The Role Of The Amino Acids Near To RGD Motif In Biological Characteristics Of FMDV

Foot-and-Mouth Disease (FMD) is an extremely contagious viral disease of many cloven-hoofedwild animals (such as cattle, sheep, pigs,et al). The disease causes significant losses to The worldhusbandry economy. FMDV (Foot-and-mouth disease virus) belongs to the genus Aphthovirus in thefamily Picornaviridae, its genome is a positive-sense single-stranded RNA which is approximately8500nt in length, including5’UTR, polyprotein coding region, and3’UTR. Polyprotein undergoesseveral proteolytic processing events to produce the three polyprotein precursors, P1(VP4、VP2、VP3、VP1), P2(2A、2B、2C) and P3(3A、3B、3C、3D). Different serotypes FMDV have a a highly conservedarginine-glycine-aspartic acid (RGD) tripeptide located on the G-H loop of VP1. RGD motif canrecognises and binds a number of integrins as receptors to initiate infection. In order to study the role ofthe amino acids near to RGD motif in biological characteristics of FMDV, FMDV O/JC/2010VP1coding region substituted the VP1coding region of O/HN/93, and then4mutations near the RGDsequence of the chimeric FMDV strain were designed (P142T, P142T/A152D, A152D/Q153P,P142T/A152D/Q153P).In this study, We used the primers to amplify1~3228nt region in O/HN/935’ treminal and cloneinto pGEM-T vector, termed pGEM-H1. The over-lap PCR product of O/JC/2010VP1and O/HN/93P2coding region, O/HN/93P3coding region inserted into pGEM-H1in turn. Then the full-length genomecDNAs of chimeric FMDV inserted into pBluescript II SK(+), named pBlue-HJ. Based on pBlue-HJ,we obtained the other four full-length genome cDNA clones which got the mutations in amino acid sites142,142/152,152/153,142/152/153of VP1G-H loop, named pBlue-HJ(142), pBlue-HJ(142+152),pBlue-HJ(152+153), pBlue-HJ(142+152+153) respectively.BHK-21cells were transfected with linearized recombinant plasmids using restrictionendonuclease NotI and plasmid expressing T7RNA polymerase. Apparent CPE were observed during50h, and The harvest time of90%BHK-21cell presenting CPE was stable after7passages of thesegenetically engineered viruses on BHK cells. The results of RT-PCR and sequencing methods identifiedthe success in rescuing these viruses. From investigation in molecular characteristics, we find there arenot significant difference in the replication of virus. The virulences of the five rescued viruses changedgradually and some of them have significant difference in the virulence, for example, the virulence ofpBlue-HJ(152+153) is over10times higher than the virulence of pBlue-HJ. Results demonstrate theimportant role of the amino acids in the bordering region of RGD sequence in FMDV binding to cells.From the result, We infer different virulences are relative to different efficiency of G-H loop usingintegrins.