mostly independent of IFN signaling, while it is partially dependent onEpo signaling in these cells. Our analysis of the ISG15 deficient mice have shown a decreased number of BFUE/CFU-E in the bone marrow, associated with an increased number of these cells in the spleen of these animals,a phenotype reminiscent of stress erythropoiesis. While ISG15-/- bone marrow and spleen-derived erythroblastsshowed a less differentiated phenotype both in vivo and in vitro, over-expression of ISG15 in erythroblasts wasfound to facilitate erythroid differentiation. At the molecular level, we have shown that important effectors oferythroid differentiation can be ISGylated, including STAT5, Globin, PLCγ and ERK2. Attempt to identify theconsequences of ISGylation has only been performed for STAT5. When studied in the context of a fusionprotein, ISGylated STAT5 was found endowed with a higher capacity to bind DNA, a property associated withup-regulation of TfR and Bcl-XL, two genes previously described as being regulated by STAT5 during erythroiddifferentiation. This establishes a new role for ISG15, in addition to its well-characterized anti-viral functions,during erythroid differentiation.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of the present study is to evaluate interferon-tau (IFNT) endocrine action in extra uterine tissues. The first approach consists of collecting samples from ewes on days 12, 13, 14 and 15 of the estrous cycle or early pregnancy. The second, consists of installing osmotic pumps to deliver a continuous concentration of recombinant ovine (ro) IFNT into the uterine vein for 24 or 72 hours. Our hypothesis is that endocrine release of IFNT into the uterine vein occurs as early as Day 13 of pregnancy. Also, that 24 and 72 hours infusion of roIFNT induces ISGs in the CL, liver and endometrium and IFNT has endocrine action on the CL to modulate major genes involved in luteolysis. Endometrium, liver, corpus luteum, uterine vein, jugular and uterine vein blood were collected from cyclic (NP) and pregnant (P) ewes on Days 12, 13, 14 and 15. Instalation of 24 and 72 hour pumps was done on Day 10 of the estrous cycle. Half were infused with BSA and the other half with roIFNT. Only in the 24 hour infusion, half of each group was challanged with a PGF injection at 12 hours following pump installation. Concentrations of progesterone in serum were similar on Days 12 and 13 and then declined (P<0.05) to less than 1 ng/ml in NP ewes between Days 14 and 15. Endometrial ISG15 mRNA increased (P<0.05) in P versus NP ewes by Day 13 and remained greater through Day 15. Endometrial ESR1 and OXTR mRNAs were up-regulated (P<0.05) in NP compared to P ewes by Day 14, and remained up-regulated on Day 15. Uterine vein ISG15 mRNA was not affected by pregnancy status. IFNAR1 and IFNAR2 mRNA were present in the CL, but did not change due to pregnancy status. ISG15 mRNA in CL and liver increased (P<0.05) by Day 14 and remained up-regulated on Day 15 in P compared to NP ewes. The expression of ESR1, OXTR, PTGFR, PTGER2, PTGER3 and PTGER4 did not change in the corpus luteum during Days 12, 13, 14 and 15 related to pregnancy status. Following the 24 hour infusion, ESR1, OXTR, PTGER2 and PTGER3 mRNA were not affected, but SLCO2A1 mRNA decreased (P<0.001) in BSA+PGF, roIFNT and roIFNT+PGF compared to BSA-infused ewes. PTGFR mRNA was down regulated (P<0.001) in roIFNT and roIFNT+PGF compared to BSA and BSA+PGF-infused ewes. PTGER4 mRNA was greater (P<0.05) in roIFNT and roIFNT+PGF compared to BSA, but not compared to BSA+PGF-infused ewes. After 72 