Enhancement Of Foot-and-Mouth Disease Virus Replication Caused By 2C T135I Substitution And Its Molecular Basis

Foot-and-mouth disease(FMD)is an acute,systemic disease affecting domestic cloven-hooved animals caused by Foot-and-mouth disease virus(FMDV),which is a positive-sense,single-stranded RNA virus that belongs to the Aphthovirus genus,Picornaviridae family.The protein 3A,the nonstructural protein of foot-and-mouth disease virus(FMDV),consists of 153 amino acids(aa),with a highly conserved N-terminus and a variable C-terminal region.The C-terminal of 3A plays an important role in viral replication,virulence and host range.It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal of 3A attenuate serotype O and C FMDVs,which replicate poorly in bovine cells but not in porcine cells,and the C-terminal of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells.It is not known whether a large deletion of the C-terminal of protein 3A could affect the replication of serotype O FMDV.In order to study the role of longer C-terminal of 3A played in the replication of serotype O FMDV,we have constructed a 3A deleted FMDV mutant on based on a serotype O FMDV O/YS/CHA/05,in which with a 60-amino acid 3A between residues 84 and 143 was deleted.The rescued virus O/YS/CHA/05-Δ3A exhibited 10-fold lower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells.These results indicated that the 60-amino acid deletion in the 3A protein impaired FMDV replication.After 14 passages in BHK-21 cells,the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a wild-type virus-like level.Then through comparative sequence analysis,two amino acid substitutions,P153 L in VP1 and T135 I in 2C,were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome,which reveals that the two amino acid substitutions may alone or together be responsible for the enhanced replication capacity of O/YS/CHA/05-Δ3A.Subsequently,the amino acid substitutions VP1 P153 L and 2C T135 I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses and to examine their growth kinetics.We found that 2C T135 I,and not VP1 P153 L,enhanced the virus replication capacity.The 2C T135 I substitution also improved the replication of the wild-type virus,which indicates that whether 3A with deletion or not,2C T135 I could enhance the replication capacity of O/YS/CHA/05.Furthermore,we determined that the T135 I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through enhanced viral RNA synthesis.In this study,our results indicate that protein 3A with 60-amino acid deletion between residues 84 and 143 could impair replication of O/YS/CHA/05,and the substitution 2C T135 I could improve the virus replication.These results also provide a new way to study the synergistic effect of 2C and 3A during the FMDV replication.