The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ' primary ' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ' cleavage ' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ' cleavage ' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA Gly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ' skip ' from one codon to the next without the formation of a peptide bond.

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2A is an oligopeptide sequence mediating a ribosome ‘skipping’ effect, producing an apparent ‘cleavage’ of polyproteins. First identified and characterized in picornaviruses, ‘2A-like’ sequences are found in other mammalian viruses and a wide range of insect viruses. Databases were analysed using a motif conserved amongst 2A/2A-like sequences. The newly identified 2A-like sequences (30 aa) were inserted into a reporter polyprotein to determine their cleavage activity. Our analyses showed that these sequences fall into two categories. The majority mediated very high (complete) cleavage to separate proteins and a few sequences mediated cleavage with lower efficiency, generating appreciable levels of the uncleaved form. Phylogenetic analyses of 2A-like sequences and RNA-dependent RNA polymerases (RdRps) indicated multiple, independent, acquisitions of these sequences at different stages during virus evolution. Within a virus family, 2A sequences are (probably) homologous, but diverge due to other evolutionary pressures. Amongst different families, however, 2A/2A-like sequences appear to be homoplasic.

The proliferation, differentiation and survival of cells of the mononuclear phagocyte system (MPS, progenitors, monocytes, macrophages and classical dendritic cells) is controlled by signals from the macrophage colony-stimulating factor 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. Here we report the development and characterization of a novel CSF1R reporter mouse. A Fusion Red (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 (HSC), arguing against a direct role for CSF1R in myeloid lineage commitment. It was highlyexpressed in marrow monocytes and common myeloid progenitors (CMP) but significantly lower in granulocyte-macrophage progenitors (GMP). 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 non-lymphoid 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.