scholarly journals Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein

2020 ◽  
Vol 14 (6) ◽  
pp. e0008283 ◽  
Author(s):  
Egor P. Tchesnokov ◽  
Ben A. Bailey-Elkin ◽  
Brian L. Mark ◽  
Matthias Götte
Keyword(s):  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Full Length ◽  
Crimean Congo Hemorrhagic Fever ◽  
L Protein ◽  
Hemorrhagic Fever Virus

scholarly journals Independent Inhibition of the Polymerase and Deubiquitinase Activities of the Crimean–Congo Hemorrhagic Fever Virus Full-Length L-Protein

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 41
Author(s):  
Egor P. Tchesnokov ◽  
Ben A. Bailey-Elkin ◽  
Brian L. Mark ◽  
Matthias Götte
Keyword(s):  
Expression System ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Full Length ◽  
World Health ◽  
Rdrp Activity ◽  
Crimean Congo Hemorrhagic Fever ◽  
L Protein ◽  
Hemorrhagic Fever Virus ◽  
Research Activities

The Crimean–Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFV as a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. We report on the expression, characterization, and inhibition of the CCHFV full-length L-protein that provides an important tool in this regard. The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. Hence, we decided to adopt a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large, with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity. Previous studies have shown that DUB activity interferes with innate immune responses and viral replication. Here, we utilized the baculovirus expression system and generated a full-length CCHFV L protein. RdRp activity was seen in the presence of divalent metal ions, and inhibition of RNA synthesis was demonstrated with nucleotide analogues. The ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. We have finally shown that RdRp and DUB activities are functionally independent. The full-length CCHFV L-protein provides an important tool for the discovery of antiviral agents. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be utilized to identify polymerase and DUB inhibitors.

scholarly journals Crimean-Congo Hemorrhagic Fever Virus-Encoded Ovarian Tumor Protease Activity Is Dispensable for Virus RNA Polymerase Function

2009 ◽  
Vol 84 (1) ◽  
pp. 216-226 ◽  
Author(s):  
Éric Bergeron ◽  
César G. Albariño ◽  
Marina L. Khristova ◽  
Stuart T. Nichol
Keyword(s):  
Protease Activity ◽  
Ovarian Tumor ◽  
Antiviral Drug ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Crimean Congo Hemorrhagic Fever ◽  
L Protein ◽  
Hemorrhagic Fever Virus ◽  
Virus Rna ◽  
N Terminus

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus (genus Nairovirus, family Bunyaviridae) associated with high case fatality disease outbreaks in regions of Africa, Europe, and Asia. The CCHFV genome consists of three negative-strand RNA segments, S, M, and L. The unusually large virus L polymerase protein and the need for biosafety level 4 (BSL-4) containment conditions for work with infectious virus have hampered the study of CCHFV replication. The L protein has an ovarian tumor (OTU) protease domain located in the N terminus, which has led to speculation that the protein may be autoproteolytically cleaved to generate the active virus L polymerase and additional functions. We report the successful development of efficient CCHFV helper virus-independent S, M, and L segment minigenome systems for analysis of virus RNA and protein features involved in replication. The virus RNA segment S, M, and L untranslated regions were found to be similar in support of replication of the respective minigenomes. In addition, the OTU domain located in the N terminus of the expressed virus L protein was shown to be a functional protease. However, no evidence of L protein autoproteolytic processing was found, and the OTU protease activity was dispensable for virus RNA replication. Finally, physiologically relevant doses of ribavirin inhibited CCHFV minigenome replication. These results demonstrated the utility of the minigenome system for use in BSL-2 laboratory settings to analyze CCHFV biology and in antiviral drug discovery programs for this important public health and bioterrorism threat.

scholarly journals Immunoglobulin-like Domain of HsFcμR as a Capture Molecule for Detection of Crimean-Congo Hemorrhagic Fever Virus- and Zika Virus-Specific IgM Antibodies

2019 ◽  
Vol 65 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Anne Rackow ◽  
Christa Ehmen ◽  
Ronald von Possel ◽  
Raquel Medialdea-Carrera ◽  
David Brown ◽  
...  
Keyword(s):  
Zika Virus ◽  
Specific Binding ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Size Exclusion ◽  
Animal Origin ◽  
Serum Samples ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Igm Antibodies

Abstract BACKGROUND The cellular surface molecule HsTOSO/FAIM3/HsFcμR has been identified as an IgM-specific Fc receptor expressed on lymphocytes. Here, we show that its extracellular immunoglobulin-like domain (HsFcμR-Igl) specifically binds to IgM/antigen immune complexes (ICs) and exploit this property for the development of novel detection systems for IgM antibodies directed against Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV). METHODS His-tagged HsFcμR-Igl was expressed in Escherichia coli and purified by affinity chromatography, oxidative refolding, and size-exclusion chromatography. Specific binding of HsFcμR-Igl to IgM/antigen ICs was confirmed, and 2 prototypic ELISAs for the detection of anti-CCHFV and anti-ZIKV IgM antibodies were developed. Thereby, patient sera and virus-specific recombinant antigens directly labeled with horseradish peroxidase (HRP) were coincubated on HsFcμR-Igl-coated ELISA plates. Bound ICs were quantified by measuring turnover of a chromogenic HRP substrate. RESULTS Assay validation was performed using paired serum samples from 15 Kosovar patients with a PCR-confirmed CCHFV infection and 28 Brazilian patients with a PCR-confirmed ZIKV infection, along with a panel of a priori CCHFV/ZIKV-IgM-negative serum samples. Both ELISAs were highly reproducible. Sensitivity and specificity were comparable with or even exceeded in-house gold standard testing and commercial kits. Furthermore, latex beads coated with HsFcμR-Igl aggregated upon coincubation with an IgM-positive serum and HRP-labeled antigen but not with either component alone, revealing a potential for use of HsFcμR-Igl as a capture molecule in aggregation-based rapid tests. CONCLUSIONS Recombinant HsFcμR-Igl is a versatile capture molecule for IgM/antigen ICs of human and animal origin and can be applied for the development of both plate- and bead-based serological tests.

scholarly journals Serological and molecular study of Crimean-Congo Hemorrhagic Fever Virus in cattle from selected districts in Uganda

2021 ◽  
Vol 290 ◽  
pp. 114075
Author(s):  
Stephen Balinandi ◽  
Claudia von Brömssen ◽  
Alex Tumusiime ◽  
Jackson Kyondo ◽  
Hyesoo Kwon ◽  
...  
Keyword(s):  
Hemorrhagic Fever ◽  
Molecular Study ◽  
Fever Virus ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus
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