Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3D^pol to membranes

by Cristina Ferrer-Orta, Diego S. Ferrero, Nuria Verdaguer

Picornavirus genome replication takes place in specialized intracellular membrane compartments that concentrate viral RNA and proteins as well as a number of host factors that also participate in the process. The core enzyme in the replication machinery is the viral RNA-dependent RNA polymerase (RdRP) 3D^pol. Replication requires the primer protein 3B (or VPg) attached to two uridine molecules. 3B uridylylation is also catalysed by 3D^pol. Another critical interaction in picornavirus replication is that between 3D^pol and the precursor 3AB, a membrane-binding protein responsible for the localization of 3D^pol to the membranous compartments at which replication occurs. Unlike other picornaviruses, the animal pathogen foot-and-mouth disease virus (FMDV), encodes three non-identical copies of the 3B (3B1, 3B2, and 3B3) that could be specialized in different functions within the replication complex. Here, we have used a combination of biophysics, molecular and structural biology approaches to characterize the functional binding of FMDV 3B1 to the base of the palm of 3D^pol. The 1.7 Å resolution crystal structure of the FMDV 3D^pol -3B1 complex shows that 3B1 simultaneously links two 3D^pol molecules by binding at the bottom of their palm subdomains in an almost symmetric way. The two 3B1 contact surfaces involve a combination of hydrophobic and basic residues at the N- (G5-P6, R9; Region I) and C-terminus (R16, L19-P20; Region II) of this small protein. Enzyme-Linked Immunosorbent Assays (ELISA) show that the two 3B1 binding sites play a role in 3D^pol binding, with region II presenting the highest affinity. ELISA assays show that 3D^pol has higher binding affinity for 3B1 than for 3B2 or 3B3. Membrane-based pull-down assays show that 3B1 region II, and to a lesser extent also region I play essential roles in mediating the interaction of 3AB with the polymerase and its recruitment to intracellular membranes.