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Virology Journal
Published in: Virology Journal 1/2019

Open Access 01-12-2019 | Research

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Authors: Di Sun, Mingshu Wang, Xingjian Wen, Sai Mao, Anchun Cheng, Renyong Jia, Qiao Yang, Ying Wu, Dekang Zhu, Shun Chen, Mafeng Liu, Xinxin Zhao, Shaqiu Zhang, Xiaoyue Chen, Yunya Liu, Yanling Yu, Ling Zhang

Published in: Virology Journal | Issue 1/2019

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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 Vmax and Km 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 Vmax and Km values were 16.52 nmol/min and 50.78 μM, respectively, using the Lineweaver–Burk plot.
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Metadata
Title
Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization
Authors
Di Sun
Mingshu Wang
Xingjian Wen
Sai Mao
Anchun Cheng
Renyong Jia
Qiao Yang
Ying Wu
Dekang Zhu
Shun Chen
Mafeng Liu
Xinxin Zhao
Shaqiu Zhang
Xiaoyue Chen
Yunya Liu
Yanling Yu
Ling Zhang
Publication date
01-12-2019
Publisher
BioMed Central
Published in
Virology Journal / Issue 1/2019
Electronic ISSN: 1743-422X
DOI
https://doi.org/10.1186/s12985-019-1155-3

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