Abstract
BackgroundThe 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.MethodsIn 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.ResultsUnder 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.ConclusionsThis 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.
Highlights
The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription
Sun et al Virology Journal (2019) 16:54 (Continued from previous page). This is the first study to examine the activity of the Duck hepatitis A virus (DHAV) 3C protease, and the activity of the DHAV 3C protease is temperature, pH- and NaCl concentration- dependent
Evolutionary analysis Based on the amino acid sequence alignment of the 3C proteases of different picornaviruses, the catalytic triad of His-Glu/Asp-Cys was highly conserved among the 3C proteases of all selected viruses (Fig. 1a)
Summary
The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. Little is known about the roles of the DHAV 3C protease plays during infection. Duck hepatitis A virus (DHAV) causes an acute, contagious, and highly fatal disease that is characterized by a swollen liver mottled with hemorrhages and effects young (less than 3 weeks old) ducklings [1]. Due to the high mortality associated with the disease, DHAV causes significant financial losses, while no public health concern has been identified [3]. DHAV infection was recently identified in mallards, goslings and pigeons [7]. A goose embryonated epithelial cell line efficiently assists with the replication of DHAV, which shows a higher virus titer compared to other duck cell lines [8]. DHAV-1 is the most common type and has spread worldwide, and the development of accurate detection methods is essential [10,11,12,13,14]
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