Human cell surface proteins selectively assembled into vesicular stomatitis virus virions

Vesicular stomatitis virus (VSV) selectively assembled proteins from human cells into progeny virions. These proteins can be surface labeled before infection with 125I, and when purified virus was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, only two or three bands of proteins (Mr around 100K) were seen. Antisera to these proteins were produced, using as immunizing antigen VSV tsO45 mutant, defective in assembly of G protein, which had been made at the nonpermissive temperature in the three human tumor cell lines, HeLa (cervical carcinoma), T47D (breast carcinoma), and HMB2 (melanoma). After absorption with wild-type VSV, each of the antisera displayed a different pattern of reactivity; at least three antigenic specificities were detected. Two of them, corresponding to antigens selected by VSV from HeLa and T47D, were to some extent related and they showed an association mainly with epithelial cell-derived gynecological tumors, but they were absent in carcinomas of lung or of digestive tract. These (or related) antigens were expressed in a lower level in some normal tissues, mainly in ovaries. Antigen(s) assembled by VSV from the melanoma cell line was entirely different and appeared to be associated with cell growth. The grounds for selective assembly of these specific proteins by VSV are not clear; they either share with viral surface glycoproteins some physical or structural properties, which are critical for incorporation into the viral envelope, or conceivably they even may represent uncleaved precursor proteins coded by env genes of incomplete genomes of endogenous human retroviruses.     

Gamma-Interferon-Induced Nerve Growth Factor Receptors in Colorectal Carcinoma Cell Lines

Abstract

Gamma-interferon (γIFN) was found to induce expression of the 150,000 M_r cell surface and the 35,000 M, chromatin receptors for nerve growth factor (NGF) in the SW1116 colorectal carcinoma cell line that does not express NGF receptors. In the SW707 colorectal carcinoma cell line that expresses a low level of NGF receptors, ylFN stimulated expression of the cell surface and the nuclear receptors. Induction of NGF receptors in SW1116 cells resulted in internalization and nuclear translocation of ¹²⁵I-NGF. When NGF bound to the chromatin, ribosomal RNA synthesis was inhibited. Two-dimensional gel electrophoresis of [³⁵S]methionine-labeled chromatin proteins indicated significant changes in chromatin protein composition in cells treated and not-treated with γIFN. γIFN effectively stimulated the expression of NGF receptors in two colorectal carcinoma cell lines, but inhibited the expression in melanoma and breast carcinoma cells. It is suggested that γIFN, by modulating the expression of NGF receptors may affect the NGF-dependent growth of some tumor cell lines.     

Assembly of xenotropic murine leukaemia virus-related antigens from the surface of mouse L cells by vesicular stomatitis virus

Two xenotropic murine leukaemia virus (XMuLV)-related proteins--a major envelope glycoprotein gp70 and a 90K protein (probably corresponding to the uncleaved envelope precursor)--were expressed on the surface of mouse L cells as demonstrated by lactoperoxidase-catalysed iodination and immunoprecipitation with anti-XMuLV serum. These two proteins out of many labelled cell surface proteins were selectively incorporated into vesicular stomatitis virus (VSV) virions. Significant differences were found in the amounts of labelled XMuLV-related proteins between L cells and two cell lines infected with XMuLV (rabbit SIRC and lamb LKC cells). The two viral antigens represented only a small proportion of radioactivity on L cells. While in XMuLV-infected SIRC and LKC cells, the gp70 was the major labelled surface protein no detectable amounts of XMuLV-related 90K protein or of cell-specific proteins were found in these cells.     