hours, concentrations of progesterone did not differ in roIFNT- compared to BSA-infused ewes. ISG15 mRNA was induced in the CL, liver and endometrium in roIFNT- compared with BSA-infused ewes. Endocrine action of IFNT occurred through up-regulation of ISG15 mRNA in CL and liver by Day 14 of pregnancy. It is concluded that IFNT has endocrine effects on the CL between Days 13 and 14 of pregnancy and may protect the CL through mechanisms that are complementary, yet independent to its paracrine effects on the OXTR endometrial pathway. / O objetivo deste trabalho foi avaliar a resposta do interferon-tau (IFNT) em tecidos extra uterinos utilizando dois modelos experimentais. O primeiro, com a utilização de amostras coletadas nos dias 12, 13, 14 e 15 do ciclo estral ou da gestação e o segundo com a instalação de bombas osmóticas de infusão contínua por 24 ou 72 horas. Formulou-se a hipótese que o IFNT é liberado na veia uterina em torno do dia 13 da gestação. Também, que após 24 ou 72 horas de infusão na veia uterina, o interferon-tau é capaz de induzir a expressão de genes estimulados pelo interferon (ISGs) no corpo lúteo, fígado e endométrio. A resposta no corpo lúteo é capaz de modular genes envolvidos na luteólise. Para isso, foram coletados endométrio, corpo lúteo, fígado, veia uterina, sangue das veias jugular e uterina, nos dias 12, 13, 14 e 15 do ciclo estral ou gestação. A instalação das bombas osmóticas de infusão por 24 e 72 horas foi realizada no dia 10 do ciclo estral, onde metade dos animais receberam infusão de albumina bovina (grupo BSA) e outra metade de IFNT recombinante ovino (ro; grupo roIFNT). Nos animais infundidos por 24 horas, metade de cada grupo BSA ou roIFNT, recebeu um desafio com prostaglandina F2 alfa (PGF; grupos BSA+PGF e roIFNT + PGF) 12 horas após a instalação das bombas. Concentrações sericas de progeterona foram iguais nos dias 12 e 13, e diminuiram (P<0,05) a menos de 1 ng/ml entre os dias 14 e 15 nos animais cíclicos. A expressão de ISG15 endometrial aumentou (P<0,05) no dia 13 em ovelhas prenhes, e permaneceu elevada até o dia 15. A expressão dos receptores de estrógeno alfa (ESR1) e de ocitocina (OXTR) aumentaram (P<0,05) em ovelhas cíclicas comparadas com prenhes no dia 14, permanecendo elevadas no dia 15. A expressão de ISG15 na veia uterina não apresentou diferença em função da gestação. A expressão dos receptores de Interferon tipo I não apresentou diferença em função da prenhez no corpo lúteo. No corpo lúteo e fígado, a expressão de ISG15 aumentou no dia 14 da gestação, permanecendo no dia 15. A expressão luteal de ESR1, OXTR, receptores de PGF (PTGFR), e E2 subtipos 2 (PTGER2), 3 (PTGER3) e 4 (PTGER4) não apresentaram diferença em função da gestação nos dias 12, 13 14, e 15. Após 24 horas de infusão, a expressão luteal de ESR1, OXTR, PTGER2 e PTGER3, não apresentaram diferenças. O transportador de prostaglandinas diminuiu nos grupos BSA+PGF, roIFNT e roIFNT+PGF (P<0,001). O receptor PTGFR diminuiu a expressão (P<0,001) nos grupos roIFNT e roIFNT+PGF, e o receptor PTGER4 aumentou (P<0,05) nos grupos roIFNT e roIFNT+PGF. Em 72 horas de infusão, não foi observada diferença na secreção de progesterona. A expressão de ISG15 nos animais do grupo roIFNT aumentou (P<0,01) no corpo lúteo, fígado e endométrio. Assim, a função endócrina do IFNT ocorre com a expressão de ISG15 no dia 14 da gestação no corpo lúteo e no fígado. Portanto, a ação endócrina do IFNT acontece entre os dias 13 e 14 da gestação e pode proteger o corpo lúteo por mecanismos complementares ao mecanismo anti luteolítico parácrino endometrial.

