Alignment of picornavirus proteinase/polymerase sequences reveals this family evolved into five ‘supergroups’. Interestingly, the nature of the 2A region of the picornavirus polyprotein is highly correlated with this phylogeny. Viruses within supergroup 4, the Paavivirinae, have complex 2A regions with many viruses encoding multiple 2ANPGP sequences. In vitro transcription/translation analyses of a synthetic polyprotein comprising green fluorescent protein (GFP) linked to β-glucuronidase (GUS) via individual 2ANPGPs showed two main phenotypes: highly active 2ANPGP sequences—similar to foot-and-mouth disease virus 2ANPGP—and, surprisingly, a novel phenotype of some 2ANPGP sequences which apparently terminate translation at the C-terminus of 2ANPGP without detectable re-initiation of downstream sequences (GUS). Probing databases with the short sequences between 2ANPGPs did not reveal any potential ‘accessory’ functions. The novel, highly active, 2A-like sequences we identified substantially expand the toolbox for biomedical/biotechnological co-expression applications.

Translational control of viral gene expression is a fundamental process essential for the vitality of all viruses. In special cases, signals encoded in the mRNA reprogram the ribosome to read the message in a different way, a process termed “translational recoding”. The 2A region of the foot-and-mouth disease virus (FMDV) encodes a short sequence, only 18 amino acids, that mediates self-processing by a novel translational effect “ribosome skipping” rather than proteolysis. Briefly, 2A interacts with the ribosome exit tunnel to inhibit peptide bond formation at the C terminus of the 2A sequence. Translation terminates at this point, but then resumes elongation, creating a second independent protein product. Thus, discrete proteins can be produced from a single transcript. The 2A sequence is particularly useful in vector strategies (AAV and retroviral vectors) where the capacity to incorporate foreign DNA is limited. Use of 2A and “2A-like” peptides to link the sequences encoding several proteins in the same open reading frame has led to their increasing use as important tools in biotechnology and biomedicine. This technology has been crucial for the visual tracking of expressed proteins, human gene therapies targeting cancer, production of induced human pluripotent stem cells for regenerative medicine, creation of transgenic animals and plants and the improvement of CRISPR-Cas9 and TALEN genome editing methods.

Both current live, attenuated, and killed virus vaccines for bovine viral diarrhea virus (BVDV) have their limitations. Here, we report the development of a BVDV subunit vaccine by (i) the expression of a secreted form of a recombinant E2 glycoprotein using BHK21 cells and (ii) determination of the immune responses in mice. The E2 glycoprotein was modified by deletion of the C-terminal transmembrane anchor domain and fusion to a V5 epitope tag. This allowed detection using anti-V5 monoclonal antibodies together with simple purification of the expressed, secreted, form of E2 from the cell media. Furthermore, we genetically fused green fluorescent protein (GFP) linked to E2 via a Thosea asigna virus 2A (T2A) ribosome skipping sequence thereby creating a self-processing polyprotein [GFP-T2A-BVDV-E2trunk-V5], producing discrete [GFP-T2A] and [E2trunk-V5] translation products: GFP fluorescence acts, therefore, as a surrogate marker of E2 expression, BALB/c mice were inoculated with [E2trunk-V5] purified from cell media and both humoral and cellular immune responses were observed. Our antigen expression system provides, therefore, both (i) a simple antigen purification protocol together with (ii) a feasible strategy for further, large-scale, production of vaccines.

Genetically-encoded biosensors are used to track biochemical activities in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. In the present article, we describe the application of genetically-encoded FRET biosensors with high content microscopy to image the signaling responses of thousands of neurons in response to drug treatments. We applied this approach to reveal intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. The striatum is largely composed of medium-spiny GABAergic neurons which are divided into two broad sub-types based in part on their expression of dopamine D1 vs. D2 receptors. Using high content FRET imaging and immunofluorescence, we identified neuronal sub-populations with unique responses to pharmacological manipulation. Focusing on dopamine- and glutamate-regulated PKA and ERK1/2 signaling in both the cytoplasm and nucleus, we identified pronounced intercellular differences, in both the magnitude and kinetics of signaling responses to drug application. Importantly, we found that a conventional “bulk” analysis that included all cells in culture yielded a different rank order of drug potency than that revealed by our single-cell analysis. The high degree of heterogeneity that we observed at the single cell level would not have been detectable using common population-level analyses, derived for example from western blotting or plate reader-based measurements. In conclusion, our single-cell analytical approach highlights the limitations of population-level analyses, and provides a novel way to study signaling biology.