Structural analysis of Rauscher virus Gp70 using monoclonal antibodies: sites of antigenicity and P15(E) linkage

A linear map of 19 monoclonal antibody-binding domains on Rauscher retroviral gp70 was generated using a technique we have designated PEC-MAP (partial enzymatic cleavage-monoclonal antibody precipitation). We used eight proteolytic enzyme preparations in limited digests to produce 39 gp70 fragments. Immune precipitation of these fragments by monoclonal antibodies from 51 cell lines allowed us to define 19 binding sites by virtue of overlapping fragments and differential precipitation patterns. The sites most accessible to proteolytic attack and antibody binding were then mapped by analyzing the apparent molecular weights of the various gp70 fragments. This analysis revealed three hyperreactive regions in gp70 located approximately within the first 2000 daltons of the amino end of gp70 as well as 18,000 and 38,000 daltons from the amino end of the M_r 47,000 deglycosylated gp70 molecule. In addition, we used monoclonal antibodies directed against the disulfide-linked p15(E) molecule to localize its linkage site to Domain XVII, estimated to be between 34,000 and 38,000 daltons from the amino end of deglycosylated gp70. These data place certain constraints on the tertiary structure of gp70 and suggest a mechanism for the generation of leukemogenic MCF recombinants.     

Analysis of the expression of spleen focus-forming virus (SFFV)-related RNA and gp55, a friend and rauscher virus-specific protein

A 55,000-dalton glycoprotein, gp55, is the major intracellular species precipitable with anti-envelope glycoprotein (gp70) sera in murine erythroleukemia cells transformed by either the anemia- or the polycythemia-inducing strains of Friend virus. Similarly, all Friend erythroleukemia cell lines studied contained similar levels of spleen focus-forming virus (SFFV)-specific RNA in the cytoplasm as assessed by hybridization to a Friend SFFV-specific eDNA probe. Neither SFFV-specific RNA nor gp55 is detectable in helper virus-infected cells or in chemically induced rat erythroleukemia cell lines. SFFV non-producer cell lines were isolated and analyzed for viral gene expression by immunoprecipitation and molecular hybridization. SFFV nonproducer cells could be grouped into two classes: those which constitutively synthesize gp55 and those which synthesize gp55 after superinfection with type-C helper virus. No gag gene-related proteins were detected in any of these nonproducer cell lines. Characterization of Friend virus-specific RNAs in SFFV nonproducer cells indicates that they can express both a 32 S and a 21 S RNA species related to Friend virus. SFFV nonproducer cells expressing 21 S SFFV-specific RNA constitutively synthesize gp55.     

Laminin production by murine melanoma cells: possible involvement in cell motility

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [³⁵S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [³⁵S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the M _r=950kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the M _r=400kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (M _r =200kD) and as a disulfide-linked B dimer (M _r=400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.     

Gene order of mouse mammary tumor virus precusor polyproteins and their interaction leading to the formation of a virus.

Mouse mammary tumor virus (MMTV) proteins are synthesized as two major precursor polyproteins; gPr75^env containing gp52 and gp36, and Pr75^gag containing p27, pp20, p14, and p10. The gene order for gPr75^env has been previously shown to be H₂N-gp52-gp36-COOH (Schochetman, et al., 1977). gag polyproteins undergo intracellular cleavage in cat cells infected with MMTV and GR mammary tumor cells. Based on immunoprecipitation studies with antisera against intermediate MMTV cleavage products we now report the gene order for Pr75^gag is H₂N-p10-pp20-p27-p14-COOH. These results were further substantiated by analyzing the binding to ssDNA of the intermediate cleavage products which contain p14. To analyze the interaction of MMTV proteins with the cell membrane leading to budding of a virus particle, we used (i) lactoperoxidase-catalyzed iodination of MMTV cell surface proteins, (ii) galactose oxidase-catalyzed radiolabeling of carbohydrates on cell surface MMTV glycoproteins, (iii) serum cytotoxicity based on [⁵¹Cr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of ¹²⁵I-labeled MMTV cell surface antigens by immune precipitation with MMTV anti-gp52, gp36, p27, p14, and p10 sera followed by SDS-PAGE revealed only ¹²⁵I-gp52. In contrast, cell surface glycoprotein labeling revealed [³H]gp52 and [³H]gp36, indicating that, although the protein portion of gp36 was buried, some carbohydrate regions were exposed. EDTA treatment of cells to alter cell membranes prior to iodination resulted in the labeling of both Pr75^gag and gp52 but not gPr75^env. Furthermore, anti-p10 but not anti-p27 serum was cytotoxic against EDTA-treated cells. Similar results were obtained when the same antisera were tested by membrane immunofluorescence, ruling out the possibility that anti-p27 serum was not cytotoxic because it was unable to fix complement. These results show that Pr75^gag molecules, presumably as MMTV cores, interact with cell membrane sites containing gp52 and gp36 via the hydrophobic p10 portion of the molecule.     

