Characterization of a YAC-1 mouse cell receptor for group B coxsackieviruses.

A receptor on YAC-1 cells, a mouse T-lymphoma cell line, bound all six serotypes of the group B coxsackieviruses (CVB). In addition, the cells produced infectious virus. Each of the CVB competed for the same receptor on YAC-1 cells. CVB3 bound relatively slowly to YAC-1 cells (k = 4 x 10(-11) min-1 cell-1), and there were only 500 attachment sites per cell. A rabbit antiserum prepared against the HeLa cell receptor protein Rp-a specifically inhibited the binding of CVB1 and CVB3. A virus-receptor complex with CVB3 could be isolated from detergent (0.5% sodium deoxycholate, 1% Triton X-100)-solubilized YAC-1 plasma membranes. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the iodinated virus-receptor complex revealed a band with the same mobility as Rp-a. The results suggested that the YAC-1 receptor for CVB resembles that of the HeLa cell receptor.     

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.     

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.     

Predominant binding of Theiler's viruses to a 34-kilodalton receptor protein on susceptible cell lines.

Western immunoblots of BHK-21 cell lysates probed with the highly virulent GDVII and the less virulent BeAn strains of Theiler's murine encephalomyelitis virus (TMEV) revealed predominant binding to a 34-kDa membrane protein and much lower levels of binding to 100- and 18-kDa membrane proteins. Complete inhibition of virus binding to both the 34- and 18-kDa membrane species by excess unlabeled TMEV demonstrated specificity of binding. Virus binding was also blocked by wheat germ agglutinin, which specifically binds to sialic acid residues and blocks TMEV binding to whole BHK-21 cells. Radiolabeled TMEV also bound to 100-, 34-, and 18-kDa membrane proteins expressed on other TMEV permissive cell lines but not on the nonpermissive cell lines tested. These data suggest that a 34-kDa cellular protein may be the primary determinant of susceptibility to TMEV infection by mediating the binding of GDVII and BeAn viruses to susceptible cells.     

Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus.

Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.     

Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus.

The attachment of lymphocytic choriomeningitis virus (LCMV) to murine and primate cell lines was quantitated by a fluorescence-activated cell sorter assay in which binding of biotinylated virus was detected with streptavidin-fluorescein isothiocyanate. Cell lines that were readily infected by LCMV (e.g., MC57, Rin, BHK, Vero, and HeLa) bound virus in a dose-dependent manner, whereas no significant binding was observed to lymphocytic cell lines (e.g., RMA and WIL 2) that were not readily infected. Binding was specific and competitively blocked by nonbiotinylated LCMV. It was also blocked by LCMV-specific antiserum and a neutralizing monoclonal antibody to the virus glycoprotein GP-1 but not by antibodies specific for GP-2, indicating that attachment was likely mediated by GP-1. Treatment of cells with any of several proteases abolished LCMV binding, whereas phospholipases including phosphatidylinositol-specific phospholipase C had no effect, indicating that one or more membrane proteins were involved in virus attachment. These proteins were characterized with a virus overlay protein blot assay. Virus bound to protein(s) with a molecular mass of 120 to 140 kDa in membranes from cell lines permissive for LCMV but not from nonpermissive cell lines. Binding was specific, since unlabeled LCMV, but not the unrelated enveloped virus herpes simplex virus type 1, competed with 125I-labeled LCMV for binding to the 120- to 140-kDa band. The proteinaceous nature of the LCMV-binding substance was confirmed by the lack of virus binding to proteinase K-treated membrane components. By contrast, glycosidase treatment of membranes did not abolish virus binding. However, in membranes treated with endoglycosidase F/N-glycosidase F, and/or neuraminidase and in membranes from cells grown in tunicamycin, the molecular mass of the LCMV-binding entity was reduced. Hence, LCMV attachment to rodent fibroblastic cell lines is mediated by a glycoprotein(s) with a molecular mass of 120 to 140 kDa, with complex N-linked sugars that are not involved in virus binding.     

