Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.

A two-step mechanism generates the spacing pattern of the ciliated cells in the skin of Xenopus embryos.

The skin of Xenopus embryos contains a population of specialized ciliated cells that are distributed in an evenly spaced pattern. Here we describe two successive steps that govern the differentiation and the generation of the spacing pattern of these ciliated cells. The first step occurs in the inner or sensorial layer of the non-neural ectoderm where a subset of cells are chosen to differentiate into ciliated-cell precursors. This choice is under the control of lateral inhibition mediated by a Suppressor of Hairless-dependent Notch signaling pathway, in which X-Delta-1 is the putative ligand driving the selection process, and a new Enhancer-of-Split-related gene is an epidermal target of Notch signaling. Because nascent ciliated-cell precursors prevent neighboring cells from taking on the same fate, a scattered pattern of these precursors is generated within the deep layer of the non-neural ectoderm. Ciliated-cell precursors then intercalate into the outer layer of cells in the epidermis. We show that the intercalation event acts as a second step to regulate the spacing of the mature ciliated cells. We propose that the differentiation of the ciliated cells is not only regulated by Notch-mediated lateral inhibition, but is also an example where differentiation is coupled to the movement of cells from one cell layer to another.

The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis.

The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, we isolated the Xenopus homolog of Suppressor of Hairless (X-Su(H)), a component of the Notch signaling pathway in Drosophila. Using animal cap assays, we show that X-Su(H) induces ESR-1 expression, perhaps directly, when modified by the addition of ankyrin repeats. Using a DNA binding mutant of X-Su(H), we show that X-Su(H) activity is required for induction of ESR-1. Finally, expression of the DNA binding mutant in embryos leads to a neurogenic phenotype as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos.

Evidence for a conformational change in a class II major histocompatibility complex molecule occurring in the same pH range where antigen binding is enhanced.

Many class II histocompatibility complex molecules bind antigenic peptides optimally at low pH, consistent with their exposure to antigen in acidic endosomal compartments. While it has been suggested that a partially unfolded state serves as an intermediate involved in peptide binding, very little evidence for such a state has been obtained. In this report, we show that the murine class II molecule IE becomes increasingly less stable to sodium dodecyl sulfate-induced dissociation since the pH is decreased in the same range that enhances antigenic peptide binding. Furthermore, at mildly acidic pH levels, IEk binds the fluorescent dye 1-anilino-naphthalene-8-sulfonic acid (ANS), a probe for exposed nonpolar sites in proteins, suggesting that protonation produces a molten globule-like state. The association of IEk with a single high-affinity peptide had only a small effect in these two assays, indicating that the changes that occur are distal to the peptide-binding groove. Circular dichroism analysis shows that a pH shift from neutral to mildly acidic pH causes subtle changes in the environment of aromatic residues but does not grossly disrupt the secondary structure of IEk. We propose a model in which perturbations in interdomain contacts outside the peptide-binding domain of IEk occur at acidic pH, producing a partially unfolded state that facilitates optimal antigen binding.

pH dependence and exchange of high and low responder peptides binding to a class II MHC molecule.

We have compared the binding kinetics of two antigenic peptides to a soluble class II MHC molecule. One of the peptides provokes a strong T cell response and the other a much weaker one. Both show greatly increased (approximately 40-fold) association rates at pH 5 in comparison to neutral pH, consistent with the low pH environment of late endosomes being most conducive to class II MHC--peptide binding. Interestingly, the weak peptide has a much faster off-rate that is significantly increased at pH 5 and it can be entirely replaced in an exchange reaction by the stronger one. This suggests that one characteristic of immunodominant peptides is that of nearly irreversible binding, such that they will be strongly selected for in the course of class II MHC transit and recycling through endosomal compartments. Modelling the parameters of this peptide exchange also suggests that a large fraction of the GPI-chimeric MHC molecules used in this study are 'empty' with respect to endogenous peptides, or else occupied with extremely weak ones, consistent with their inability to load processed peptides intracellularly.

Expression of a class II major histocompatibility complex (MHC) heterodimer in a lipid-linked form with enhanced peptide/soluble MHC complex formation at low pH.

A murine class II major histocompatibility complex (MHC) heterodimer, Ek, expressed as a glycan-phosphatidyl inositol-anchored chimera on Chinese Hamster Ovary cells, can present peptides, but not processed antigen to T cells. This chimeric MHC requires a 100-times higher peptide concentration to achieve a two- to four-times lower level of T cell stimulation. Cleavage with phosphatidylinositol-specific phospholipase C and purification result in large quantities of heterodimer in a water-soluble form. Plates coated with this material and then incubated with peptide can efficiently stimulate the appropriate T cell hybridomas. This stimulation is significantly enhanced when peptides are preincubated with the plate-bound MHC molecules in a pH range (5.0-5.5) similar to that of late endosomes. More than half of the soluble Ek molecules can form a specific complex with cytochrome c peptides in this pH range. This suggests that class II MHC molecules undergo distinct conformational changes in endosomal compartments that render them more capable of forming functional complexes with peptide antigens, irrespective of other cell components.

T-cell receptor delta gene rearrangements in early thymocytes.

The T-cell receptor delta-chain variable region can be assembled from as many as four distinct gene segments, V, D1, D2 and J, more than any other antigen-receptor gene. In fetal thymocytes V----D joinings are as common as D----J or VDJ rearrangements and one V gene segment predominates. Analysis of rearrangements at TCR gamma and delta loci during fetal ontogeny suggests abrupt changes and possible coordinate control in the rearrangement and expression of these loci.