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The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from “helper” replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the “helper” replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy., Author summary Positive-strand RNA viruses are generally typified by a small genome size. To compensate for this limitation they have evolved ways of multiplying the functions achievable from a small genome, thus increasing ‘genetic economy’. In the picornavirus family of RNA viruses the viral proteins are produced via a series of transient precursors, which are believed to possess functions required for virus replication which are additional to those of the fully processed proteins. In this report, we show that the small 3B3 protein from foot-and-mouth disease virus, an economically important animal pathogen, is key in directing the processing pathways of the P3 polyprotein to facilitate release of the viral polymerase. This is in addition to, and independent from, the role of 3B3 as a primer for replication. This study exemplifies how multiple functions within one small viral protein can control viral replication and demonstrates a new level of genetic economy in the picornaviruses. Furthermore, systems that provide ‘help’ in trans to assist replication have the potential to be exploited for the production of attenuated vaccines (e.g. using “helper’ cell lines). We are now in the process of investigating how mutations at polyprotein processing boundaries may be used for producing attenuated vaccines.

Foot-and-mouth disease (FMD) is one of the most economically important infectious diseases of production animals globally. Vaccination can help to control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in mammalian cell culture under high containment. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a “single cycle” packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. In cattle vaccinated once with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge. Following challenge with FMDV, the cattle were protected against disease and no FMDV RNA was detected in their sera. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed and clinical disease occurred. This prime-boost system, using reagents that can be generated outside of high-containment facilities, offers significant advantages to achieve control of FMD by vaccination.

IntroductionFoot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can help to control this disease, however, current vaccines based on chemically inactivated FMDV, are imperfect and there is a need for new, safe and effective vaccines to control FMD. There is no cross protection between the 7 serotypes but serotype O is the most abundant globally.Material and methodsThe FMDV capsid protein precursor (P1-2A) of strain O1 Manisa has been expressed with the FMDV 3C protease (3Cpro) using a “single cycle” packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). Purified O1 Manisa empty capsid particles (ECs) have been prepared using a recombinant vaccinia virus expression system. Cattle have been vaccinated with the SFV-FMDV vectors and boosted subsequently with the ECs and then challenged with serotype O FMDV. The immune response against FMDV achieved by vaccination and infection status following challenge has been determined. ResultsIn cattle vaccinated once with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge than in naïve animals. In subsequent experiments, cattle were sequentially vaccinated with the rSFV-FMDV followed by recombinant FMDV empty capsid particles prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge. Following challenge with serotype O FMDV, the cattle were protected against disease and no FMDV RNA was detected in their sera. DiscussionThis prime-boost system, using reagents that can be generated outside of high-containment facilities, offers significant advantages to achieve control of FMD by vaccination.

Sulfur is vital for primary and secondary metabolism in plant roots. To understand the molecular and morphogenetic changes associated with loss of this key macronutrient, we grew Arabidopsis (Arabidopsis thaliana) seedlings in low-sulfur conditions. These conditions induced a cascade of cellular events that converged to produce a profound intracellular phenotype defined by large cytoplasmic inclusions. The inclusions, termed low-sulfur Pox, show cell type- and developmental zone-specific localization. Transcriptome analysis suggested that low sulfur causes dysfunction of the glutathione/ascorbate cycle, which reduces flavonoids. Genetic and biochemical evidence indicated that low-sulfur Pox are the result of peroxidase-catalyzed oxidation of quercetin in roots grown under sulfur-depleted conditions.

Highlights • FMDV replication can be studied outwith high disease secure facilities. • FMDV replicon genomes encoding GFP are replication competent. • These FMDV replicon systems can be used to study replication by live-cell imaging/image analyses., The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious ‘replicon’ systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (Lpro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.