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.     

Identification and properties of chlamydial polypeptides that bind eucaryotic cell surface components.

An electroblotting technique was used to identify proteins of Chlamydia that bound surface-radioiodinated and Triton X-100-solubilized HeLa cell extracts. Two proteins, with apparent molecular masses of 18 and 32 kilodaltons (kDa), that bound HeLa cell surface components were identified on Chlamydia trachomatis L2 elementary bodies (EBs). Radioiodinated heparin, which disrupts chlamydial association with cultured cells, was also bound by these proteins. These two proteins were found on EBs but were absent or were present in reduced amounts on the noninfectious reticulate bodies. All C. trachomatis strains tested displayed two such proteins, although the apparent molecular weight of the larger protein varied with serotype in correlation with biotype and the disease that it caused. Two Chlamydia psittaci strains examined displayed only a single binding protein in the range of 17 to 19 kDa. All of the binding proteins stained intensely and distinctively on silver-stained sodium dodecyl sulfate-polyacrylamide gels and displayed an unusual sensitivity to reducing agents. The 32-kDa protein was not seen and did not bind 125I-labeled HeLa cell components if the EBs were solubilized in the presence of 2-mercaptoethanol. The 32-kDa protein was not affected by dithiothreitol, however. Similar to the effect of 2-mercaptoethanol, the 32-kDa protein was not visualized after treatment of EBs with the protease inhibitors tosyl-phenylalanine chloromethyl ketone (TPCK) or tosyl-lysine chloromethyl ketone (TLCK). TPCK and TLCK also abolished infectivity as did the alkylating agents N-ethylmaleimide and iodoacetamide, yet the latter two agents did not affect the appearance of the 32-kDa protein. These proteins were not detected in immunoblots with either rabbit antisera to C. trachomatis L2 EBs or by serum from a patient with lymphogranuloma venereum. The role of these proteins in the interaction of chlamydiae with host cells is not clear, but the binding of eucaryotic cell surface components and heparin, presence only during the infectious stage of the life cycle, variation between serotypes in correlation with disease, and sensitivity to reducing agents or protease inhibitors, collectively, suggest a role for these proteins in parasite-host interactions.     

Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor

Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.     

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.     

The identification and characterization of collagen receptors involved in HeLa cell-substratum adhesion

Four proteins of molecular mass 102, 87, 45, and 38 kDa were isolated from plasma membrane preparations by affinity chromatography. The 102-, 87-, and 38-kDa proteins were shown to be collagen receptors involved in the adhesion of HeLa cells to a gelatin substratum. All four proteins were eluted by high salt from affinity columns made of either types I or IV collagen or type I gelatin. Generally, a total of six major proteins were found in the high salt eluates, although the relative amounts of each varied among experiments. Immunoprecipitation, immunoblotting, and limited peptide mapping indicated that the 102-kDa protein was most sensitive to proteolysis leading to the formation of proteins of molecular mass 58 and 54 kDa. Even in the presence of a mixture of protease inhibitors the 58-kDa fragment was usually the more abundant species. Lectin binding indicated that the 102-, 87-, and 38-kDa proteins contain carbohydrate. Phase-partitioning with Triton X-114 and the need to solubilize the proteins in Triton X-100 indicated that the 102-, 87-, 45-, and 38-kDa proteins have a hydrophobic domain. The 87-kDa protein partitioned exclusively with the detergent-rich phase, suggesting that it is the most hydrophobic. Cell surface labeling with 125I indicated that the four proteins have an extracellular domain. Four criteria were used to determine which of the four proteins are collagen receptors mediating cell-substrate adhesion: 1) during HeLa cell adhesion, proteins with Mr values similar to all four proteins or their peptide fragments were cross-linked to a gelatin substratum derivatized with a photoactivatable probe; 2) a pentapeptide containing the Arg-Gly-Asp cell recognition sequence eluted the same four proteins as those found by high salt elution of collagen affinity columns; 3) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, inhibited the spreading of HeLa cells on a gelatin substratum; 4) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, bound to culture dishes substituted for gelatin in mediating the spreading of HeLa cells. Taken together, the data suggest that the 102-, 87-, and 38-kDa proteins are collagen receptors involved in HeLa cell adhesion. Although the 45-kDa protein has two of the characteristics of a collagen receptor defined here, it does not fit the criteria for one involved in cell-substratum adhesion.     

