Study On The Mechanism Of Porcine CYTH2 Gene Mediating Influenza Virus Endocytosis

The influenza A virus has caused huge economic losses to the breeding industry worldwide,and the transmission between different species has posed a serious public health risk.Because the swine trachea have both avian influenza virus and human influenza virus binding receptors,thus as a "mixer" of influenza viruses,swine can reassemble the different subtypes of influenza viruses into new influenza viruses in vivo,thus increasing the risk of virus immune escape and cross-species transmission.The process of influenza virus infecting the host requires the participation of host protein.Therefore,the study of interaction mechanism between influenza virus and host protein is contribute to the discovery of targets for drugs and breeding resistance.Previous research group studies have found that the genome-wide knockout library of clustered regular interspersed short palindromic repeats(CRISPR)was used to identify the essential host factors related to H7N9 infection and proliferation in Human lung adenocarcinoma cells(A549).Among them,Cytohesin 2(cyth2)involved the endocytosis of the virus in infection that contribut to fusion of the viral envelope and endosomal membrane.In order to explore the mechanism of CYTH2 mediating the endocytosis of influenza virus and the cyth2 gene was evaluated as a target for breeding resistance.Therefore,this research purpose to explore the effect of cyth2 gene on the proliferation of influenza virus in newborn pig trachea epithelial NPTr and porcine macrophage cell 3D4 and analyze the mechanism of CYTH2 mediating the endocytosis of influenza virus.The research content and the result are as follows:1 The swine cyth2 gene homology analysisIn previous experiments we found that human cyth2 gene can mediate influenza virus infection.In order to evaluate the influence of swine cyth2 gene on influenza virus infection.Firstly,through the homology analysis of the cyth2 genes of humans and swine,The amino acid homology of swine CYTH2 and human CYTH2 was higher(99.50%)and consisted with exactly same domains,which play a function of mediating downstream Arf protein signal transduction(Casanova 2007).Therefore,we studied whether the swine cyth2 gene can also play a role in mediating influenza virus infection in swine cells.2 The effect of swine cyth2 gene on the proliferation of influenza virusIn order to explore the effects of swine cyth2 gene on the proliferation of influenza virus.The cyth2 gene knockout cell line(NPTr-?CYTH2)was constructed on NPTr cells using CRISPR/Cas9,and the cyth2 gene complement cell line(NPTr-C-?CYTH2)was constructed on NPTr-?CYTH2 using recombinant retroviral.Firstly,by detecting the cell supernatant lactate dehydrogenase(Lactate Dehydrogenase,LDH),we found that cyth2 gene knockout and complement did not affect the viability of NPTr cells.further,NPTrnon-target,NPTr-?CYTH2,NPTr-C-?CYTH2 cells were infected with influenza virus,the results of the proliferation curve found that cyth2 gene knockout on NPTr cells can inhibit the proliferation of influenza virus.While this inhibition effect disappeared after cyth2 gene complement.Finally,we construct cyth2 gene knockout cell lines and complement cell lines on pig 3D4 cells by same way.After influenza virus infection,it was found that gene knockout can also inhibit the proliferation of influenza virus on 3D4 cells.3 The effect of swine CYTH2 on the endocytosis of influenza virusPrevious experiments showed that in A549 with cyth2 gene knocked out,viral particles would stay in the endosomal vesicles,and the viral capsid membrane could not fuse with the endosomal membrane,thus inhibiting the release of viral particles and unable to infection.In order to further explore the effect of swine cyth2 gene knockout on influenza virus endocytosis,fluorescence labeled virus was used to trace and confirm that swine cyth2 gene knockout on NPTr cells did not affect the binding and internalization of influenza virus firstly.Then fluorescence quantitative detection of v RNA and m RNA of influenza virus in cells found that the knockout of swine cyth2 gene inhibited the process of influenza virus transfer from endosomes to replication and transcription.Further acidification test found that treated with acidic PBS(p H=5)to bypass the stage of viral endosome transport and release virus particles in advance.We found that swine cyth2 gene affects influenza virus endosomes 's transfer.Finally,confocal analyse of the co-localization of influenza virus with Rab5(early endosomal marker)and Rab7(late endosomal marker)in NPTr-?CYTH2 cells,we found that knockout of swine cyth2 gene inhibited the transportion from early endosome to late endosomes.In conclusion,this research found that swine CYTH2 in NPTr cells mediates the proliferation of influenza viruses by involved the process of influenza virus transportion from early endosomes to late endosomes.