Infektionen lösen eine Immunantwort aus, welche u.a. durch Induktion von Interferonen (IFN) für die Expression von antibakteriell oder antiviral wirkenden Proteinen sorgt. Eines der am stärksten induzierten Gene nach Typ I IFN Stimulation ist das Interferon stimulated gene 15 (ISG15). ISG15 gehört zu den Ubiquitin-ähnlichen Proteinen und wird über eine Enzymkaskade kovalent an zu modifizierende Proteine konjugiert. Sowohl freiem ISG15, als auch ISG15-Konjugaten wurden antivirale Funktionen nachgewiesen. In der vorliegenden Arbeit wurde die Regulation von ISG15, der ISGylierung und der an der ISGylierung beteiligten Enzyme vergleichend nach Typ I und Typ II IFN Stimulation untersucht. In HeLa Zellen zeigte sich, dass ISG15 und ISG15-Konjugate sowohl nach Typ I, als auch nach Typ II IFN Stimulation gebildet werden. Die Unterschiede in der Menge der ISGylierten Proteine konnten in Zusammenhang mit der unterschiedlich starken Expression der an der ISGylierung beteiligten Enzyme gebracht werden. Durch Inhibition der Expression der einzelnen Enzyme wurde weiterhin nachgewiesen, dass nach Typ I und Typ II IFN Stimulation das gleiche E2-Enzym (UBE2L6), aber unterschiedliche E3-Ligasen an der ISGylierung beteiligt sind. Während die ISGylierung nach Typ I IFN Stimulation größtenteils von der E3-Ligase Herc5 abhängt, zeigte sich, dass nach Typ II IFN Stimulation weitere noch nicht identifizierte ISG15 E3-Ligasen an dem Prozess beteiligt sind. Microarry- und siRNA-Analysen führten zur Identifizierung der potentiellen ISG15 E3-Ligasen UBE3A und ARIH1. Dabei zeigte sich, dass UBE3A nach Typ I und ARIH1 nach Typ II IFN Stimulation einen Einfluss auf die ISGylierung von Proteinen haben. Diese Ergebnisse machen deutlich, dass in HeLa Zellen Typ I und Typ II Interferone die ISGylierung induzieren, wobei sich die Enzymkaskade zwischen den Interferonen auf Ebene der E3-Ligasen unterscheidet. Diese Ergebnisse geben neue Einblicke in die Regulation der IFN vermittelten ISGylierung. / Infections initiate the induction of an immune response that lead to induction of interferons followed by expression of antibacterial and antiviral proteins. One of the most upregulated genes after type I interferon (IFN) stimulation is the Interferon stimulated gene 15 (ISG15). ISG15 belongs to the family of ubiquitin-like modifiers and is covalently attached to target proteins via an enzyme cascade. It was shown that free ISG15 as well as ISG15-conjugates exhibit antiviral functions. In this thesis the regulation of ISG15, ISGylation and the involvement of enzymes after type I and type II IFN treatment were analysed. It could be demonstrated that ISG15 and ISG15-conjugates are generated after type I as well as type II IFN treatment in HeLa cells. Differences in the amount of ISGylated proteins were shown to correlate with the expression of proteins of the enzyme cascade. Inhibition of these enzymes demonstrated that the E2-enzyme UBE2L6 is involved in the ISGylation process after both types of IFN stimulation, whereas different E3-ligases are implicated in ISGylation of proteins after type I and type II IFN stimulation. It could be shown that the E3-ligase Herc5 is involved in type I IFN mediated ISGylation, whereas other not yet identified ISG15 E3-ligases are involved in type II IFN mediated ISGylation. Microarry- and siRNA-assays led to the identification of the putative ISG15 E3-ligases UBE3A and ARIH1, whereas UBE3A had an influence on type I IFN mediated ISGylation and ARIH1 affected the type II IFN mediated ISGylation. In this thesis it could be demonstrated that both types of interferons are able to induce ISGylation in HeLa cells, but different E3 ligases are involved in conjugation of ISG15 to its target proteins after type I and type II IFN induction. These observations give new insights into the regulation of IFN mediated ISGylation.