Renal cell carcinomas and pancreatic adenocarcinomas produce nidogen in vitro and in vivo

The production of nidogen by four renal cell carcinoma (RCC) and three pancreatic adenocarcinoma (PAc) cell lines has been studied in cell culture and in xenografted tumours in nude mice. In RCC cells, immunoreactivity for nidogen was seen only after exposure to monensin to induce cytoplasmic accumulation of secretory proteins. In PAc cells, immunoreaction was also detectable in control cells. Immunoblotting of control and monensin-exposed cells and immunoprecipitation of culture media of radioactively labelled cells demonstrated the production of nidogen polypeptide of M_r ca. 150000 by six of the seven cell lines. Basement membranes (BMs) and stroma of the xenografted tumours derived from these six cell lines demonstrated immunoreactivity for both human and mouse nidogen, as revealed with species-specific antibodies. The ability of the cells to produce nidogen in vitro and deposit in vivo was positively correlated with high histological grade of the xenografted tumours, although the small number of cell lines studied calls for further studies to confirm this. The distribution of nidogen in human RCC and PAc specimens was also studied by immunohistochemistry. There was strong immunoreactivity for nidogen in tumour stroma, BM of carcinoma cell nests, and endothelial basal lamina, but no conclusions could be drawn regarding histological grade and immunostaining patterns, because stromal production could not be ruled out. The results show that nidogen is produced by human carcinoma cells both in vitro and in vivo. Copyright © 1999 John Wiley & Sons, Ltd.     

ER-aminopeptidase 1 determines the processing and presentation of an immunotherapy-relevant melanoma epitope

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100_209-217 epitope, which is liberated from the melanoma differentiation antigen gp100^PMEL17, is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)—but not ERAP2—defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100_209-217-containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.     

Varied responses of human B-lymphoblastoid cell lines to infection with vesicular stomatitis virus

Previous studies carried out with a limited number of human lymphoblastoid cell lines of B-cell origin concluded that B cells were relatively insensitive to cell killing by vesicular stomatitis virus (VSV) (Nowakowski et al., J. Virol.12, 1272–1278, 1973; Creager et al., Virology111, 211–222, 1981). The B-cell lines employed in those studies all contained endogenous Epstein-Barr virus (EBV). The present investigation was carried out using additional B-cell lines with and without endogenous EBV genomes. It was found that the presence of EBV in human B-lymphoblastoid cells has a marked influence on the outcome of infection by VSV. In the EBV-negative B-cell lines, few, if any, cells survived VSV infection. The EBV-positive B-cell lines responded quite differently: much less cytopathology occurred, and persistent infections invariably were established. These findings suggest that EBV may play a critical role in the response of human B-lymphoblastoid cell lines to VSV infection, especially in regard to cell killing and the establishment of VSV persistence. Although there was a striking difference in the ability of VSV to kill EBV-positive and EBV-negative B-cell lines, there was no correlation between cell destruction and the amount of viral RNA or progeny virus synthesized.     

