| Avian infectious bronchitis virus(IBV),a member of the genus γ Coronavirus in the family Coronaviridae,is the etiological agent of chicken infectious bronchitis,which has caused the great economic losses in poultry industry around the world.IBV genome is a positive single-stranded RNA,which encodes four major structural proteins: Spike glycoprotein(S),Envelope protein(E),Membrane protein(M),and Nucleocapsid protein(N).Among them,N protein is composed of 409 amino acids and is a highly alkaline phosphorylated protein,which plays an important role in viral RNA replication,protein translation,particle formation and immune regulation.Structurally,SR-region is a serine-and arginine-rich linker(residues 161-217)located between two structural domains of IBV N protein: an N-terminal RNA binding domain(NTD,residues 29-160)and a C-terminal dimerization domain(CTD,residues 218-329).The SR region is reported to be involved in the multimerization of SARS-Co V N protein,interacting with unstructured protein 3 in mouse hepatitis virus(MHV).In this study,the effects of arginine residues in SR region on IBV replication was investigated by mutagenesis and reverse genetics.The main research results are as follows:(1)IBV reverse genetics research indicated that the deletions of the entire and partial SR region(Δ 161-217,Δ193-217 and Δ209-217aa)lead to the failure in the recovery of IBV,implying that IBV-SR region is necessary for IBV replication.(2)In order to study the effects of arginine residues in the SR region on virus replication,arginine(R)was mutated into alanine(A),and mutants with 1,2,and 4 point mutations named R162 A,R164A,R167 A,M-2R/A(R178A/R182A),and R-4R/A(R182A/R186A/188A/R189A)were constructed,respectively.In addition,the SR region of SARS-Co V was used to replace the IBV-SR region(residues161-191)to construct the corresponding mutant.IBV reverse genetic study showed that these IBV mutants were recovered,suggesting these arginine residues were not necessary for IBV replication,and the SR region of SARS-Co V could replace the IBV-SR region to perform its function.(3)Plaque assay showed that r IBV,R162 A,R164A,and R167 A had no significant differences in the average diameters of plaque,indicating that the changes of arginine residue 162,164,167 in SR region had no or insignificant impact on the IBV pathogenicity.However,the diameter of plaque formed by the mutant M-2R/A was significantly smaller than that of r IBV,indicating that the simultaneous mutation of R178 and R182 significantly reduced the IBV pathogenicity and the mutations simultaneous mutation of R182,R186,R188 and R189 also affected the pathogenicity.Otherwise,replacing the IBV-SR region with the SR region of SARS-Co V resulted in smaller plaque,indicating that the SR region of IBV-N protein is relatively conservative in function and can be replaced by the SR region of SARS-Co V.(4)To explore the influences of the arginine residues in the SR region on IBV replication and transcription,RT-q PCR detection of the syntheses of the IBV genomic negative-stranded RNA and subgenomic RNA(sgm RNA)of S gene were performed.The results revealed that the amounts of the negative-stranded RNA in Vero cells infected with R-4R/A and M-2R/A were 0.49 and 0.12 times of those in r IBV-infected cells 20 h post infection respectively,and the amounts of S-sgm RNA were 0.44 times and 0.07 times,respectively,while the amounts of negative stranded RNA and Ssgm RNA mutant SARS-SR were 1.77 and 1.09 times of those in r IBV-infected cells,respectively.These results indicated that mutations of arginine residues at four sites(R182A/R186A/R188A/R189A)and mutations of arginine residues at two sites(R178A/R182A)both resulted in the decreased syntheses of IBV genomic RNA and sgm RNA,but the latter had a stronger inhibitory effect on IBV replication.In contrast,replacing 161-191 amino acids in the IBV-SR region with 31 amino acids in the SR region of SARS-Co V increased the syntheses of IBV genomic RNA and sgm RNA,promoting the IBV replication(5)Western blot was used to detect the expression levels of N and S proteins at different time courses of virus infection.The results showed that,at 12,16 and 20 h post infection,the expression levels of N proteins in mutant R-4R/A infected cells were 0.28,0.48 and 0.41 times of those in r IBV infected cells,respectively.The expression levels of N protein in M-2R/A infected cells were 0.17,0.07 and 0.24 times of those in r IBV infected cells,respectively.The N protein expression levels in the mutant SARS-SR infected cells were 7.68,1.95 and 0.96 times of those in r IBV infected cells,respectively.Similarly,Western blot analysis on the expression level of virus S protein indicated that the expression level of S protein in mutant R-4R/A and M-2R/A infected cells both decreased at 16 and 20 hours of infection,but the effect of M-2R/A was more significant.At the time of infection,the S protein in the mutant SARS-SR infected cells was significantly higher than that in r IBV infected cells.These results suggested that mutations of arginine residues at four sites(R182A/R186A/R188A/R189A)and mutations of arginine residues at two sites(R178A/R182A)both resulted in decreased expression of IBV-N and S proteins,but the latter had a stronger inhibitory effect on the synthesis of IBV-N and S proteins.In contrast,the substitution of 31 amino acids in SR region of SARS-Co V for amino acids at sites 161-191 in IBV-SR region significantly increased the expression of IBV-N and S proteins.In summary,the results showed that the IBV-SR region is necessary for virus replication.Mutations of arginine residues at four sites(R182A/R186A/R188A/R189A)and arginine residues at two sites(R178A/R182A)could inhibit the IBV replication,but the latter had a stronger inhibitory effect.In contrast,the substitution of 31 amino acids in the SR region of sars-cov for amino acids in the IBV-SR region can significantly improve the replication level of IBV,revealing the conserved function of coronavirus SR region. |
PDF Full Text Request