Hepatitis C Virus (HCV) infects 170 million people worldwide. The current treatment regimen, which is combination therapy with pegylated interferon (PegIFN) and Ribavirin (Rib), cures only 50% of the patients infected with the most prevalent HCV genotype. Therefore, there is a pressing need to understand the molecular mechanism of interferon resistance and to develop a prognostic tool to predict who will respond to treatment before initiation of therapy. It has been firmly established that the virus-host interaction plays an important role in determining treatment outcomes. My thesis investigated the host factors that are involved in interferon resistance with an aim to provide insights into the molecular mechanism of IFN resistance. cDNA microarray analysis identified 18 differentially expressed hepatic genes from pretreatment liver tissues of responders (Rs) and non-responders (NRs). Based on the differential expression levels of these 18 genes, a prognostic tool was developed to predict who will respond to therapy, with a positive predicting value (PPV) of 96%. Most of these 18 genes are interferon stimulated genes (ISGs) and they are more highly expressed in NR livers, indicating that preactivation of interferon signaling in the pre-treatment liver tissues contributes to NR. 3 out of the 18 genes are involved in an ubiquitin-like ISG15/USP18 signaling pathway that plays an important role in interferon response. Over-expression of USP18 and ISG15 in the pretreatment liver tissues of NR promotes HCV production and blunts interferon anti-HCV activity. There exists a distinct cell-type specific ISG activation in the pretreatment liver tissues of Rs and NRs. Up-regulation of the two ISGs that I tested (ISG15 and MxA) was found mainly in hepatocytes in NRs while ISG activation was preferentially observed in macrophages in Rs. Taking all these data together, pre-activation of interferon signaling and cell-type specific gene activation in the pretreatment liver tissues of patients infected with HCV are associated with treatment non-response. HCV exploits the host interferon system to favour its persistence by enhanced replication /secretion stimulated by a few ISGs (ISG15, USP18) in response to IFN. The developed prognostic tool can be used to stratify patients for treatment and the novel insights of the molecular mechanism of IFN resistance in HCV patients offer potential drug targets for future development.

Hepatitis C Virus (HCV) infection represents a worldwide pandemic and is currently the leading cause of cirrhosis and liver transplantation. After transplantation recurrence is almost universal with 96% of patients testing positive for viral RNA and exhibiting histological evidence of infection within the first year. Type I interferons (IFN) and interferon inducible genes are responsible for the innate antiviral state and single nucleotide polymorphisms (SNPs) within these genes may affect the patients ability to respond post-transplantation. We hypothesize the elucidation of associations between SNPs in Type-I Interferon and Interferon inducible genes and HCV recurrence post-liver transplantation might help to identify HCV patients with different prognosis and improve liver transplant recipient selection. 100 HCV positive patients were genotyped using Allelic Discrimination on an ABI Prism 7700 sequence detector (Applied Biosystems) for SNPs in IFNB1, OAS-1, and ISG-15 to establish a relationship between SNPs and clinical complications post-transplantation. Quantitative real-time polymerase chain reaction (QPCR) was also run to determine the relationship between SNPs or disease state and the level of RNA expression. Results were collected and analyzed using Fishers exact test, Kaplan-Meir method, and the log-rank test. Results obtained indicated that SNPs in OAS-1 are associated with HCV recurrence within 12 months post-orthotopic liver transplantation (OLT) and OAS-1 SNP genotypes were significantly associated with the development of fibrosis within the first year. Additionally we observed an association between the SNP genotypes of OAS-1 and ISG-15 and CMV infection post-OLT. A significant distribution of ISG-15 genotypes was also found to correlate with acute rejection. These findings might help identify patients at high risk of developing complications within the first year.