The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus

We have characterizedsrc proteins encoded by approximately 30 nonconditional transformation-defective mutants of avian sarcoma virus (ASV) and by several back mutants which reestablish a transformed phenotype. We used gel electrophoresis of immunoprecipitated proteins labeled with³²PO₄ or [³⁵S]methionine to assess size, stability, and phosphorylation; partial digestion with staphylococcal V8 protease to determine structure; and an immune complex assay to measure protein kinase activity. The mutants were all isolated as phenotypic revertants of the B31 line of B77-ASV transformed rat cells, each revertant cell bearing a single provirus without appreciable deletions, as described in the accompanying report (Varmuset al., 1980). In several instancesm the mutant proteins were examined both in the revertant rat cells and in chicken cells infected with transformation-defective viruses rescued from the nonpermissive rat cells. In addition, secondary mutations to restore a transformed phenotype (back mutations) occurred in some cases, in the original rat cells and/or chicken cells infected with rescued viruses. Three categories of mutants were identified by this survey. The largest group (Class I) encodedsrc proteins of normal size (60,000M_r); these proteins were hypophosphorylated and exhibited little or no protein kinase activity.Class II mutants displayed immunoprecipitablesrc proteins of less than normal size. In three cases, the shortsrc related proteins were mapped to the amino terminus of wild-type pp60^src and may be the result of nonsense mutations; in two cases, the short proteins were mapped to the car?yl terminus. Most of Class II mutants lacked protein kinase activity, but the 45,000M_r protein in line 000 exhibited moderate levels of activity, thereby mapping the enzymatically active site to the car?yl terminal three-fourths of pp60^src. The smallest group of mutants (Class III) did not produce detectablesrc proteins. Some of the mutant proteins behaved differently in permissive and nonpermissive hosts; in particular, the product of mutant L produced fusiform transformation and was highly phosphorylated and associated with wild-type levels of protein kinase activity in chicken cells, but was nontransforming, hypo-phosphorylated, and associated with low levels of protein kinase activity in rat cells. In all cases, back mutation to a transformed phenotype was accompanied by a restoration of wild-type (or near wild type) levels of protein kinase activity, further documenting the functional significance of the enzymatic activity. Some of the back mutants, however, encoded proteins of atypical size, either smaller or larger than pp60^src. The active proteins larger than pp60^src ranged up to 68,000M_r in size and were altered at or near the amino terminus. In one case (a retransformed derivative of the Class II revertant 000), the generation of a functionalsrc protein of 68,000M_r coincided with the appearance of an insert of ca. 200 base pairs into the ASV provirus, within or adjacent to the coding region for the amino terminus ofsrc. The diversity of reagents, both mutants and back mutants, derived from the single provirus in B31 cells indicates that this system will be useful for correlation of functional and structural attributes ofsrc.     

Detection of VSV(MuLV) pseudotypes by an immunobiochemical technique

Vesicular stomatitis virus (murine leukemia virus) (VSV(MuLV)) pseudotypes containing a [³H]uridine-labeled VSV RNA genome and MuLV envelope glycoproteins (gp70) were produced by phenotypic mixing of the two viruses. In order to better detect such pseudotypes, an immunobiochemical (IB) technique was developed. [³H]Uridine-labeled virus progeny of the dual virus infection was immunoprecipitated by monospecific MuLV gp70 antibodies complexed with fixed Staphylococcus aureus. The immunoprecipitated ³H-labeled genomic RNA was identified as that of VSV by its sedimentation coefficient, by the lack of polyadenylate, and by molecular hybridization with complementary VSV RNA. By the IB technique, approximately 11% of the progeny of the dual virus infection were found to be VSV(MuLV). By neutralization and other biological assays, however, only 0.1% of the progeny were found to be VSV(MuLV) pseudotypes. Apparently, the IB technique is capable of detecting VSV pseudotypes encapsidated with only a few molecules of MuLV gp70. The IB technique, therefore, offers a quantitative and molecular technique for the detection of VSV(MuLV) pseudotypes and can be modified to detect other viral pseudotypes when other assays are lacking. In spite of its sensitivity however, the IB technique did not detect the formation of MuLV (VSV) pseudovirions among the virus progeny of the dual virus infection. These results confirmed a similar observation made previously using immunoelectron microscopy.     

Tritiation of protein antigens of Plasmodium knowlesi schizont-infected erythrocytes using pyridoxal phosphate-sodium boro[3H]hydride

