Abstract
Infection of susceptible cells with foot and mouth disease virus (FMDV) results in multiplication of the RNA genome and assembly of mature virions. The entire process of genome replication is completed in a few hours and encompasses many intracellular events. Like other picornaviruses, FMDV uses a peptide primed RNA replication mechanism. The factors that are required to uridylylate each of the three FMDV VPg peptides and the role of the FMDV cis-acting replication element (cre) or 3B Uridylylation Site (bus) in VPg uridylylation have been determined. The native N-terminus of the FMDV 3Dpol enzyme is a pre-requisite for VPg uridylylation in vitro and the effects of mutations in the RNA template are consistent with a slide-back mechanism. The role of the poly(A) tail in uridylylating VPg was insignificant using full-length FMDV RNA transcripts suggesting the possibility of an alternative mechanism of VPg incorporation into negative strand RNA. The optimal RNA sequences required for VPg uridylylation were found to be within the 5' non-coding region (NCR). Furthermore, the results also showed evidence for RNA-RNA interactions between distinct structures from within the 5' NCR that influence VPg uridylylation. The polymerase precursor 3CDpro is also a prerequisite for uridylylation of each of the FMDV VPg peptides. However BCpro alone can substitute for 3 CD, but is much less efficient. It also appeared that the overall charge of the VPg peptides determines their recognition by the FMDV 3Dpol. The RNA binding activity of the 3C was found to be required for its stimulatory effects on VPg uridylylation. Unlike the poliovirus cloverleaf, the FMDV S-fragment (at the 5' end of the genome) does not interact with the FMDV 3CD precursor protein; however it binds specifically to a cellular factor p48. The crude replication complexes (CRCs) isolated from FMDV-infected cells were found to synthesize viral RNA very efficiently and an in vitro RNA replication system developed using these CRCs can be used to study the complete RNA replication events of FMDV.