The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

SUMMARY IL-1 has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified a novel isoform of the IL-1R Accessory Protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1 receptor but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.

To identify novel genes that are involved in positive selection of thymocytes, we performed polymerase chain reaction (PCR)-based subtractive hybridization between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) null background are efficiently selected into the CD8 lineage in H-2b mice (RAG-2−/−OT-1, selecting thymi), but are not selected on a transporter associated with antigen processing (TAP) null background (RAG-2−/−TAP-1−/−OT-1, nonselecting thymi). We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus. The expression pattern of ITM2A in thymocyte subsets correlates with upregulation during positive selection. In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation. ITM2A expression was also induced in RAG-2−/− thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent Mr of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surface. Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial downregulation of CD8 in CD4+CD8+ double positive (DP) thymocytes, and a corresponding increase in the number of CD4+CD8lo thymocytes. Possible roles for this novel activation marker in thymocyte development are discussed.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Tyl, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no PI position is occupied by an amino acid with a branched beta carbon.

We have identified a novel LIM gene encoding the thymus LIM protein (TLP), expressed specifically in the thymus in a subset of cortical epithelial cells. TLP was identified as a gene product which is upregulated in a thymus in which selection of T cells is occurring (Rag ؊/؊ OT-1) compared to its expression in a thymus in which selection is blocked at the CD4 ؉ CD8 ؉ stage of T-cell development (Rag ؊/؊ Tap ؊/؊ OT-1). TLP has an apparent molecular mass of 23 kDa and exists as two isomers (TLP-A and TLP-B), which are generated by alternative splicing of the message. The sequences of TLP-A and TLP-B are identical except for the C-terminal 19 or 20 amino acids. Based on protein sequence alignment, TLP is most closely related to the cysteine-rich proteins, a subclass of the family of LIM-only proteins. In both medullary and cortical thymic epithelial cell lines transduced with TLP, the protein localizes to the cytoplasm but does not appear to be strongly associated with actin. In immunohistochemical studies, TLP seems to be localized in a subset of epithelial cells in the cortex and is most abundant near the corticomedullary junction. We generated mice with a targeted disruption of the Tlp locus. In the absence of TLP, thymocyte development and thymus architecture appear to be normal but thymocyte cellularity is reduced by approximately 30%, with a proportional reduction in each subpopulation. T-cell development requires many interactions between thymocytes and thymic stromal cells. As thymocytes develop, they begin as CD4Ϫ CD8 Ϫ double-negative (DN) cells which proliferate and rearrange their T-cell receptor (TCR) beta chain genes. The DN thymocytes, which express a pre-TCR, progress to CD4 ϩ CD8 ϩ double-positive (DP) cells, which rearrange TCR alpha chain genes and express TCR. A small percentage of the DP thymocytes are positively selected into the CD4 single-positive (SP) or CD8 SP lineage. Fully mature SP cells then emigrate to the periphery. Thymic stromal cells, a heterogeneous population of cells including epithelial cells, fibroblasts, mesenchyme cells, and the bone marrow-derived dendritic cells (DCs) and macrophages, are involved at each step in T-cell development (reviewed in references 4, 54, and 74). The DN-to-DP transition requires cell-cell interactions between early precursor thymocytes and both thymic epithelial cells (TECs) and fibroblasts (3, 57). In addition, cytokines that are necessary for the early expansion of DN cells and at later thymocyte developmental stages are provided by TECs (21, 46). For example, interleukin-7 (80) and stem cell factor (52) each appear to have an important role in the proliferation of thymocytes (29,32,49,76). Positive selection of thymocytes is mediated by cortical TECs (cTECs) expressing the major histocompatibility complex (MHC)/peptide ligand for TCR (3,63). cTECs are unique in this capacity, providing more than just the selecting ligand, since other cell types expressing the ligand are unable to substitute for them (5,16,47). DCs in the medulla and at the cort...

Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa 3-7 -His-Xaa 23-32 -Cys-Xaa 2 -Cys and Asp, Asp-Xaa 35 -Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3 end 2 bp that are removed prior to or during integration. Such 3-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3 ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule.

AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab')(2) or human immunoglobulin (IVIG). AMG X F(ab')(2) did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.

Ty3 encodes structural proteins in its upstream open reading frame (GAG3) and catalytic proteins in an overlapping open reading frame (POL3). As is the case for retroviruses, high levels of structural protein versus catalytic proteins are synthesized and we show here that catalytic proteins are derived from a GAG3-POL3 fusion polyprotein. To evaluate the relative contributions of structural and catalytic components of the Ty3 particle, we perturbed the balance of these proteins by fusing the GAG3 and POL3 frames. This fusion Ty3 was capable of complementing low levels of transposition of a donor Ty3 which contained only cis-acting sequences required for transposition. Examination of extracts of cells expressing the GAG3-POL3 fusion mutant showed that particle formation differed qualitatively and quantitatively from viruslike partide formation by wild-type Ty3. Surprisingly, expression of 238 codons of GAG3, encoding only capsid protein, complemented transposition and particle formation defects of the fusion mutant, showing that the limiting deficiency was in capsid, and not in nucleocapsid, function. In addition, protein containing the capsid domain expressed alone accumulated in the same particulate fraction as viruslike particles, showing that it was sufficient for particle formation. The activity of the Ty3 fusion mutant contrasts with the inviability of mutant retroviruses in which gag and pol frames were fused and argues that retrotransposons tolerate considerable variation in the nucleoprotein complexes that permit replication and integration.