Numb is an endocytic protein.

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.

Differential patterns of expression of Eps15 and Eps15R during mouse embryogenesis.

Eps15 and Eps15R are related tyrosine kinase substrates, which have been implicated in endocytosis and synaptic vesicle recycling. Through the protein:protein interaction abilities of their EH domains, they establish a complex network of interactions with several proteins, including Numb, a protein necessary for neuronal cell fate specification. We analyzed the expression of Eps15 and Eps15R during murine development, at the time of active neurogenesis. The most striking difference was at the level of subcellular localization, with Eps15 present in the cytosol and on the plasma membrane, while Eps15R exhibited mainly a nuclear localization. Interesting topographical differences also emerged. In the 12.5 days post coitum neuroepithelium, Eps15 was expressed in the ventricular zone, which contains proliferating neuroblasts, whereas Eps15R was found only in postmitotic neurons. Conversely, both proteins were expressed in sensory and cranial ganglia. At later times, the expression of Eps15 and Eps15R was widely maintained in neuronal structures. In other tissues, Eps15 was first seen in the liver primordium and at low levels in choroid plexus, lung, kidney and intestine; later on the expression was maintained at high levels in epithelia. Nuclear staining of Eps15R was present in kidney, intestine, lung and liver, as well as in heart and pancreas.

The EH network.

The EH domain is an evolutionary conserved protein-protein interaction domain present in a growing number of proteins from yeast to mammals. Even though the domain was discovered just 5 years ago, a great deal has been learned regarding its three-dimensional structure and binding specificities. Moreover, a number of cellular ligands of the domain have been identified and demonstrated to define a complex network of protein-protein interactions in the eukaryotic cell. Interestingly, many of the EH-containing and EH-binding proteins display characteristics of endocytic "accessory" proteins, suggesting that the principal function of the EH network is to regulate various steps in endocytosis. In addition, recent evidence suggests that the EH network might work as an "integrator" of signals controlling cellular pathways as diverse as endocytosis, nucleocytosolic export, and ultimately cell proliferation.

Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module.

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.

In vitro study of the NS2-3 protease of hepatitis C virus.

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.

The NS2 protein of hepatitis C virus is a transmembrane polypeptide.

The NS2 protein of hepatitis C virus (HCV) is released from its polyprotein precursor by two proteolytic cleavages. The N terminus of this protein is separated from the E2/p7 polypeptide by a cleavage thought to be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize the biogenesis of NS2 encoded by the BK strain of HCV, we have defined the minimal region of the polyprotein required for efficient cleavage at the NS2-3 site and analyzed the interaction of the mature polypeptide with the membrane of the endoplasmic reticulum (ER). We have observed that although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing varies among different HCV strains and that host proteins located on the ER membrane, and in particular the signal recognition particle receptor, are required to sustain efficient proteolysis. By means of sedimentation analysis, protease protection assay, and site-directed mutagenesis, we also demonstrate that the NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol.

Biosynthesis and biochemical properties of the hepatitis C virus core protein.

The biosynthesis and biochemical properties of the putative nucleocapsid protein of hepatitis C virus (HCV) were investigated. RNA transcripts for cell-free translation were prepared from truncated form of the cDNA construct encoding the structural proteins of HCV. Processing of the translation products was dependent on microsomal membranes and signal recognition particle, suggesting that release of the 21-kDa core protein from the polyprotein precursor is mediated solely by the signal peptidase of the endoplasmic reticulum (ER) and is achieved by the removal of a putative signal sequence of approximately 18 residues located at its C terminus. The core protein was found to bind membranes in vitro and in transfected cells, as shown by centrifugation analysis of in vitro translation products and transfected-cell lysates. Immunofluorescence of transfected cells showed that the core protein colocalized with the E2 glycoprotein as well as with a cellular ER membrane marker. The nucleocapsid protein expressed by in vitro translation in rabbit reticulocyte lysates cosedimented with the large ribosomal subunit in sucrose gradients. The ribosome binding domain was mapped to the N-terminal region of the core protein. Moreover, the same region was shown to bind RNA in vitro, suggesting that cosedimentation of core protein with ribosomes may be mediated by the RNA binding of the nucleocapsid protein of HCV. These studies indicate that the HCV core protein is a cytoplasmic protein associated with the ER membranes and possesses RNA binding activity.

NS3 is a serine protease required for processing of hepatitis C virus polyprotein.

Hepatitis C virus (HCV) possesses a positive-sense RNA genome which encodes a large polyprotein of 3,010 amino acids. Previous data and sequence analysis have indicated that this polyprotein is processed by cellular proteases and possibly by a virally encoded serine protease localized in the N-terminal domain of nonstructural protein NS3. To characterize the molecular aspects of HCV protein biogenesis and to clearly identify the protein products derived from the HCV genome, we have examined HCV polyprotein expression by using the vaccinia virus T7 transient expression system in transfected cells and by cell-free translation studies. HCV proteins were identified by immunoprecipitation with region-specific antisera. Here we show that the amino-terminal region of the HCV polyprotein is processed in vitro by cellular proteases releasing three structural proteins: p21 (core), gp37 (E1), and gp61 (E2). Processing of the nonstructural region of HCV was evident in transfected cells. Two proteins of 24 and 68 kDa were immunoprecipitated with anti-NS2 and NS3 antisera, respectively. Antiserum against NS4 recognized three proteins of 6, 26, and 31 kDa, while antisera specific for NS5 immunoprecipitated two polypeptides of 56 and 65 kDa, indicating that each of these two genes encodes at least two different proteins. When the NS3 protease domain was inactivated by replacing the proposed catalytic Ser-1165 with Ala, processing at several sites was abolished. When Ser-1164 was mutated to Ala, no effect on the processing was observed. Cleavage activities at three of the four sites affected by NS3 were shown to occur in trans, while processing at the carboxy terminus of NS3 could not be mediated in trans. These results provide a detailed description of the protein products obtained from the processing of the HCV polyprotein. Furthermore, the data obtained implicate NS3 as a serine protease and demonstrate that a catalytically active NS3 is necessary for cleavage of the nonstructural region of HCV.

Epstein-Barr virus DNA recombines via latent origin of replication with the human genome in the lymphoblastoid cell line RGN1.

We show here that in a lymphoblastoid cell line Epstein-Barr virus DNA recombines with the human genome. The genetic exchange involves the oriP region of the virus. A junction between viral and human DNA from this line has been cloned and sequenced. The results indicate that the integration of Epstein-Barr virus DNA involves a region of the human genome which contains internal short repetition. An 800-bp probe has been isolated from the human part of the junction. This probe has been used to show that the human region exists as a duplication in normal cells.