Mason-Pfizer monkey virus: analysis and localization of virion proteins and glycoproteins.

The polypeptide composition of Mason-Pfizer monkey virus was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six major polypeptides of molecular weights 68,000, 27,000, 20,000, 14,000, 12,000, and 10,000 were resolved regardless of the cell type (i.e., two human and two rhesus) in which the virus was grown. Protein gp68 (68,000) represented the major virus glycoprotein and protein gp20 (20,000) represented a minor glycoprotein of the virion, again regardless of the cell type of origin of the virus. Protein gp68 appears to be located on the outer surface of the viral envelope, as demonstrated by lactoperoxidase catalyzed iodination of intact virions. Additional glycoproteins were shown to be virion associated; their presence depended, however, on the cell type in which the virus was propagated.     

Radioiodination of the envelope proteins of newcastle disease virus

Lactoperoxidase-catalyzed iodination selectively labels the two glycoproteins (VP1 and VP2) of Newcastle disease virus. The low-molecular-weight, nonglycosylated major viral protein, VP6, was not iodinated in the intact virus but was iodinated in disrupted virions, suggesting a localization on the inner, rather than the outer envelope surface. Studies on the distribution of virion proteins labeled with ¹²⁵I and ³H-isoleucine between detergent-soluble and detergent-insoluble fractions show that the virion proteins VP4, VP5, and VP6 are solubilized to a much lesser extent than are VP1 and VP2.     

Localization of major group-specific polypeptides in the avian RNA tumor viruses

Chloramine T iodination of intact avian myeloblastosis virus revealed, that the four major group-specific polypeptides were labeled unequally, with the largest polypeptide 4 (m.w. 23,000) being preferentially iodinated, when the labeling time was limited. By contrast, in enzymatic iodination of intact virus only major virion glycoprotein and glycolipids were extensively iodinated. These observations suggest that the protein 4 surrounds other group-specific polypeptides and is itself enveloped by the bilayer of glycolipids.     

Analysis of the envelope of Rauscher murine oncornavirus: in vitro labeling of glycopeptides.

The identity and localization of the oligosaccharides of Rauscher murine type C viral glycoproteins have been examined by techniques of in vitro labeling. Terminal sialic acid was labeled with tritium by borohydride reduction after selective periodate oxidation, and galactose was labeled by borohydride reduction after specific enzymatic oxidation of the nonreducing terminal of the sugar. The results were compared with those of protein surface labeling with pyridoxal phosphate or lactoperoxidase catalyzed radioiodination. Examination of the labeled reaction products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that in every case the major component labeled was a glycoprotein of about 70,000 daltons. The identity of this glycoprotein as the virion envelope component was confirmed by immunoprecipitation with mono-specific antiserum prepared against purified Rauscher virus glycopeptides of 69,000 and 71,000 daltons. No other protein or glycoprotein on the surface of the virion was detected, and disruption of virions-before labeling did not reveal additional distinctive glycoproteins. There was minor labeling of sugar residues of other components, but these remain to be characterized and are not now identified as other viral proteins. Studies of the structural organization of virion proteins using the cross-linking reagent methyl-4-mercaptobutyrimidate showed only linkage of the virion envelope or core proteins to themselves. These results indicate that most, if not all, of the oligosaccharides at the surface of Rauscher virus are entities of the 69,000- and 71,000-dalton glycopeptides and that they contain a terminal sialic acid and galactose and a subterminal galactose.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis.

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts. Two-dimensional patterns of ghost proteins were not heavily contaminated (less than or equal to 25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8--10 fold over whole cells. Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

Isolation and immunological characterization of an iron-regulated,transformation-sensitive cell surface protein of normal rat kidney cells

