No abstract

The frequencies of human cytomegalovirus (HCMV) protein‐specific CD8 T cells, identified by the presence of intracellular IFN‐γ, were measured by flow cytometry following stimulation of freshly isolated peripheral blood mononuclear cells (PBMC) with comprehensive peptide pools. These pools spanned the entire amino acid sequences of the HCMV pp65 and major immediate early (IE‐1) proteins and consisted of 15‐amino acid peptides with at least nine overlaps between neighboring peptides. As a result all potential CD8 T cell epitopes contained in these proteins were provided by the complete pools and, therefore, unlike with single epitopes, testing was independent of donor HLA type. Individual stimulating peptides from the same pools were identified in parallel experiments. Thus we found that our results with the complete pools using PBMC from 26 healthy HCMV‐seropositive donors were 100 % sensitive and specific with respect to predicting the presence of recognized epitopes in the respective proteins. In addition, cells from 15 renal transplant patients were tested with complete pools alone. While our results confirmed our previous contention that HCMV IE‐1 is an important CD8 T cell target, the technical improvement we made in order to address this question has clearly wider implications. Similar pools may be applied to examine the role of proteins from other pathogens, in autoimmune disease or following vaccination.

Maturation of eukaryotic 20S proteasomes involves the processing of beta‐subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta‐subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly‐1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild‐type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome‐specific inhibitor. While the processing of inhibitor‐treated wild‐type proprotein was completely prevented, the site‐directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8–10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self‐activation of proteasomal beta‐subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta‐subunits proceeds via a novel ordered two‐step mechanism involving autocatalysis.

STRAP is a comfortable and extensible tool for the generation and refinement of multiple alignments of protein sequences. Various sequence ordered input file formats are supported. These are the SwissProt-,GenBank-, EMBL-, DSSP- PDB-, MSF-, and plain ASCII text format. The special feature of STRAP is the simple visualization of spatial distances C(alpha)-atoms within the alignment. Thus structural information can easily be incorporated into the sequence alignment and can guide the alignment process in cases of low sequence similarities. Further STRAP is able to manage huge alignments comprising a lot of sequences. The protein viewers and modeling programs INSIGHT, RASMOL and WEBMOL are embedded into STRAP. STRAP is written in JAVA: The well-documented source code can be adapted easily to special requirements. STRAP may become the basis for complex alignment tools in the future.