Identification And Functional Study Of Positive Selection Sites In MERS-CoV Genome

Middle East respiratory syndrome coronavirus(MERS-CoV)is the pathogen of Middle East respiratory syndrome,which emerging in the Middle East in 2012.MERS-CoV is a typical animal-derived virus,and dromedary camels are the direct animal source of it.Coronavirus,as the known RNA virus with the largest genome,has a high mutation frequency.Previous studies have shown that there are differences in MERS-CoV genome sequences isolated from different species,periods and locations,hence it is speculated that there are important variation sites of positive selection during the transmission of MERS-CoV from camel to human and from human to human.The functional study of the mutation site showed that the variation of S protein of the virus prevalent in Korea in 2015 gave it the ability to resist neutralizing antibody,resulting in the enhancement of virulence,suggesting that the mutation site may affect the function of the virus gene and lead to alter viral pathogenicity.The host innate immune system is one of the main factors of virus gene selection pressure,and a large number of studies have shown that MERS-CoV ORF4 a,ORF4b,ORF8 b,N and many other genes have the function of regulating host innate immunity.Therefore,the purpose of this study is to screen significant positive selective sites in the MERS-CoV genome and to explore whether mutations affect host innate immunity.We used PAML software to screen several significant positive selection mutation sites of MERS-CoV genome,and selected the mutation at amino acid site 86 of ORF3 gene with high mutation frequency,that is,the mutation from proline(P)to phenylalanine(F)and leucine(L).And the mutation of 69 amino acid site of M gene,that is,the mutation from Valine(V)to F and isoleucine(I),to study the effect of the mutation on host innate immunity regulated by ORF3 and M protein.According to the MERS-CoV genome sequence(Genbank:JX869059.2),the eukaryotic expression vectors of ORF3,M full-length genes and the above-mentioned single amino acid mutations were constructed.In the model of 293 T cells,the effects of the positive selective mutations on host innate immunity regulated by ORF3 and M protein were studied by quantitative PCR.The results showed that compared with the wild type M protein,the V69 F and V69 I mutants significantly inhibited the expression of type I interferon IFN-? and downstream ISGs induced by Se V,while there was no significant difference between the P86 F and P86 L mutants on the expression of Se V induced type I interferon IFN-?compared with the wild type ORF3 protein.This study preliminarily revealed the positive selection site of MERS-CoV during epidemic and transmission,and preliminarily proved that the single amino acid mutation at site 69 of M protein of MERS-CoV significantly enhanced the ability of M protein to antagonize the IFN-? signal pathway of host type I interferon.At the same time,it was proved that the single amino acid mutation at site 86 of ORF3 had no significant effect on the antagonistic effect on the signal pathway of I interferon IFN-?.The results of the study are helpful to reveal the transmission and evolution features of MERS-CoV,may help for the prevention and control of MERS-CoV and may also provide new ideas for novel coronavirus research.