Les interférons (IFN) de type I et type III sont des cytokines induites par des pathogènes. L'IFN de type I (IFN α/β)se fixe à un récepteur constitué des chaînes IFNAR1 et IFNAR2. L'IFN de type III (3 λs) se fixe à un récepteur constitué des chaines IFNLR1 et IL-10R2. La liaison de ces IFNs à leur récepteur active la voie Jak/Stat, induit les mêmes gènes et des réponses cellulaires communes essentielles à la protection antivirale. L'IFN de type I joue un rôle pléiotropique et de ce fait la réponse cellulaire aux IFNs doit être contrôlée dans le temps et dans l'espace. Certains régulateurs négatifs tels que les SOCS ou les ubiquitine ligases ciblant la sous-unité IFNAR1 vont agir rapidement après la stimulation, alors que d'autres agissent à des temps plus tardifs, tels qu'USP18. USP18 est une cystéine protéase induite par l'IFN, elle clive ISG15, une molécule semblable à l'ubiquitine, à partir de protéines ISGylées. J'ai étudié comment une stimulation prolongée avec de l'IFN de type I ou III interfère avec la capacité de ces cellules à répondre à une re-stimulation par les IFN α, tout en maintenant leur sensibilité à l'IFN β et λ . Ce phénomène de désensibilisation différentielle n'est pas dû à une diminution des récepteurs à la surface des cellules mais à l'induction de la forme catalytiquement active d'USP18. Lors de traitements prolongés à l'IFN, l'accumulation d'USP18, dont l'expression est régulée par ISG15, inhibe progressivement la signalisation induite par l'IFN α. En conclusion, ces études montrent qu'USP18 fait partie intégrante des signaux transmis lors d'une stimulation par les IFN de type I et III et définit le seuil d'activité des différents sous-types α/β.

Influenza B virus causes a respiratory disease in people with a compromised immune system. The NS1B protein of influenza B virus is essential for virus growth and plays a crucial role in inhibiting the anti-viral responses mounted by the infected host cell. The N terminal 104 amino acids of NS1B bind a cellular protein called ISG15. ISG15 is an interferon induced 'ubiquitin-like' protein, and upon interferon induction, is conjugated to hundreds of targets. It has been found that both ISG15 and its conjugation inhibit many viruses. The focus of the current study was to characterize the interaction between NS1B and ISG15. Study of a recombinant influenza B virus which encoded a mutant NS1B protein that is unable to bind ISG15 revealed that ISG15 is mis-localized in cells infected with wild type but not the mutant influenza B virus. Further, such a mutant virus is attenuated in growth as compared to wild type virus in human cell lines but is not attenuated in canine cell lines. This result led to the discovery of the species specific nature of the interaction between NS1B and ISG15. Specifically, NS1B was found to bind ISG15 homologs from human and old world monkeys like Rhesus macaques and African green monkeys but not those from mouse or canines. These findings were extended by identifying the hinge between the N and C terminal domains of ISG15 as one of the major determinants of species specificity. These results highlight the importance of using human or primate cell culture models to study the effect of ISG15 on influenza B virus, and raises new possibilities on differences in the function of the ISG15 system in different species. / text

ISG15 is an interferon-induced and anti-viral ubiquitin-like protein (Ubl). Ube1L, UbcH8, and Herc5 have been identified as the E1-E2-E3 enzymes for ISG15 conjugation, and, like ISG15, their expression is induced by type I interferons. Although Herc5 is the major E3 for ISG15, over 300 proteins have been identified as ISG15 target proteins in interferon-stimulated cells. In this work, I address two aspects of the human ISG15 conjugation system: 1) the specificity of the Ube1L-UbcH8 interaction and 2), the basis of substrate recognition by Herc5. Regarding the selection of UbcH8 by Ube1L, my experiments show that although UbcH8 had been reported to function as an E2 for both Ub and ISG15, UbcH8 is preferentially activated by Ube1L compared to Ube1 (E1[superscript Ub]). The basis of this preference is a result of specific interactions between the ubiquitin-fold domain (UFD) of Ube1L and the amino-terminal [alpha]1 helix and [beta]1 [beta]2 region within UbcH8. Examination of the interferon-induced and transfected expression levels of UbcH8, combined with the kinetic constants, suggest that UbcH8 is unlikely to function as a Ub E2 in most cell lines. In examining the selection of target proteins by Herc5, I show that the range of substrates extends far beyond the proteins identified in proteomics studies and includes many exogenously expressed foreign proteins. Furthermore, I show that ISG15 conjugation is restricted to newly synthesized pools of proteins and Herc5 is associated with polyribosomes. I propose a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a co-translational manner and suggest that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, I show that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses. These discoveries have greatly increased our understanding of the mechanism of ISG15 pathway and provide a framework for establishing an in vitro ISG15 conjugation system and further examination of the anti-viral function of ISG15. / text

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.