Possible regulatory function of the Saccharomyces cerevisiae Ty1 retrotransposon core protein.

The yeast Ty1 retrotransposon encodes proteins and RNA that assemble into virus-like particles (VLPs) as part of the life cycle of the retro-element. The Tya protein, which is equivalent to the retroviral Gag, is the major structural component of these particles. In this work, we demonstrate that Tya proteins fulfil other functions apart from their structural role. We show that Tya interacts in vitro with the Ty1 RNA domain required for RNA packaging, suggesting that this RNA-protein interaction may direct the packaging process. Furthermore, the overexpression of both Tya proteins, i.e. p1, the primary translation product, and p2, the mature form, increases endogenous Ty1 RNA levels in trans without increasing translation significantly. These observations suggest that Tya may exert a regulatory function during transposition. Interestingly, however, only p2, the mature form of Tya, trans-activates transposition of a marked genomic Ty element. This confirms that processing is required for transposition.

The yeast Ty virus-like particles.

Virus-like particle (VLP) assembly is a crucial step of the life cycle of retrotransposons. The S. cerevisiae Ty elements represent an interesting model for the analysis of these particles and thus have been studied extensively. Our current knowledge of the organisation and assembly of Ty1 and Ty3 VLPs is reviewed here. This includes the mechanism of assembly, the role of the Tya core protein during VLP formation and the RNA packaging process. The physical properties of Ty1 VLPs are also described and the latest three-dimensional Ty1 VLP reconstructions are shown. In addition, the relevance of these studies is discussed in the context of retro-element biology.

Expression and purification of recombinant neurotensin in Escherichia coli.

An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies. A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein. The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein. Purification of recombinant neurotensin was performed by reverse-phase HPLC. This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (<15 amino acids) with isotopes for NMR or other biophysical techniques.

Yeast Ty retrotransposons assemble into virus-like particles whose T-numbers depend on the C-terminal length of the capsid protein.

The virus-like particles (VLPs) produced by the yeast Ty retrotransposons are structurally and functionally related to retroviral cores. Using cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction, we have examined the structures of VLPs assembled from full-length and truncated forms of the capsid structural protein. The VLPs are highly polydisperse in their radius distribution. We have found that the length of the C-terminal region of the capsid structural protein dictates the T -number, and thus the size, of the assembled particles. Each construct studied appears to assemble into at least two or three size classes, with shorter C termini giving rise to smaller particles. This assembly property provides a model for understanding the variable assembly of retroviral core proteins. The particles are assembled from trimer-clustered units and there are holes in the capsid shells.

Prediction of murine MHC class I epitopes in a major house dust mite allergen and induction of T1-type CD8+ T cell responses.

The group I (Der p 1) allergen of Dermatophagoides pteronyssinus (house dust mite, HDM) contains several T helper (Th) epitopes recognized by C57BL/6 mice, with the peptide (111-139) containing a dominant MHC class II-restricted epitope (113-127). Since CD8+ T cells are thought to play a role in the regulation of allergic disease, we examined the Der p 1 sequence for potential MHC class I-binding motifs and observed that residues 111-119 (FGISNYCQI) contain motifs for H-2Db and Kb. Furthermore, immunization of C57BL/6 mice with unadjuvanted Ty virus-like particles (VLP) carrying Der p 1 (111-139), a method known to induce murine cytotoxic T lymphocyte (CTL) responses, primed Der p 1 (111-119)-specific Db-restricted CTL which produce high levels of IFN-gamma and low levels of IL-5 and IL-6 in vitro (T1-type CTL). VLP carrying the minimal epitope (FGISNYCQI) also induced a CTL response following immunization without adjuvant by various routes. Der p 1 (111-139)-VLP adjuvanted with alum did not prime CTL in C57BL/6 mice but were found to prime Th1-type CD4+ T cells that recognize the overlapping peptide (113-127) and native protein. The ability to successfully predict allergen-specific CD8+ T cell epitopes and prime CD8+ and/or CD4+ T cell responses provides an opportunity to dissect the relative roles of these T cells in the regulation of allergic responses and may offer therapeutic potential for reprogramming Th2-type allergic responses.