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

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About: Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein Goto Sponge NotDistinct Permalink

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Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
has title	Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein
Creator	Bailey-Elkin, Ben Mark, Brian Tchesnokov, Egor Gö Tte Id, Matthias
Source	Medline; PMC
abstract	BACKGROUND: 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 CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted 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, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity. METHODOLOGY/PRINCIPLE FINDINGS: Recombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2’-deoxy-2’-fluoro-CTP (FdC) and 2’-deoxy-2’-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, 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. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions. CONCLUSIONS/SIGNIFICANCE: The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors.
has issue date	2020-06-04(xsd:dateTime)
bibo:doi	10.1371/journal.pntd.0008283
bibo:pmid	32497085
has license	cc-by
sha1sum (hex)	c968914cb5b96df390a411241efa9ca7db083db6
schema:url	https://doi.org/10.1371/journal.pntd.0008283
resource representing a document's title	Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein
has PubMed Central identifier	PMC7271988
has PubMed identifier	32497085
schema:publication	PLoS Negl Trop Dis
resource representing a document's body	covid:c968914cb5b96df390a411241efa9ca7db083db6#body_text
is schema:about of	named entity 'disease' named entity 'viral' named entity 'RNA' named entity 'Independent' named entity 'inhibition' named entity 'LENGTH' named entity 'RDRP' named entity 'EXPRESSION' covid:arg/c968914cb5b96df390a411241efa9ca7db083db6 named entity 'protein' named entity 'World Health Organization (WHO)' named entity 'polymerase' named entity 'targets' named entity 'RNA virus' named entity 'activities' named entity 'listed' named entity 'human disease' named entity 'RdRp' named entity 'virus' named entity 'biochemical' named entity 'CCHFV' named entity 'World Health Organization' named entity 'RNA-dependent RNA polymerase' named entity 'CCHFV' named entity 'protein' named entity 'RdRp' named entity 'Rift Valley fever virus' named entity 'peptide' named entity 'long primer' named entity 'antiviral drug' named entity 'nucleotide' named entity 'RdRp' named entity 'San Jose' named entity 'nucleotides' named entity 'ribavirin-TP' named entity 'favipiravir' named entity 'nucleotide analogues' named entity 'biochemical' named entity 'divalent' named entity 'HCV' named entity 'UTP' named entity 'RNA synthesis' named entity 'ribavirin' named entity 'SDS PAGE' named entity '2'-hydroxyl' named entity 'phosphorylated' named entity 'RNA' named entity 'cytidine' named entity 'cysteine protease' named entity 'RNA synthesis' named entity 'RNA viruses' named entity 'Indianapolis' named entity 'metal ions' named entity 'fluorogenic' named entity 'Coomassie Brilliant Blue G-250' named entity 'CCHFV' named entity 'kDa' named entity 'N-terminal' named entity 'base-pairing' named entity 'off-target activities' named entity 'protein' named entity 'RNA synthesis' named entity 'protein' named entity 'GTP' named entity 'RNA-dependent RNA polymerase' named entity 'ATP' named entity 'Crimean-Congo hemorrhagic fever' named entity 'mice models' named entity 'AMC' named entity 'cap-snatching' named entity 'CCHFV' named entity 'phosphorylated' named entity 'Crimean-Congo Hemorrhagic Fever' named entity 'endonuclease' named entity 'mutation' named entity 'plasmid' named entity 'Austria' named entity 'UTP'

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