Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

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.

ProteinEvolverABC: coestimation of recombination and substitution rates in protein sequences by approximate Bayesian computation

“…Concerning the aspartyl protease protein family, it presented a high recombination rate that agrees with previous studies (e.g. Speranskaya et al , 2012 ; Sun et al , 2019 ).…”

“…The results ( Table 1 ) showed that both recombination and substitution rates are heterogeneous among coronavirus protein families and that the aspartyl protease protein family presents a high recombination rate in agreement with previous observations (e.g. Speranskaya et al , 2012 ; Sun et al , 2019 ).…”

A viroporin-like 2B protein of duck hepatitis A virus 1 that induces incomplete autophagy in DEF cells

“…The procedure was performed as described previously and modification slightly ( Sun et al, 2019b ). Briefly, to construct eukaryotic recombinant plasmids pCAGGS-2B-HA and pCAGGS-FLAG-2B, DHAV-1 genomic RNA was extracted from the culture supernatants of DEF cells that were infected with DHAV-1.…”

Duck Hepatitis A Virus Type 1 Induces eIF2α Phosphorylation-Dependent Cellular Translation Shutoff via PERK/GCN2

“…The eukaryotic expression vector pCMV-Myc was purchased from TaKaRa. pCAGGs-VP3-Flag and pEGFP-N1-3C were also stocks in our laboratory (Lai et al, 2019;Sun et al, 2019). Duck-derived G3BP1 gene was cloned into the pEGFP-C2 vector with the DNA Ligation Kit (TaKaRa).…”