Programmed −1 ribosomal frameshifting (−1 PRF) is a geneexpression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of −2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on −1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via −2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSVinfected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient −2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that −2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.Nidovirales | virology | genetic recoding | overlapping gene | translation

JHFF and MG are inventors on a pending patent for the nonoptimized and optimized TRAV and TRBV sequences of TCRs for mutated NPM1 as well as cells containing these TCR sequences (no. 2019156).

Purpose: In human leukocyte antigen (HLA)–matched stem cell transplantation (SCT), it has been shown that beneficial immune response mediating graft-versus-tumor (GVT) responses can be separated from graft-versus-host disease (GVHD) immune responses. In this study, we investigated whether it would be possible to dissect the beneficial immune response of allo-HLA–reactive T cells with potent antitumor reactivity from GVHD-inducing T cells present in the detrimental immune response after HLA-mismatched SCT. Experimental Design: The presence of specific tumor-reactive T cells in the allo-HLA repertoire was analyzed at the time of severe GVHD after HLA-mismatched SCT, using tetramers composed of different tumor-associated antigens (TAA). Results: High-avidity allo-HLA-restricted T cells specific for the TAA preferentially expressed antigen on melanomas (PRAME) were identified that exerted highly single-peptide–specific reactivity. The T cells recognized multiple different tumor cell lines and leukemic cells, whereas no reactivity against a large panel of nonmalignant cells was observed. These T cells, however, also exerted low reactivity against mature dendritic cells (DC) and kidney epithelial cells, which was shown to be because of low PRAME expression. Conclusions: On the basis of potential beneficial specificity and high reactivity, the T-cell receptors of these PRAME-specific T cells may be effective tools for adoptive T-cell therapy. Clinical studies have to determine the significance of the reactivity observed against mature DCs and kidney epithelial cells. Clin Cancer Res; 17(17); 5615–25. ©2011 AACR.

The presented HLA class I ligands are the products of the intracellular processing machinery, with its continuous cycle of protein synthesis and degradation (3). Much is known about the proteins involved in antigen processing, but high fidelity ligand/epitope predictions are at present not possible. The discovery of additional involved enzymes (3, 4) and the exciting discovery of peptide splicing (5) have shown that antigen processing is even more complex than was previously thought. Moreover, gene expression studies have shown many nonstandard, unexpected protein products, including the production of antigens derived from aberrant protein fragments as a result of expression in alternative reading frames (6). Several studies report the identification of HLA ligands (7-10). Many results have been collected and discussed in a recent review on the large-scale analysis of HLA class I ligands (11). Collectively, these reports illustrate the need for in-depth elucidation of the HLA ligandome.Elucidation of T cell epitopes has traditionally been achieved with the use of a forward immunological approach, as pioneered by Hunt and coworkers (12,13). In this approach, the cognate peptide of T cells with the appropriate activity profile is elucidated via repeated rounds of chromatographic separation in combination with T cell recognition assays. Because T cells are not always available from the start, reverse immunological approaches (14 -17) have been developed to predict T cell epitopes through a combination of bioinformatics and in vitro proteasome digests. Predicted epitopes are synthesized and tested for their capability to activate T cells. The main disadvantage of this approach is that less than 0.1% of the peptides that survive intracellular processing are presented on HLA class I molecules (3).Therefore, we developed a large-scale peptidomics approach that is a reverse immunology approach based not on From the ‡Department of Immunohematology and Blood Transfusion,