Effects Of VP3 Protein On DHAV-1 Adsorption And Apoptosis Induction And Identification Of VP3 Interaction Protein

Duck hepatitis A virus type 1?DHAV-1?VP3^pro,is one of the capsid protein encoded by VP3 gene.As a structural protein,its amino acid side chain is exposed on the surface of capsid.The main research contents and results are as follows that how does VP3 affect the adsorption of DHAV-1 on host cells and induce apoptosis?1 The antibody blocking effect on virus particle adsorption of rabbit anti-VP3 IgG100?g of purified rabbit anti-VP3 IgG,rabbit anti-DHAV-1 IgG,and rabbit-negative IgG were used to incubate with 200?L of DHAV-1(5×10^6.6 copies/?L)virus respectively at37?for an hour.Then the antibody processed virus particles were added to DEF,and adsorbed subjects for 0 min,10 min,60 min,90 min and 120 min at 4?.After incubation,the virus particles were placed at 37?for 2 min to penetration.Cell samples were collected to detect the copy number of virus particles on the surface of DEF.The results showed that the adsorption trend of DHAV-1 gradually increased within 0⁶0 min,and the maximum adsorption capacity was reached at 60 min.Then the adsorption amount of virus particles tended to be stable in 60¹20 min.The results showed that the adsorption capacity of virus particles treated with rabbit anti-VP3 IgG and rabbit anti-DHAV-1 IgG significantly lower than untreated virus particles.When DHAV-1 was treated with different doses of IgG?10?g,100?g,and 200?g?,the results showed that the amount of virus adsorbed by rabbit anti-VP3 IgG and rabbit anti-DHAV-1 IgG was significantly reduced,and the rabbit anti-VP3IgG maximum blocking dose was 100?g at which the blocking effect was weaker than rabbit anti-DHAV-1 IgG under the same conditions.2 The competitive adsorption inhibition of exogenous VP3 protein to DHAV-1DEF were incubated with different doses?10?g,100?g,and 200?g?of purified GST-VP3 soluble fusion protein,GST-tagged protein,and BSA for 1 h at 4?.The protein processed DEF were then incubated with 200?L DHAV-1(5×10^6.6 copies/?L)for 1 h at 4?.Then the number of virus particles on the surface of DEF cells was detected after 2 min at37?for virus infiltration.The results showed that the amount of DEF virus adsorbed by the GST-VP3 protein treatment group was significantly reduced?P<0.05?.100?g of added GST-VP3 protein had the greatest competitive blocking effect but could not block the adsorption completely,which suggested that VP3 protein was not the only protein in DHAV-1 that could compete to block the adsorption of virus particles.3 Exogenous DHAV-1 VP3 protein activated the transcription of apoptosis factors caspase-3/8/9 in DEF cellsRT-qPCR method was used to detect the transcriptional levels of apoptosis factors caspase-3/8/9 after reaction for 2 h,4 h and 8 h at 4°C.The results showed that the transcription levels of apoptotic cytokines caspase-3/8/9 were significantly upregulated after8 h at 4°C,caspase-3/8/9 was up-regulated about 4 fold,2 fold,and 1 fold,respectively.It proved that VP3 protein could activate the transcription of cytokines caspase-3/8/9 in vitro,and mainly activate caspase-3.4 Induction of DEF apoptosis by eukaryotic expression of VP3 recombinant proteinWestern blot and IFA showed that VP3-FLAG protein was successfully expressed in DEF after 48 h of eukaryotic plasmid pCAGGS/VP3 transfection.DAPI nuclear staining could observe the phenomenon of nuclear migration,shrinkage,and fragmentation.After transfection,the tunel assay detected the staining of yellow-brown nuclei,and the ratio of total apoptotic cells detected by flow cytometry was significantly higher than that of control group?P<0.05?.RT-qPCR detected transcription levels of apoptotic cytokines caspase-3,caspase-8,and caspase-9 up about 9-fold,3-fold,and 5-fold,respectively.The enzyme activities of caspase-3/8/9 were all increased in which caspase-3 had the largest increase in enzyme activity,caspase-9 was the middle,and caspase-8 was the lowest in activity change.Therefore,the results showed that VP3 could induce DEF apoptosis mainly through activating caspase-3 after it was expressed in DEF.5 Screening and identification of DEF host cell interacting proteins with DHAV-1 VP3proteinThe pCAGGS/VP3 cell lysates were immunoprecipitated with rabbit anti-FLAG IgG and normal rabbit IgG.After empty pCAGGS cell lysates were immunoprecipitated with rabbit anti-FLAG IgG,the protein strips were cut and analyzed by LC-MS/MS mass spectrometry.And then co-immunoprecipitation was validated with rabbit anti-human Myosin X polyclonal antibody and rabbit anti-DHAV-1 VP3 polyclonal antibody.The results showed that DHAV-1 VP3 protein interacts with Myosin X protein in DEF.