Crimean-Congo Hemorrhagic Fever virus (CCHFV; family Nairoviridae) is an extremely pathogenic member of the Bunyavirales order. Previous studies have shown that the N-terminal domain of the CCHFV polymerase (L) contains an ovarian tumor-type protease (OTU) domain with the capability to remove both ubiquitin and ISG15 molecules from proteins. The approximately 200 amino acids-long OTU domain, if ectopically expressed, can interfere with both the induction of antiviral type I interferons (IFN) as well as the IFN-stimulated signaling. A OTU protease mutant (C40A), by contrast, was inactive in that respect. However, the effect of the OTU protease activity in the context of the full-length L protein (approximately 4000 amino acids) is only poorly characterized, and recombinant CCHFV with the C40A mutation could not be rescued. Here, we employed transcriptionally active virus-like particles (tc-VLPs) to investigate the interaction between the L-embedded OTU protease and the IFN system. Our data show a cis requirement of the OTU protease for optimal CCHFV polymerase activity in human HuH-7 cells. The L-embedded OTU did not influence IFN signaling, the sensitivity to IFN, or IFN induction. Moreover, the attenuation of OTU C40A-mutated L could not be relieved by inactivating the IFN response, but after overexpression of conjugation-competent ISG15 the polymerase activity recovered to wild-type levels. Consequently, ISG15 was used to produce OTU-deficient tc-VLPs, a potential vaccine candidate. Our data thus indicate that in the context of full-length L the OTU domain is important for the regulation of CCHFV polymerase by ISG15. Author summary: Tick-transmitted Crimean-Congo Hemorrhagic Fever virus (CCHFV) causes serious and potentially fatal disease in humans. The CCHFV polymerase possesses an N-terminal ovarian tumor-type protease (OTU) domain that cleaves ubiquitin and ISG15 modifiers from target proteins. Previous studies demonstrated that the ectopically expressed OTU domain can inhibit antiviral type I interferon responses. Hence, cleavage-negative OTU mutants of virus or transcriptionally active virus-like particles (tc-VLPs) are expected to exhibit elevated immunogenicity and would be candidates for a live vaccine. For unknown reasons, however, recombinant virus with just the OTU minus mutation cannot be generated. Using tc-VLPs, we show that in human HuH-7 cells the activity of the OTU minus polymerase is reduced by more than 80%. Curiously, the attenuation could not be compensated by inactivating the interferon system or by adding the OTU domain in trans. However, a complete reversion of the OTU minus phenotype was achieved by transcomplementation with ISG15, whereas the other OTU substrate, ubiquitin, had no such positive influence. Our data thus indicate a role of cis OTU in CCHFV polymerase regulation that is independent of an anti-interferon activity but connected to ISG15. Transcomplementation with ISG15 may be a means to rescue the OTU minus CCHV vaccine candidate. [ABSTRACT FROM AUTHOR]