Recycled wastewater is widely used owing to the potential shortage of water resources for drinking purposes, recreational activities, and irrigation. However, gut microbiomes of both human beings and animals negatively affect this water quality. Wastewater contamination is continuously monitored, using fecal contamination indicators or microbial source tracking approaches, to oppose arising enteric infections. Viral gastroenteritis is considered a principal manifestation of waterborne pathogenic virome-mediated infections, which are mainly transmitted via the fecal-oral route. Furthermore, acquired enteric viromes are the common cause of infantile acute diarrhea. Moreover, public exposure to wastewater via wastewater discharge or treated wastewater reuse has led to a significant surge of public health concerns. In this review, we discussed the etiology of waterborne enteric viromes, notably gastrointestinal virus infections, and public exposure to municipal wastewater. Conclusively, the early human virome is affected mainly by birth mode, dietary behavior, and maternal health, and could provide a signature of disease incidence, however, more virome diversification is acquired in adulthood. A multi-phase treatment approach offered an effective means for the elimination of wastewater reuse mediated public risks. The insights highlighted in this paper offer essential information for defining probable etiologies and assessing risks related to exposure to discharged or reused wastewater.

Some RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 46 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the nonstructural proteins, only 3 are essential for replication of an FMDV subgenomic replicon.

AIMS:Vaccines for bovine ephemeral fever virus (BEFV) are available but are difficult to produce, expensive or suffer from genetic instability. Therefore, we designed constructs encoding C-terminally truncated forms (transmembrane anchoring region deleted) of glycoproteins G and GNS such that they were secreted from the cell into the media to achieve high-level antigen expression, correct glycosylation pattern and enable further simple purification with the V5 epitope tag.METHODS AND RESULTS:In this study, synthetic biology was employed to create membrane-bound and secreted forms of G and GNS glycoprotein. Mammalian cell culture was employed as an antigen expression platform, and the secreted forms of G and GNS protein were easily purified from media using a highly effective, single-step method. The V5 epitope tag was genetically fused to the C-termini of the proteins, enabling detection of the antigen through immunoblotting and immunomicroscopy. Our data demonstrated that the C-terminally truncated form of the G glycoprotein was efficiently secreted from cells into the cell media. Moreover the immunogenicity was confirmed in mice test.CONCLUSIONS:The immuno-dot blots showed that the truncated G glycoprotein was present in the total cell extract, and was clearly secreted into the media, consistent with the western blotting data and live-cell images. Our strategy presented the expression of secreted, epitope-tagged, forms of the BEFV glycoproteins such that appropriately glycosylated forms of BEFV G protein was secreted from the BHK-21 cells. This indicates that high-level expression of secreted G glycoprotein is a feasible strategy for large-scale production of vaccines and improving vaccine efficacy.SIGNIFICANCE AND IMPACT OF THE STUDY:The antigen expression strategy designed in this study can produce high-quality recombinant protein and reduce the amount of antigen used in the vaccine.

