Functional advantages of triplication of the 3B coding region of the FMDV genome

For gene duplication to be maintained, particularly in the small genomes of RNA viruses, this should offer some advantages. We have investigated the functions of a small protein termed VPg or 3B, which acts as a primer in the replication of foot‐and‐mouth disease virus (FMDV). Many related picornaviruses encode a single copy but uniquely the FMDV genome includes three (nonidentical) copies of the 3B coding region. Using sub‐genomic replicons incorporating nonfunctional 3Bs and 3B fusion products in competition and complementation assays, we investigated the contributions of individual 3Bs to replication and the structural requirements for functionality. We showed that a free N‐terminus is required for 3B to function as a primer and although a single 3B can support genome replication, additional copies provide a competitive advantage. However, a fourth copy confers no further advantage. Furthermore, we find that a minimum of two 3Bs is necessary for trans replication of FMDV replicons, which is unlike other picornaviruses where a single 3B can be used for both cis and trans replication. Our data are consistent with a model in which 3B copy number expansion within the FMDV genome has allowed evolution of separate cis and trans acting functions, providing selective pressure to maintain multiple copies of 3B.