The Role Of Host Protein GBP1 In Pseudorabies Virus Infection

Pseudorabies virus?PRV?belongs to varicella herpesvirus genus.It is a double-stranded linear DNA virus with a wide range of hosts.Its genome size is about 143 kb and contains more than 70 functional genes.Pseudorabies?PR?caused by PRV is an acute infectious disease,mainly for pregnancy failure,boar fertility,newborn piglets difficulty breathing,stunted growth,and nervous system symptoms.PR spreads quickly with many ways of transmission,wide range of prevalence and high mortality,which brings the pig industry huge losses.Guanylate binding protein 1?GBP1?belongs to the Guanylate binding proteins family?GBPs?,which is the member of the interferon-induced GTPases superfamily?65-72 k Da?.It can inhibit bacterial proliferation,viral replication and promote the formation of inflammatory response.It plays an important role in the antipathogen reaction.However,the role of GBP1in PRV infection has not been reported.A strain of PRV was isolated successfully in this study.Wild-type PK-15 cells were infected with the CRISPR-Cas9 lentivirus knockout system targeting the porcine gbp1 gene and screened with an appropriate concentration of puromycin to obtain positive clones.The gbp1 deleted cells were obtained.Respectively infected with wild-type PK-15 cells,gbp1gene deleted cells by PRV,real-time PCR and western blotting were used to detect the changes of host protein GBP1 and virus copy numbers in PRV infected cells,so as to clarify the role of GBP1 in PRV infection.The following experimental results were obtained:1.After PCR detection,plaque purification and expanded cell culture,a PRV isolated strain was successfully isolated and named SNBL1 strain.After PCR amplification and sequencing of the full length of g B,g C and g E genes,it was compared with the g B,g C and g E of other 29 PRV strains in Gen Bank.Phylogenetic trees and homology analysis of nucleic acid showed that PRV?SNBL1?strain belonged to the same branch as the domestic epidemic strains,which had high homology.Compared with the isolates from China after 2012,the homology was between 95.9%and 100.0%.The analysis of amino acid residues showed that the amino acid residues at position 472 of PRV?SNBL1?g B was mutated to glycine and the amino acid residues at position 761 was mutated to serine.The amino acid residues at position461 of PRV?SNBL1?g C was mutated to alanine,and the amino acid residues at positions477-487 were mutated.The amino acid sequence of PRV?SNBL1?g E did not mutate.At the same time,PRV g C recombinant prokaryotic expression vector was constructed.The recombinant protein was induced and purified,and the Balb/c mice aged 6 weeks were immunized.When the antiserum titer reached 1:10,000?1:100,000 by ELISA,whole blood was collected to get the serum.Western blotting results verified that the polyclonal antibody could detect the change of PRV g C protein level successfully.2.gbp1 gene knockout cells were successfully obtained by CRISPR-Cas9 lentivirus knockout system.Western blotting results showed that GBP1 protein expression could not be detected in knockout cells targeted at 278 of gbp1 gene.According to the genome sequencing results the CDS area 270-276 bases of gbp1 gene deleted cells targeted to 278 were missing,which caused the gene sequence shift,GBP1 90-93 amino acid residues mutation,and the 94amino acid residues shift termination.3.1 MOI PRV was used to infect wild-type PK-15 cells,23PK-15^gbp1+/+cells,109PK-15^gbp1+/+cells,and 278PK-15^gbp1-/-cells respectively.Cells in each group were collected at 24 h and 48 h after infection.Real-time PCR was used to detect the PRV DNA copy number in each group and the results showed that PRV DNA copies of 278PK-15^gbp1-/-cell group was significantly higher than that of the other three groups at 24 h and 48 h after PRV infection.TCID₅₀ results showed that the virus level in the 278PK-15^gbp1-/-cell group was significantly higher than that in the other three groups at 24 and 48 h after PRV infection.Meanwhile,western blotting results showed that the PRV g C protein of the 278PK-15^gbp1-/-group was significantly more than that of the other three groups.The effect of GBP1overexpression on PRV replication was further detected,and the results showed that the PRV infection after 24 h and 48 h,PRV DNA copies of PK-15^GBP1 cells group was significantly lower than that of the other two groups;TCID₅₀ results showed that virus level of PK-15^GBP1cells group was significantly lower than that of the other two groups.Meanwhile,western blotting results showed that the PRV g C protein of p K-15^GBP1 cells group was significantly less than that of the other two groups.Based on the above results,this study successfully isolated a PRV strain,prepared polyclonal antibodies that can be used to detect PRV g C protein,and constructed p K-15 cells with gbp1 gene delected.By comparing the PRV DNA copy numbers,TCID₅₀ and PRV g C protein levels of wild-type PK-15 cells and p K-15 cells with gbp1 gene delected,which were infected by PRV,the antiviral effect of host protein GBP1 in PK-15 cells infected with PRV was clarified,and it was determined that gbp1 deletion would lead to the increase of PRV replication level.The results provide a theoretical basis for further research on the formation mechanism of PRV resistance in pigs and gbp1 as a candidate disease resistance gene.