Viroporin Activity Of Type 1 Duck Hepatitis Virus Non-Structural Protein 2B

Viroporin is a small molecular weight hydrophobic transmembrane protein encoded by a virus containing at least one hydrophobic transmembrane region,which has the functions of increasing host cell membrane permeability,disturbing host cell ion balance,inducing host cell apoptosis and autophagy to disturbs the physiological function of the host cell to ensure that the virus completes its life cycle.The picornavirus 2B protein is a non-structural protein,some of which have been confirmed to belong to the type II viloporin family.The structural characteristics and biological functions of the type 1 duck hepatitis virus?DHAV-1?2B protein are still unclear.This paper has carried out related research and obtained the following results:1.Structural characteristics of DHAV-1 2BThe 2B gene has a total of 357 bases encoding 119 amino acids.It is predicted by biological software that it contains a transmembrane region from amino acid 42 to amino acid 63,which is highly hydrophobic and does not contain a cationic amphiphilic alpha helix.The 2B complete sequence and the truncated sequence of the removal of the intracellular domain and the transmembrane region were prokaryotic expression,and no expression was detected in the 2B full sequence,while the 2B extracellular domain truncated prokaryotic expression vector was at a final concentration of IPTG of 0.6 mmol/L,37°C.The supernatant was expressed at 8 h after induction and was recognized by DHAV-1 infected duck serum.The prokaryotic expression of the 2B truncated protein was purified by gelatinization and the mouse anti-2B polyclonal antibody was obtained.The antibody recognizes DHAV-1infection and eukaryotic transient expression of 2B,and can be used for the detection of DHAV-1 and its 2B.Positioning and other research.HA and FLAG tags were added to the C-terminus and N-terminus of the 2B protein to construct eukaryotic expression vectors pCAGGS-2B-HA and pCAGGS-FLAG-2B,which were expressed in duck embryo fibroblast?DEF?.Indirect immunofluorescence was performed after permeabilization of0.25%Triton X-100,which can permeabilize cell membrane and organelle membrane,and digitonin?0.01 mg/mL?which can only permeabilize cell membrane.The result was 0.2%Triton X-100 after treatment with pCAGGS-Both 2B-HA and pCAGGS-FLAG-2B were able to detect fluorescence.After treatment with digitonin,only pCAGGS-2B-HA detected fluorescence,and it was confirmed that the 2B protein transmembrane mode was consistent with IA type viroporin.2.Biological function of DHAV-1 2BpCAGGS-2B-HA was transfected into DEF cells for transient expression.As a result,the expression of 2B protein disturbed the Ca²⁺balance of the host cells and caused the intracellular Ca²⁺concentration to be up-regulated.Nuclear fragmentation,nuclear lateral migration and nuclear shrinkage were observed by DAPI.In other cases,the fragmented DNA fragment was observed by DNA Ladder,and the brown nuclei were observed by the Tunnel method,and the enzyme activity of the apoptotic factor caspase-8 was detected to be unchanged and caspase-9 was significantly increased,indicating that the expression of 2B protein leads to apoptosis in host cells and apoptosis is most likely caused by caspase-9-related mitochondrial pathway;expression of 2B protein causes an increase in LC3-II/LC3-I and an increase in autophagosomes in host cells,but does not lead to autophagy.The substrate SQSTM1/p62 protein was significantly degraded,and autophagosomes and lysosomes did not co-localize,which confirmed that the expression of 2B protein caused autophagy in the host cells,but the autophagic flow was incomplete.In conclusion,the non-structural protein 2B of DHAV-1 has the biological characteristics of viroporin which can cause the increase of Ca²⁺in the host cell,induce apoptosis of host cells and autophagy.Finally,2B is an IA type viroporin.