ISG15 overexpression compensates the defect of Crimean-Congo hemorrhagic fever virus polymerase bearing a protease-inactive ovarian tumor domain

Abstract

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.

Comparison of Crimean-Congo Hemorrhagic Fever Virus and Aigai Virus in Life Cycle Modeling Systems Reveals a Difference in L Protein Activity

“…The CCHFV L protein is known to have a number of nontraditional polymerase functions. Although not all of these functions have been characterized, the most studied region is the N-terminal OTU domain and its activity in counteracting the activity of type I interferon (IFN) ( 7 , 9 , 20 ). It is possible that the difference in the activity of CCHFV and AIGV L proteins is due to differences in the ability of these two proteins to modulate the IFN response.…”

“…Indeed, some evidence exists to suggest this may in fact be the case. While not fully elucidated, these functions involve the deconjugation and turnover of ubiquitin and ISG15 ( 8 , 9 ). The OTU domain of AIGV is less sensitive than the CCHFV domain to a modified ubiquitin variant (Ubv-CC4) ( 8 ).…”

“…In alignment with the other L proteins of the Bunyavirales , significant expansions exist between all the conserved regions ( 6 ). The most studied of these expansions is an N-terminal extension, which contains an ovarian tumor-type protease (OTU) domain that cleaves ubiquitin and ISG15, which functions in counteracting the innate immune response ( 7 ) and regulating polymerase activity ( 8 , 9 ). In addition to the activity of the OTU domain and traditional vRdRp roles, it is believed that the uncharacterized regions of the L protein may have as yet undescribed additional functions.…”

HERC5 and the ISGylation Pathway: Critical Modulators of the Antiviral Immune Response

“…Interestingly, the most fatal virus in humans, CCHFV, is capable of both deubiquitylating and deISGylating target proteins, likely resulting in subversion of both the antiviral and inflammatory responses [126,130]. The inhibition of the CCHFV vOTU resulted in impaired viral replication and infectivity [131,132], suggesting that this deISGylase may be a prime target for further therapeutics to treat CCHFV.…”

Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures

“…Indeed, a reduced activation of key components of the innate immune response (including RIG-I) was observed upon infection with wild-type CCHFV, while infection with a mutated CCHFV whose OTU lacked DUB activity led to the establishment of an antiviral state in infected cells [ 159 ]. However, when associated with the full-length L protein, the ability of the OTU domain to suppress the type I IFN-mediated immune response seems to be reduced [ 155 , 156 , 157 ] and a study using a CCHFV VLP system with a protease-negative OTU mutant in immunocompetent cells revealed that IFN induction was not impacted upon infection [ 166 ], adding more complexity to the understanding of the contribution of the OTU domain in the antiviral response.…”