The proliferation, differentiation, and survival of cells of the mononuclear phagocyte system (MPS; progenitors, monocytes, macrophages, and classical dendritic cells) are controlled by signals from the M-CSF receptor (CSF1R). Cells of the MPS lineage have been identified using numerous surface markers and transgenic reporters, but none is both universal and lineage restricted. In this article, we report the development and characterization of a CSF1R reporter mouse. A FusionRed (FRed) cassette was inserted in-frame with the C terminus of CSF1R, separated by a T2A-cleavable linker. The insertion had no effect of CSF1R expression or function. CSF1R-FRed was expressed in monocytes and macrophages and absent from granulocytes and lymphocytes. In bone marrow, CSF1R-FRed was absent in lineage-negative hematopoietic stem cells, arguing against a direct role for CSF1R in myeloid lineage commitment. It was highly expressed in marrow monocytes and common myeloid progenitors but significantly lower in granulocyte-macrophage progenitors. In sections of bone marrow, CSF1R-FRed was also detected in osteoclasts, CD169(+) resident macrophages, and, consistent with previous mRNA analysis, in megakaryocytes. In lymphoid tissues, CSF1R-FRed highlighted diverse MPS populations, including classical dendritic cells. Whole mount imaging of nonlymphoid tissues in mice with combined CSF1R-FRed/Csf1r-EGFP confirmed the restriction of CSF1R expression to MPS cells. The two markers highlight the remarkable abundance and regular distribution of tissue MPS cells, including novel macrophage populations within tendon and skeletal muscle and underlying the mesothelial/serosal/capsular surfaces of every major organ. The CSF1R-FRed mouse provides a novel reporter with exquisite specificity for cells of the MPS.

Foot-and-mouth disease virus encodes all of its proteins in the form of a polyprotein. The full-length translation product (some 2,330 amino acids) is not observed within infected cells, however, due to processing of the polyprotein. The polyprotein undergoes three co-translational, intramolecular, or 'primary', cleavages mediated by the virus-encoded proteinases L and 3C, and a short oligopeptide sequence (2A). 2A-mediated 'cleavage' is now thought to be a translational effect: an unusual ribosome 'skipping' activity. The polyprotein primary cleavage products then undergo 'secondary' proteolytic processing by a combination of inter- and intramolecular cleavages to produce the mature processing products. The aphthoviruses are unique in possessing a proteinase (Lpro) at the N-terminus of the polyprotein. The L and 3C proteinases serve not only to cleave the virus polyprotein, but to degrade certain host-cell proteins thereby greatly enhancing virus replication. The strategy of encoding proteins as polyproteins comprising virus-encoded proteinases lies, therefore, at the core of the replication strategy of these viruses.

SummaryOligopeptide “2A” and “2A‐like” sequences (“2As”; 18‐25aa) are found in a range of RNA virus genomes controlling protein biogenesis through “recoding” of the host‐cell translational apparatus. Insertion of multiple 2As within a single open reading frame (ORF) produces multiple proteins; hence, 2As have been used in a very wide range of biotechnological and biomedical applications. During translation, these 2A peptide sequences mediate a eukaryote‐specific, self‐“cleaving” event, termed “ribosome skipping” with very high efficiency. A particular advantage of using 2As is the ability to simultaneously translate a number of proteins at an equal level in all eukaryotic systems although, naturally, final steady‐state levels depend upon other factors—notably protein stability. By contrast, the use of internal ribosome entry site elements for co‐expression results in an unbalanced expression due to the relative inefficiency of internal initiation. For example, a 1:1 ratio is of particular importance for the biosynthesis of the heavy‐chain and light‐chain components of antibodies: highly valuable as therapeutic proteins. Furthermore, each component of these “artificial polyprotein” systems can be independently targeted to different sub‐cellular sites. The potential of this system was vividly demonstrated by concatenating multiple gene sequences, linked via 2A sequences, into a single, long, ORF—a polycistronic construct. Here, ORFs comprising the biosynthetic pathways for violacein (five gene sequences) and β‐carotene (four gene sequences) were concatenated into a single cistron such that all components were co‐expressed in the yeast Pichia pastoris. In this review, we provide useful information on 2As to serve as a guide for future utilities of this co‐expression technology in basic research, biotechnology, and clinical applications.

To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

Foot-and-mouth disease virus encodes all of its proteins in a single, long, open reading frame which encodes a polyprotein. The full-length translation product (similar to 2,330 amino acids) is not observed within infected cells, however, due to 'processing' of this polyprotein. The polyprotein undergoes extremely rapid co-translational, intramolecular, or `primary, cleavages at three sites by the activities of the virus-encoded proteinases L and 3C, and a short oligopeptide sequence (2A) which mediates a ribosome 'skipping' activity a translational `recoding' event. The primary cleavage products then undergo 'secondary' proteolytic processing by a combination of inter-and intramolecular cleavages to produce the mature processing products. The L and 3C proteinases serve not only to cleave the virus polyprotein, but also to degrade specific host cell proteins thereby greatly enhancing virus replication and suppressing the innate immune response to infection.

