Stable Occupancy of the Crimean-Congo Hemorrhagic Fever Virus-Encoded Deubiquitinase Blocks Viral Infection

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) infection can result in a severe hemorrhagic syndrome for which there are no antiviral interventions available to date. Certain RNA viruses, such as CCHFV, encode cysteine proteases of the ovarian tumor (OTU) family that antagonize interferon (IFN) production by deconjugating ubiquitin (Ub). The OTU of CCHFV, a negative-strand RNA virus, is dispensable for replication of the viral genome, despite being part of the large viral RNA polymerase. Here, we show that mutations that prevent binding of the OTU to cellular ubiquitin are required for the generation of recombinant CCHFV containing a mutated catalytic cysteine. Similarly, the high-affinity binding of a synthetic ubiquitin variant (UbV-CC4) to CCHFV OTU strongly inhibits viral growth. UbV-CC4 inhibits CCHFV infection even in the absence of intact IFN signaling, suggesting that its antiviral activity is not due to blocking the OTU’s immunosuppressive function. Instead, the prolonged occupancy of the OTU with UbV-CC4 directly targets viral replication by interfering with CCHFV RNA synthesis. Together, our data provide mechanistic details supporting the development of antivirals targeting viral OTUs. IMPORTANCE Crimean-Congo hemorrhagic fever virus is an important human pathogen with a wide global distribution for which no therapeutic interventions are available. CCHFV encodes a cysteine protease belonging to the ovarian tumor (OTU) family which is involved in host immune suppression. Here we demonstrate that artificially prolonged binding of the OTU to a substrate inhibits virus infection. This provides novel insights into CCHFV OTU function during the viral replicative cycle and highlights the OTU as a potential antiviral target.

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

“…For the wt L the subsequent cleavage of ISGylation may allow binding of ubiquitin as a counterbalancing negative regulator. Such a role of bound ubiquitin was proposed recently [31], and is confirmed by our observation that ubiquitin overexpression has a negative effect on L activity in human cells. These regulatory events may be indirectly connected to the IFN system via ISG15.…”

“…Here, we show that the CCHFV L polymerase mutant C40A, lacking the OTU protease activity, is attenuated in human cells, perhaps explaining the lethal phenotype of recombinant C40A virus [30,31]. Although the L attenuation is absent in the IFN-deficient BHK cells, it could not be relieved by blocking the IFN system in human cells.…”

“…Moreover, viruses or tc-VLPs with catalytically inactive OTU domains may be prime candidates for live vaccines. Attempts to rescue a C40A mutated CCHFV have failed [30,31], and the corresponding VLPs only reach low yields (see above). We took advantage of the complementation effect described above, and produced C40A mutant tc-VLPs in the presence of ISG15.…”

“…Inactivating the OTU protease activity, by contrast, resulted in a lethal phenotype, i.e. such a mutant could not be rescued from cDNA, except if ubiquitin binding was additionally destroyed [30,31]. Thus, the protease-negative L mutant of CCHFV, mutated exclusively in the catalytically active site of the OTU (C40A), could not be studied in the viral context.…”

Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in Ifnar-/- mice

“…The development of a reverse genetics system for CCHFV [25] has been instrumental for advanced pathogenesis studies [26,27], the development of high-throughput screening systems [11] and therapeutic candidates [28,29]. Here, we find that a recombinant CCHFV expressing ZsG is uniformly lethal in Ifnar -/mice, with a disease course comparable to that described for the parental virus (Fig 1).…”