We have analyzed the surface proteins of cultured normal rat kidney (NRK) cells and virus-transfromed NRK cells subjected to iron deprivation. Such a treatment specifically induces two transformation-sensitive plasma membrane-associated glycoproteins with a subunit molecular wegiht of 160,000 (160 K) and 130,000 (130 K) daltons in NRK cells. In these cells the 160 K glycoprotein is readily available to lactoperoxidase-mediated iodination, and the 130 K is apparently inaccessible to iodination. Major differences were revealed when iodinated membrane proteins of normal and virus-transformed cells subjected to iron deprivation were compared. In Kirsten sarcoma virus-transformed NRK cells the 160 K glycoprotein was weakly labeled. In two clones of simian virus 40-transformed NRK cells the 160 K glycoprotein was weakly labeled or not at all. The 130 K glycoprotein was inaccessible to iodination in all the virus-transformed cell lines. The 160 K and 130 K glycoproteins were isolated form plasma membranes of NRK cells using preparative SDS gel electrophoresis. Antibodies generated against these glycoproteins stained the external surfaces of NRK cells and induced antigen redistribution. Evidence presented suggests that 160 K and 130 K are plasma membrane-associated procollagen molecules. A possible interaction of these proteins with transferrin is also described. The data suggest that these proteins may have an important role in the sequence of events leading to transformation.     

Cell surface proteins of NIL1 hamster fibroblasts labeled by a galactose oxidase, tritiated borohydride method

Cell surface glycoproteins of normal and virus transformed NIL1 hamster fibroblasts have been labeled by incubation with galactose oxidase followed by reduction with tritiated borohydride. A high molecular weight protein labeled on normal but not transformed cells is the same protein previously detected by lactoperoxidase catalysed iodination.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells

In polarized epithelial cells measles virus (MV) is predominantly released at the apical cell surface, irrespective of the sorting of its two envelope glycoproteins F and H. It has been reported previously that the viral matrix (M) protein modulates the fusogenic capacity of the viral envelope glycoproteins. Here, extant MV mutants and chimeras were used to determine the role of M protein in the transport of viral glycoproteins and release of progeny virions in polarized epithelial CaCo2 cells. In the absence of M, envelope glycoproteins are sorted to the basolateral surface, suggesting that they possess intrinsic basolateral sorting signals. However, interactions of M with the glycoprotein cytoplasmic tails allow M-glycoprotein co-segregation to the apical surface, suggesting a vectorial function of M to retarget the glycoproteins for apical virion release. Whereas this may allow virus airway shedding, the intrinsic sorting of the glycoproteins to the basolateral surface may account for systemic host infection by allowing efficient cell-cell fusion.     

Lactoperoxidase-catalyzed iodination of cell cultures infected with mycoplasma

Hamster BHK cells or secondary cultures of mouse embryo fibroblasts are not iodinated by lactoperoxidase in the absence of hydrogen peroxide. When such cell cultures are infected with a noncultivable strain of M. hyorhinis, endogenous peroxide generation is sufficient to permit nearly maximal iodination. The SDS-polyacrylamide gel pattern of iodinated cell surface polypeptides is essentially the same regardless of the source of peroxide and whether or not the cultures are infected with mycoplasma.     

Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins.

1. Human tumour KB cells growing in suspension culture were labelled by lactoperoxidase-catalysed iodination. Several major radioactively labelled proteins were detected by poly-acrylamide-gel electrophoresis in sodium dodecyl sulphate. 2. After reduction with 2-mercaptoethanol the major radioactive electrophoretic bands migrated as substances with apparent molecular weights of about 90,000, 70,000, 60,000, 50,000 and 34,000 and corresponded closely to the positions at which the major glycosylated polypeptide subunits of KB-cell homogenates migrated during electrophoresis under the same conditions. 3. All the iodinated protein bands except one were present in purified preparations of KB plasma membranes. 4. Most of the 50,000-molecular-weight species, supposedly a surface protein component labelled during iodination of intact and viable KB cells by a non-penetrating enzyme reagent, appeared in a crude nuclear pellet during fractionation. 5. The glyco-protein nature of the major external iodinated species of KB cells was confirmed by adsorption chromatography of these substances, dissolved in low concentrations of Triton X-100, on a lectin-Sepharose column. Two major enzyme markers of the KB plasma membrane, 5'-nucleotidase and alkaline phosphatase were also found to be glycoproteins. 6. Enzyme-catalysed incorporation of radioactive iodine into a fraction of low molecular weight and soluble in chloroform-methanol mixtures also occurred during lactoperoxidase treatment of intact KB cells. The partial characterization of this fraction is briefly described.     