The authors gratefully acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC) for the work on cellular 2A-like sequences.

We report the initial characterization of an N-terminal oligopeptide 2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting.

A substantial proportion (~39%) of all human proteins are either secreted from the cell, located within the lumen/ membranes of cytoplasmic vesicular structures, or, are plasma membrane proteins. Given that such high proportion of proteins are initially translocated into the endoplasmic reticulum (ER), many therapeutic strategies rely on the ability to co-express multiple proteins – some, or all of which, might be targeted to such sites. This directly applies to in vivo gene therapy strategies, or, when therapeutic proteins may need to be co-expressed with selectable markers (e.g. ex vivo gene therapies). As was shown before, Picornavirus 2A (foot-and-mouth disease virus 2A; F2A) and ‘2A-like’ sequences are powerful tools that allow multiple proteins to be translated and co-expressed from a single transcript mRNA under the control of only one promoter. When 2A is positioned between sequences encoding two, or more, genes, it mediates a co-translational ‘cleavage’ at its own C-terminus. A major problem with co-expression of certain proteins targeted to, or transiting through, the ER is that the ‘cleavage’ activity of short F2As can be greatly inhibited by sequences immediately upstream leading to aberrant sub-cellular localisation of some proteins. We have also discovered a number of active cellular 2A-like sequences, associated with non-long terminal repeat (non-LTR) retrotransposons, but also with structural and metabolic proteins: ankyrin repeats, sodium dependent neutral amino acid transporters, and NOD-like receptor (NLR) proteins. Examination of the surrounding protein and gene structure revealed that in the majority of cases these 2A sequences occurred as N-terminal features. Interestingly, using SignalP, many of these novel 2As scored highly as N-terminal signal peptides. Here, we present our latest findings on 2A sequences. A series of test proteins (eGFP and mCherry) were expressed i) followed by ‘hybrid’ ‘self-cleaving’ F2A sequences (with different upstream contexts) or ii) downstream of a putative signal 2A. We demonstrate that inhibition of F2A-mediated cleavage in shorter sequences can be overcome by introduction of mutations upstream of 2A changing the context of the sequence between the C-terminus of the upstream protein and 2A sequence. In the case of N-terminal – cellular – (NLR) 2As, ‘uncleaved’ 2A indeed can act as a signal peptide. If 2A does not ‘cleave’, it directs a proportion of the newly synthesised reporter protein to the exocytic pathway: if 2A ‘cleaves’, the protein downstream is localised to the cytoplasm. This type of 2A mediates, therefore, a newly discovered form of dual protein targeting.

Shen et al. (1) describe the modification of three regions of the Dengue virus type 2 (DENV-2) genome to match the codon pair use of insect genes rather than those of mammals. We have previously shown that such recoding also modifies frequencies of CpG and UpA dinucleotides and have proposed it is this, rather than codon pair use, that restricts replication in mammalian cells (2). We argue that DENV-2 mutants are attenuated for cell culture and in vivo replication in mice through the same mechanism. Our conclusions are based on the following observations.

The combination, ‘pyramiding’ or ‘stacking’ of multiple genes in plants is a fundamental aspect of modern plant research and biotechnology. The most widely adopted stacked traits (herbi‐ cide tolerance and insect protection) provide growers with benefits of increased crop yield, simplified management of weed control and reduced insecticide use. The global acreage of stacked traits or more precisely genetically modified organisms bearing stacked traits is expected to increase rapidly in the near future, with the introduction of nutritional and/or industrial traits to satisfy the needs of consumers and producers [1]. Several approaches have been used to stack multiple genes into plant genomes and then to coordinate expression [2-4]. Stacking approaches include sexual crossing between plants carrying distinct transgenes [5,6], sequential re-transformation [7], and single-plasmid [8] or multiple-plasmid co-transforma‐ tion [9]. These strategies, however, suffer from the inherent weakness that co-expression of the heterologous proteins is unreliable.

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.