Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicasetranscriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3 35 direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2 -O-methylated RNA substrates and required divalent metal ions for activity. A range of 5 -labeled ssRNA substrates were processed to final products of Ϸ8 -12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5 -labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.replication ͉ ribonuclease ͉ severe acute respiratory syndrome

Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PL pro s). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/ pp1ab region by two paralogous PL pro activities. Reverse-genetics data revealed that replacement of the PL2 pro active-site cysteine was lethal. By contrast, the PL1 pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1 pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1 pro activity. Further experiments showed that both PL1 pro and PL2 pro were able to cleave the nsp1-nsp2 cleavage site, with PL2 pro cleaving the site less efficiently. The PL1 pro -negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1 pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PL pro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2 pro plays a universal and essential proteolytic role that appears to be assisted by the PL1 pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.Expression of positive-strand RNA virus genomes generally starts with the translation of the incoming viral RNA to produce large precursor proteins that are co-and posttranslationally processed by viral and, in some cases, cellular proteases. The proteolytic release of intermediate and mature processing products, which may have diverse functions in different phases of viral replication, is spatially and temporally coordinated. Protease activities can therefore be viewed as key regulators of the life cycles of positive-strand RNA viruses.The positive-strand RNA coronaviruses (CoVs) have evolved a most complex pattern of polyprotein processing and regulation (64, 66). The 5Ј-terminal two-thirds of the giant 30-kb genome is occupied by two overlapping open reading frames (ORFs), called 1a and 1b, which together form the viral replicase gene and whose translation results in the production of two large polyproteins. ORF1a encodes polyprotein 1a (pp1a), and ORFs 1a and 1b encode pp1ab. The biosynthesis of pp1ab involves a ribosomal frameshift at the ORF1a-ORF1b junction during translation (9, 10). As many as 15 or 16 processing end products, which are called nonstructural proteins (nsps), are proteolytically released from pp1a/pp1ab (64,66...

BackgroundMany biomedical applications require the expression or production of therapeutic hetero-multimeric proteins/protein complexes: in most cases only accomplished by co-ordinated co-expression within the same cell. Foot-and-mouth disease virus 2A (F2A) and ‘2A-like’ sequences are now widely used for this purpose. Since 2A mediates a co-translational ‘cleavage’ at its own C-terminus, sequences encoding multiple proteins (linked via 2As) can be concatenated into a single ORF: a single transgene. It has been shown that in some cases, however, the cleavage efficiency of shorter versions of F2A may be inhibited by the C-terminus of certain gene sequences immediately upstream of F2A. This paper describes further work to optimise F2A for co-expression strategies.ResultsWe have inserted F2A of various lengths in between GFP and CherryFP ‘reporter’ proteins (in reciprocal or tandem arrangements). The co-expression of these proteins and cleavage efficiencies of F2As of various lengths were studied by in vitro coupled transcription and translation in rabbit reticulocyte lysates, western blotting of HeLa cell lysates and fluorescence microscopy.ConclusionsOptimal and suboptimal lengths of F2A sequences were identified as a result of detailed ‘fine-tuning’ of the F2A sequence. Based on our data and the model according to which 2A activity is a product of its interaction with the exit tunnel of the ribosome, we suggest the length of the F2A sequence which is not ‘sensitive’ to the C-terminus of the upstream protein that can be successfully used for co-expression of two proteins for biomedical applications.

Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A “cleavage” may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites) used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.

Regulatory T cells (Treg) play a critical role in immune tolerance. The scarcity of Treg therapy clinical trials in humans has been largely due to the difficulty in obtaining sufficient Treg numbers. We performed a preclinical investigation on the potential of mesenchymal stromal cells (MSCs) to expand Treg in vitro to support future clinical trials. Human peripheral blood mononuclear cells from healthy donors were cocultured with allogeneic bone marrow-derived MSCs expanded under xenogeneicfree conditions. Our data show an increase in the counts and frequency of CD4 + CD25 high Foxp3 + CD127 low Treg cells (4-and 6-fold, respectively) after a 14-day coculture. However, natural Treg do not proliferate in coculture with MSCs. When purified conventional CD4 T cells (Tcon) are cocultured with MSCs, only cells that acquire a Treg-like phenotype proliferate. These MSC-induced Treg-like cells also resemble Treg functionally, since they suppress autologous Tcon proliferation. Importantly, the DNA methylation profile of MSC-induced Treg-like cells more closely resembles that of natural Treg than of Tcon, indicating that this population is stable. The expression of PD-1 is higher in Treg-like cells than in Tcon, whereas the frequency of PDL-1 increases in MSCs after coculture. TGF-β levels are also significantly increased MSC cocultures. Overall, our data suggest that Treg enrichment by MSCs results from Tcon conversion into Treg-like cells, rather than to expansion of natural Treg, possibly through mechanisms involving TGF-β and/or PD-1/PDL-1 expression. This MSC-induced Treg population closely resembles natural Treg in terms of phenotype, suppressive ability, and methylation profile.