Study On The Protease Activity Of 3C Protein In Duck Hepatitis A Virus Type 1

Duck hepatitis A virus type 1(DHAV-1)is a major pathogen for duck viral hepatitis,which mainly infects young ducklings(less than three-week-old).The mortality rate of infected ducklings is as high as 100%.To prevent and cure this disease,attenuated vaccines and egg yolk immunoglobulins have been used,but there is no effective antiviral drug.3C protein is one of the most conserved proteins encoded by picornaviruses,and it is involved in the polyprotein processing and the cleavage of host factors,which plays significant roles in viral replication,translation,immune escape and release.However,the function of DHAV-13C protein has not been reported.Here,we have done a series of studies on the protease activity,the intracellular localization of 3C protein,and its effect on the translation of host cells.The main contents and results are as follows:1 The protease activity of DHAV-1 3C proteinBased on the sequence of DHAV-1 X strain(JQ316452.1),the p ET32a-DHAV-1 3C recombinant plasmid for prokaryotic expression and its expression strains were constructed.Purified 3C protein and a peptide substrate(Dabcyl-ASFMNQSKVRRFE-Edans)were used in vitro cleavage assay.The results showed that DHAV-1 3C protein could recognize and cleave the amino acid sequence between the P2 region and the P3 region within polyprotein.The optimal temperature for the activity of 3C protease was regarded as 37?,and optimal p H was p H 7.0.The maximum protease activity was observed at a concentration of 150 m M Na Cl.The kinetic analysis was calculated,and V_maxand K_mvalues were 16.52 nmol/min and50.78?M,respectively.A method was constructed with optimal cleavage reaction conditions,which revealed AG7088 exhibited inhibitory effect on the protease activity of DHAV-1 3C protein.2 The identification of key amino acids in DHAV-1 3C proteinBased on the amino acid sequence alignment of the DHAV-1 3C protein,the catalytic triad of His₃₈-Asp₆₉-Cys₁₄₄and a conserved Gx CG motif were observed.Recombinant vectors of 3C gene for eukaryotic expression were constructed and transfected into duck embryo fibroblast cells and 293T cells,respectively.We detected the fusion protein,EGFP protein and 3C protein,which indicates DHAV-1 3C protein was expressed with protease activity leading to the release of EGFP protein and 3C protein.The fusion protein was detected when DHAV-1 3C mutants were expressed(histidine at position 38 or cysteine at position 144 of 3C protein was substituted with an alanine).3 The subcellular localization of DHAV-1 3C protein and its effect on protein synthesis of host cellsMice and rabbits were injected with the purified 3C protein to generate anti-3C polyclonal antibody for indirect immunofluorescence experiments.The localization of 3C protein was found in the cytoplasm and nucleus,when duck embryo fibroblast cells were infected with DHAV-1 or transfected with the recombinant plasmids containing 3C gene.The substitution of His₃₈or Cys₁₄₄by Ala caused the accumulation of 3C protein in the cytoplasm,indicating that its capability to enter the nucleus depends on its protease activity.It showed that the infection of DHAV-1 could inhibit the protein synthesis of host cells by puromycin labeling,but AG7088,an inhibitor of picornaviral 3C protein,could block the inhibition of host cell protein synthesis induced by DHAV-1,which indicated that 3C protein was involved in DHAV-1-induced the inhibition of the protein translation in DEF cells depending on its protease activity.To determine the inhibition mechanism of 3C protein,the3C gene recombinant plasmid was transfected into 293T cells.We detected the expression of host proteins involved in translation.There was no significant difference in the expression level of these translation-related cellular proteins,including e IF4G,e IF5B and PTB,when the 3C protein was expressed in 293T cells.However,the expression of 3C protein resulted in a significant decrease in the content of PABP,indicating that PABP might be a target for3C protein to inhibit cellular protein synthesis.4 DHAV-1 3C protein cleaves PABP proteinThe protein level of PABP was detected during infection by Western blot,when the total protein of duck embryo fibroblast cells infected with DHAV-1 at different time points was collected.The results showed that the content of PABP protein gradually decreased from 1 to6 hpi.Based on in vitro cleavage assays,the cell extract of uninfected DEF cells and recombinant PABP of prokaryotic expression were incubated with purified 3C protein.The results showed that 3C protein could cleave PABP protein generating two cleavage fragments(around 37-40 k Da)without the assistance of other viral proteins or host proteins.Recombinant plasmids of p ET32-Flag-PABP_WT-HA,p ET32a-Flag-PABP_Q341A-HA,p ET32a-Flag-PABP_Q367N-HA and p ET32a-Flag-PABP_G368N-HA were constructed.Duck PABP and PABP mutants were expressed and used in cleavage assay,in which results indicated PABP_WT,PABP_Q341Amutant,and PABP_G368Nmutant could be cleaved by 3C protein.3C protein could not cleave PABP_Q367Nmutant,showing 3C protein could cleave WTAQ₃₆₇-G₃₆₈in duck PABP.Moreover,the knockdown of PABP inhibited the production of viral RNA,while the overexpression of PABP increased the amount of viral RNA.Further research found that lower viral copy number was detected in DEF cells expressing the PABP_Q367Nmutant than cells expressing PABP_WT.Comparing to empty vector,there was no increase in viral replication when cells were expressed the N-terminal domain of PABP_1-367,which indicated full-length PABP could benefit viral replication.The C-terminal domain of PABP_368-557had a negative effect on viral replication.These results showed that PABP protein could promote the replication of DHAV-1,while N-terminal cleavage product produced by 3C protein could not promote the viral replication and C-terminal product could inhibit the viral replication.5 The interaction between DHAV-1 and the apoptosis of duck embryo fibroblast cellThe results of the DNA ladder confirmed that DHAV-1 infection induced apoptosis in duck embryo fibroblast cells,and transfection of 3C protein alone could induce apoptosis.During viral infection,m RNA expression levels of apoptosis-related factors were detected by q RT-PCR at different time points.At 48 hpi,caspase-3,caspase-8 and caspase-9 were up-regulated.The results also showed that m RNA of Cyt-c was significantly up-regulated at 60hpi.At 72 hpi,the m RNA expression of these apoptosis-related factors all reached a peak.Caspase-8 and Cyt-c were up-regulated higher than other factors.When DHAV-1 infected duck embryo fibroblast cells,there was a decrease in cellular RIPK1.Eukaryotic expression of DHAV-1 3C protein resulted in a reduction of intracellular RIPK1.However,in vitro cleavage assay showed that 3C protein could not cleave RIPK1,which indicated the decrease of RIPK1 might result from inhibition of protein synthesis but not depend on the protease activity of 3C protein directly.In conclusion,DHAV-1 encodes 3C protein with protease activity,whose active sites contain H38 and C144.It shows that 3C protein can recognize substrates that require glutamine at P1 and serine,glycine or asparagine at P1'.And the 3C protein prefers phenylalanine or tyrosine at P4.Depending on protease activity,3C protein can enter into the nucleus during viral infection or transfection alone.3C protein can cleave PABP protein required for eukaryotic translation initiation at Q367-G368 site to participate in the DHAV-1-induced translational inhibition.Besides,3C protein is also a significant protein involving in apoptosis triggered by DHAV-1.