Roles Of CNX,CRT And ERp57 In The Expression Of Influenza Virus Hemagglutinin

In this study,in order to study the influence of endoplasmic reticulum(ER)chaperone CNX,CRT and erp57 on thebiosynthesis of influenza virus hemagglutinin(HA),expression of H7 subtype influenza virus HA of was determined in CNX,CRT and erp57 genetic knock out cells.Results of current study will lay the theoretical foundation for the maturation process of influenza virus HA protein in the endoplasmic reticulum:(1)Five N-glycosylation sites,including three N-terminal and two C-terminal glycosylation sites,were determined according to the prediction of the glycosylation sites of H7 HA.These glycosylation sites were subjected to mutations indifferent combination,i.e.,individual,double or triple glycosites mutations were constructed through site-directed mutations.(2)The expression of N-terminal glycosylation site mutation plasmids in wild type 293 T cells was detected by Western blot.(3)HA is a glycosylated protein,which requires the assistant of chaperons in the ER to reach maturation state.In order to study the functions of ER chaperons,H7HA-flag plasmid was transfected into293 T cells in which CNX or CRT gene was knocked out by the method of CRISPR/Cas9.(4)To study the relationship between CRT and influenza virus HA,wild type and N-terminal glycosylation site mutated HA-expressing plasmids were transfected into 293 T and CNX knockout 293 T cells,respectively,and detected by Western blot and co-immunoprecipitation.(5)To further confirm the interaction between H7 HA and CRT,H7HA-flag and N-terminal glycosylation site mutation plasmids with CRT-myc plasmids were transfected into CRT knockout 293 T cellsand detected by Western blot and co-immunoprecipitation.(6)In order to study the mechanism under which ERp57 modulates the maturation of HA,we first screened the erp57 gene-deficient human lung adenocarcinoma cell line(A549)by CRISPR/Cas9 system.p Cas9-EGFPwas co-transfected with erp57 g RNA-carrying recombinant plasmids into A549.Monoclonal cell lines were screened through limited dilution and flow cytometry.Genetic knocking out cells were detected by nucleotide sequencing and Western blot analysis.(7)To figure out the influence of erp57 on influenza virus.The wild type and erp57 knock out cells were infected with H1N1 influenza virus and detected viral titer.Results:(1)N-terminal and two C-terminal glycosylation sites were mutations in different combination,i.e.,individual,double or triple glycosites mutations were successful constructed through site-directed mutations.(2)It was found that the expression of HA0 in the third glycosylation site mutation decreased and the expression of HA0 and HA2 cannot be detected in when the C-terminal glycans were eliminated.(3)Results of Western blot and flow cytometry showed that expression of H7 HA protein significantly decreased in CNX/CRT double knock out 293 T cells.(4)Results of Western blot and co-immunoprecipitation demonstrated that endogenous CRT formed a complex with H7 HA,and removing of N-terminal glycosylation sitedidn'tinfluence the interaction between H7 HA and CRT.(5)It was shown that exogenous CRT interacted with H7 HA,and N-terminal glycans elimination of H7 HA didn'tinfluence the interaction between H7 HA and CRT.(6)Successful genetic knocking out was confirmed by nucleotide sequencing and Western blot analysis.Results indicated that the erp57 knockout cell line exhibited similar growth characteristicswith wild type cells.(7)It was found that the viral replication was significantly inhibited in erp57 knock out cells,suggesting that ERp57 play important roles in viral replication.In conclusion,results of current study indicated that(1)The glycosylation of number 3 site of the N-terminal glycosylation sites of H7 HA reduced the expression of HA0(2)C-terminal glycosylation site mutation abolished the expression of HA0 and HA2 plasmids(3)expression of H7 HA protein decreased significantly in CNX/CRT knock out cells(4)H7HA interacted with CRT and N-terminal glycosylation site mutate of H7 HA didn't influence the connection between H7 HA and CRT,and(5)the viral replication was significantly inhibited in erp57 knock out cells.The current study provides useful information for the development of new anti-viral strategies.