Isolation and partial characterization of high affinity laminin receptors in neural cells

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.     

Isoform-specific transforming growth factor-beta binding proteins with membrane attachments sensitive to phosphatidylinositol-specific phospholipase C.

We report the identification of cell surface glycoproteins that bind transforming growth factor-beta (TGF-beta) in an isoform-specific manner, and are distinct from TGF-beta receptors I and II or the TGF-beta binding proteoglycan beta-glycan. The novel TGF-beta binding proteins have been identified in various cell lines including fetal bovine heart endothelial cells and MG-63 human osteosarcoma cells. They include proteins of 90-100 and 180 kDa that preferentially bind TGF-beta 1 (KD 0.1-0.2 nM) and proteins of 60 and 140 kDa that preferentially bind TGF-beta 2 (KD 0.5-1 nM). The 180-kDa TGF-beta 1 binding protein and the 60- and 140-kDa TGF-beta 2 binding proteins can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that these proteins are attached to the plasma membrane through a phosphatidylinositol anchor. The expression of these three proteins as well as their sensitivity to phosphatidylinositol-specific phospholipase C is cell line-dependent. The 90-100-kDa TGF-beta 1 binding proteins are components of a 190-kDa disulfide-linked complex. The structural properties of these proteins and their high affinity and selectivity for different TGF-beta isoforms defines them as a novel class of cell surface TGF-beta binding proteins.     

Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain.

A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a approximately 60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.     

The human cytomegalovirus receptor on fibroblasts is a 30-kilodalton membrane protein.

Previous studies have demonstrated that human cytomegalovirus (HCMV) binding to human foreskin fibroblasts (HFF) is mediated by a single type of molecule, likely a glycoprotein, which serves as a specific receptor for the virus. In the present experiments, HCMV was found to bind to an HFF membrane protein with an approximate molecular mass of 30 kilodaltons (kDa); weak binding to 28- and 92-kDa membrane components was also observed. Binding was specific, as it was inhibited by excess unlabeled HCMV. Radiolabeled HCMV also bound selectively to Raji and Daudi lymphoblastoid cell membrane proteins of the same molecular masses. The 30-kDa radiolabeled HFF membrane protein bound to HCMV in solution; this binding was also specific, as it was blocked by an excess of HCMV. These data suggest that a membrane protein with a molecular mass of approximately 30 kDa mediates HCMV binding to several cell types.     

The two proteins of the erythropoietin receptor are structurally similar

The structure of the erythropoietin receptor has been identified in this laboratory as two proteins of 100 and 85 kDa by cross-linking 125I-erythropoietin (125I-EP) to the surface of erythroid cells purified from the spleens of mice infected with the anemia strain of Friend virus. This study investigates the relatedness of these two proteins and the possibility that these proteins are subunits of the functional receptor for EP. Other workers have claimed that the 100- and 85-kDa proteins are bridged by disulfide bonds. This most likely is an artifact due to the insolubility of the cross-linked membrane. Proteolytic digestion by the method of Cleveland (Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106) resulted in identical fragments from the 100- and 85-kDa proteins, which strongly suggests that the primary amino acid sequence of these two proteins is similar if not identical. Increasing the number of protease inhibitors during the preparation of membranes and the binding and cross-linking steps increased the ratio of 100-kDa protein labeled compared to the 85-kDa protein. Together these results suggest that the 85-kDa protein is derived by proteolytic cleavage of the 100-kDa receptor for EP. It is not clear whether the 100-kDa protein can bind EP in the absence of the 85-kDa protein.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.