Isolation and charaterization of surface glycoproteins from L-1210, P-388 and HeLa cells

A method is described that permits the rapid extration of the cell surface glycoproteins of two murine leukemic cells, the P-388 and the L-1210 cells as well as those of the human adenocarcinoma cells, the HeLa cells.Proof of the surface location of these glycoproteins is provided by labeling the intact cells; (a) with ¹²⁵I by the lactoperoxidase iodination technique; (b) with ³H by the galactose oxidase-reductive tritiation method. Most of these glycoproteins were also shown to incorporate radioactive glucosamine and fucose. By these criteria as well as by the distribution of molecular weights, the surface glycoproteins of the two murine cells are indistinguishable; however, they differ markedly from the surface glycoproteins of HeLa cells. The extracts of the murine cells wee shown to contain lectin receptor activity as determined by their ability to inhibit the lectin-induced agglutination of the intact cells.     

Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture

Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.     

Lactoperoxidase-catalyzed iodinaton of cell cultures infected with mycoplasma

Summary Hamster BHK cells or secondary cultures of mouse embryo fibroblasts are not iodinated by lactoperoxidase in the absence of hydrogen peroxide. When such cell cultures are infected with a noncultivable strain ofM. hyorhinis, endogenous peroxide generation is sufficient to permit nearly maximal iodination. The SDS-polyacrylamide gel pattern of iodinated cell surface polypeptides is essentially the same regardless of the source of peroxide and whether or not the cultures are infected with mycoplasma.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Lactoperoxidase-catalyzed iodination of surface membrane lipids

Lactoperoxidase-catalyzed iodination of intact cells, is known to label predominantly, if not exclusively, the exposed tyrosine residues of cell surface proteins. The present study demonstrates that during this iodination process surface membrane lipids are also iodinated through an enzyme-dependent step. Phosphatidylcholine, cholesterol-phosphatidylcholine liposomes and confluent secondary cultures of chick embryo cells were iodinated by the lactoperoxidase-glucose oxidase-glucose [¹²⁵I] procedure. Liposomes were efficiently labeled. In the cells, 20–30% of the radioactivity was found in proteins and 20–30% in the lipids. Both neutral and polar lipids were found to bind [¹²⁵I] covalently. Controls in which lactoperoxidase was omitted showed < 6% of the radioactivity found in liposomes or cells labeled with the enzyme.     

Biosynthesis of the Sindbis Virus Carbohydrates

The sequence in which sugars are added to the Sindbis virus glycoproteins was studied. Infected cells contain three glycosylated virus-specific proteins: the two virion glycoproteins and the immediate precursor to the smaller virion glycoprotein. Larger Sindbis-specific proteins are not glycosylated. The cell-associated forms of both of the virion glycoproteins contain glucosamine, mannose, galactose, and fucose. The glycosylated precursor contains only glucosamine, mannose, and some galactose. The conversion of precursor to virion protein involves both the addition of galactose and fucose and the loss of mannose. The apparent extent of glycosylation of each virus-specific protein is not influenced by the host cell.     

Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein.

Transformed cells lack a large, external, transformation-sensitive (LETS) glycoprotein which is a major surface component of their normal counterparts. Addition of LETS glycoprotein isolated from normal cells to transfomed cells restores certain morphological features and adhesive properties characteristic of normal cells. LETS protein is detected on the cell surface both by iodination using lactoperoxidase and by immunofluorescent staining. The surface distribution pattern detected by immunofluorescence is strikingly similar to that of normal cells. After addition of LETS protein, transformed cells also exhibit well defined actin cables which are not seen in untreated, transformed cells. All these alterations can be blocked by treating LETS protein with specific antisera or by subjecting it to mild trypsinization prior to addition to transformed cells. The effects are rapidly reversible by mild trypsinization, which removes the added LETS protein. The high rate of uptake of 2-deoxyglucose, characteristic of transformed cells, is not affected by LETS protein. These results suggest that LETS protein may have a role in cell attachment and spreading, and affect the organization of cytoskeleton.     

Cell cycle dependent exposure of a high molecular weight protein on the surface of mouse L cells

The non-penetrating lactoperoxidase iodination probe has been employed in conjunction with synchronously dividing populations of mouse L cells to identify a high molecular weight protein which is preferentially exposed on the L cell surface during G1 phase of the cell cycle. Progression of cells from G1 to S is accompanied by a marked decrease in the availability of this structure, called band 1, to lactoperoxidase-catalyzed iodination and it remains unavailable until cells re-enter G1. It is suggested that the band 1 polypeptide may be functionally involved in the regulation of L cell growth.