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.

Natural products produced by members of the phylum Actinobacteria underpin many industrially and medically important compounds; however, the majority of the ~30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. Understanding the diversity of regulatory strategies controlling the expression of these pathways is therefore critical if their biosynthetic potential is to be explored for new drug leads. Our findings reveal that the candicidin cluster-situated regulator FscRI coordinately controls the biosynthesis of both candicidin and antimycin, which is the first observation of cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products. We anticipate that this will emerge as a major strategy by which members of the phylum Actinobacteria coordinately produce natural products, which will advance our understanding of how the expression of secondary metabolism is controlled and will aid the pursuit of “silent” biosynthetic pathway activation.

Discovering new antibiotics is vital to combat the growing threat of antimicrobial resistance. Most currently used antibiotics originate from the natural products of actinomycete bacteria, particularly Streptomyces species, that were discovered over 60 years ago. However, genome sequencing has revealed that most antibiotic-producing microorganisms encode many more natural products than previously thought. Biosynthesis of these natural products is tightly regulated by global and cluster situated regulators (CSRs), most of which respond to unknown environmental stimuli, and this likely explains why many biosynthetic gene clusters (BGCs) are not expressed under laboratory conditions. One approach towards novel natural product discovery is to awaken these cryptic BGCs by re-wiring the regulatory control mechanism(s). Most CSRs bind intergenic regions of DNA in their own BGC to control compound biosynthesis, but some CSRs can control the biosynthesis of multiple natural products by binding to several different BGCs. These cross-cluster regulators present an opportunity for natural product discovery, as the expression of multiple BGCs can be affected through the manipulation of a single regulator. This review describes examples of these different mechanisms, including specific examples of cross-cluster regulation, and assesses the impact that this knowledge may have on the discovery of novel natural products.