About: Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

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About: Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : wasabi.inria.fr associated with source document(s)

Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
title	Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization
Creator	Zhang, Ling Wu, Ying Cheng, Anchun Wang, Mingshu Chen, Shun Jia, Renyong Sun, Di Chen, Xiaoyue Liu, Mafeng Yang, Qiao Zhu, Dekang Liu, Yunya Mao, Sai Wen, Xingjian Yu, Yanling Zhang, Shaqiu Zhao, Xinxin
source	Medline; PMC
abstract	BACKGROUND: The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. METHODS: In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. RESULTS: Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and V(max) and K(m) values were determined to be 16.52 nmol/min and 50.78 μM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. CONCLUSIONS: This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the V(max) and K(m) values were 16.52 nmol/min and 50.78 μM, respectively, using the Lineweaver–Burk plot.
has issue date	2019-04-29(xsd:dateTime)
bibo:doi	10.1186/s12985-019-1155-3
bibo:pmid	31036013
has license	cc-by
sha1sum (hex)	636fb28e842b058bff497388c5444fc1f53cb61c
schema:url	https://doi.org/10.1186/s12985-019-1155-3
resource representing a document's title	Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization
has PubMed Central identifier	PMC6489322
has PubMed identifier	31036013
schema:publication	Virol J
resource representing a document's body	covid:636fb28e842b058bff497388c5444fc1f53cb61c#body_text
is schema:about of	named entity 'Duck' named entity 'mediates' named entity 'maturation' named entity 'translation' named entity 'MINIMAL' covid:arg/636fb28e842b058bff497388c5444fc1f53cb61c named entity 'ANTIVIRAL DRUG' named entity 'DIFFERENT' named entity 'BIOCHEMICAL CHARACTERIZATION' named entity 'STRUCTURAL' named entity 'ABOUT' named entity 'ROLES' named entity 'PLAYS' named entity 'MULTIPLE' named entity 'ITS' named entity 'DEVELOPMENT' named entity 'PROTEASE' named entity 'ONGOING' named entity 'PROTEINS' named entity 'RECOMBINANT' named entity 'LOCALIZATION' named entity 'ITS' named entity 'VALID' named entity 'SEQUENCE SIMILARITY' named entity 'TRANSCRIPTION' named entity 'DATE' named entity 'THREAT' named entity 'PROTEASE' named entity 'HOST' named entity 'PICORNAVIRUSES' named entity 'FAMILY' named entity 'MODULATE' named entity 'MATURATION' named entity 'INFECTION' named entity 'DUE TO' named entity 'BACKGROUND' named entity 'POLYPROTEIN' named entity 'TARGET' named entity 'IS A' named entity 'MEDIATES' named entity 'INHIBITORS' named entity 'A MAJOR' named entity 'VIRAL' named entity 'LITTLE' named entity 'SIMILARITY' named entity 'AVIHEPATOVIRUS' named entity 'KNOWN' named entity 'VIRAL TRANSLATION' named entity 'DUCK HEPATITIS A VIRUS' named entity 'POULTRY INDUSTRY' named entity 'structural similarity' named entity 'development' named entity 'viral' named entity 'plays' named entity 'poultry industry' named entity 'Avihepatovirus' named entity 'protease' named entity 'plasmid' named entity 'fluorogenic' named entity 'lysis buffer' named entity 'protease' named entity 'China' named entity 'Avihepatovirus' named entity 'protease' named entity 'histidine'

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