Insights into Polyprotein Processing and RNA-Protein Interactions in Foot-and-Mouth Disease Virus Genome Replication.

Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; Lpro and P1, P2, and P3 (also termed 1ABCD, 2BC, and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate the proteins 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 intermolecular 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 has an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural 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. IMPORTANCE Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans alternative pathways (i.e., intra- and intermolecular proteolysis). The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest 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 RNA genome replication.

VelcroVax: a "Bolt-On" Vaccine Platform for Glycoprotein Display.

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and newly emerging pathogens. Virus-like particles (VLPs) form the basis of two of the most successful licensed vaccines (against hepatitis B virus [HBV] and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which assemble into immunogenic nanoparticles. VLPs can be modified to present unrelated antigens, and here we describe a universal "bolt-on" platform (termed VelcroVax) where the capturing VLP and the target antigen are produced separately. We utilize a modified HBV core (HBcAg) VLP with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus Junín virus (JUNV). Using this model system, we have solved the first high-resolution structures of VelcroVax VLPs and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the noncomplexed viral protein. We propose that this system could be modified to present a range of antigens and therefore form the foundation of future rapid-response vaccination strategies. IMPORTANCE The hepatitis B core protein (HBc) forms noninfectious virus-like particles, which can be modified to present a capturing molecule, allowing suitably tagged antigens to be bound on their surface. This system can be adapted and provides the foundation for a universal "bolt-on" vaccine platform (termed VelcroVax) that can be easily and rapidly modified to generate nanoparticle vaccine candidates.