Insights into polyprotein processing and RNA-protein interactions in foot-and-mouth disease virus genome replication.

Abstract

Foot-and-mouth disease affects cloven hoofed animals and is caused by foot-and-mouth disease virus (FMDV), a picornavirus with a positive-sense RNA genome. The FMDV genome contains a single open reading frame, which is translated to produce a polyprotein that is cleaved by viral proteases to produce the viral structural and non-structural proteins. Initial processing of the polyprotein occurs at three main junctions to generate four primary products; Lpro and the P1, P2 and P3 precursors (also termed 1ABCD, 2BC and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate non-structural proteins that are required for viral replication, including the enzymes 2C, 3Cpro and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and inter-molecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction had an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single point mutation at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this point mutation permitted production of some non-enzymatic non-structural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. ImportanceFoot-and-mouth disease virus (FMDV) is an economically important pathogen of animals that is responsible for foot-and-mouth disease (FMD). FMD is endemic in many parts of the world and can results in major economic losses. Replication of the virus is a highly coordinated event that occurs within membrane-associated compartments in infected cells and requires the viral non-structural proteins. These are all initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans pathways (i.e., intra- and inter-molecular proteolysis). Alternative processing pathways can provide a mechanism to help coordinate viral replication by providing temporal control to protein production. Here, we analyse the consequences of mutations that change temporal control of FMDV polyprotein processing. Our data suggests that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of FMDV genome replication.