The protein antigens of Plasmodium knowlesi schizont-infected red blood cells (SI-RBCs) and normal RBCs were compared using the pyridoxal phosphate/NaB³H₄ method. Permeation of the outer SI-RBC membrane by the pyridoxal phosphate anion was enhanced since unlike normal RBCs it was not possible to exclusively label surface membrane proteins without concurrent haemoglobin labelling. Under conditions of minimal haemoglobin labelling a subset of total susceptible SI-RBC proteins (M_r 125 000, 50 000, 45 000 and 30 000) were labelled that were absent from normal RBCs and which may be surface proteins. The M_r 125 000 band labels much more readily than Band 3, the normal anion transporter, suggesting that it may be a new anion transporter in the SI-RBC membrane. At higher pyridoxal phosphate concentrations additional bands (M_r 230 000, 180 000, 165 000, 145 000, 107 000 and 72 000) were labelled exclusively with SI-RBCs. The new pyridoxal phosphate-labelled proteins had altered electrophoretic mobility and reduced Coomassie Blue staining, both properties assisting in their identification. Antigen analysis using Protein A-Sepharose and sera from infected monkeys demonstrated that all new ³H-labelled proteins were SI-RBC-specific antigens. One very high M_r antigen (> 250 000) recognized only by homologous strain antisera may represent a strain-specific antigen.     

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

Translation of tick-borne encephalitis virus (flavivirus) genome in vitro: Synthesis of two structural polypeptides

RNA isolated from tick-borne encephalitis virus (TBEV) was translated in an mRNA-dependent cell-free system derived from Krebs-2 cells, producing a set of polypeptides with M_r ranging from ca. 13,000 to 160,000 and higher. Two of these polypeptides with M_r of 13,000 (p13) and 53,000 (p53) were identified as TBEV core polypeptide V2 and the polypeptide moiety of envelope glycoprotein, V3, respectively. The amino acid sequences of p13 and p53 were also shown to be contained in a high-molecular-weight polypeptide (M_r ～ 118,000). The label from the initiator tRNA species, f-[³⁵S]Met-tRNA_f^Met, could be incorporated into p13 but not into p53, suggesting that p13 is encoded in a region of the viral genome immediately adjacent to the site at which the translation in vitro is initiated; on the other hand, the p53 coding segment appears to be located further from the initiation point. The two structural polypeptides of TBEV were accumulated in vitro with dissimilar kinetics and both differed from the majority of other translation products in that their synthesis was relatively resistant to an increase in KCl concentration in the incubation mixture. Possible modes of synthesis of TBEV structural proteins and post-translational processing of their precursor are discussed.     

The use of vesicular stomatitis virus pseudotype production in the study of a temperature-sensitive murine leukemia virus.

A mutant of Moloney murine leukemia virus (MoLV), designated ts₃ was recently shown to have a temperature-sensitive defect associated with the release of mature virus particles budding from the cell membrane [Wong, P. K. Y., and McCarter, J. A., Virology58, 396–408 (1974)]. In an attempt to determine whether the defective function resides in an envelope component of the virion, the formation of pseudotypes between VSV and ts₃ was studied under nonpermissive and permissive conditions of ts₃ infection. Whereas similar levels of phenotypic mixing were observed between VSV and wild-type MoLV at both 39 and 34°, the level of pseudotypes formed between VSV and ts₃ was found to be considerably lower at 39° (nonpermissive temperature) than at 34° (permissive temperature). The results of temperature-shift experiments indicate that two separate blocks to VSV ts₃ pseudotype production may occur depending on the length of time ts₃-infected cells are incubated at the nonpermissive temperature. Preincubation of ts₃-infected cells for 24 hr at 39°, prior to superinfection with VSV at 39°, prevents pseudotype formation. In contrast, brief incubation at 39°C, coincident with VSV infection, introduces a reversible block on the release of VSV (ts₃) pseudotypes from the cell membrane. Complementation of ts₃ through ts₃ (VSV) pseudotype production was not detected at the nonpermissive temperature.     

Expression and disposition of the murine mammary tumor virus (MuMTV) envelope gene products by murine mammary tumor cells

Three murine mammary tumor virus (MuMTV)-producing epithelial cell lines derived from murine mammary tumors were examined in order to identify the MuMTV-specific cell surface antigens and their distribution on the cell surface, to study the kinetics of the MuMTV envelope precursor processing, virus assembly, and release, and to characterize the soluble MuMTV antigens that are shed into culture medium. Cell surface labeling experiments showed that only the mature MuMTV envelope glycoproteins gp52 and gp36 were exposed on the cell surface, and that gp52 was more abundant than gp36. In cells producing large quantities of MuMTV, expression of gp52 on the cell surface was shown by immunoelectron microscopy to be localized predominantly on the surface of budding virions and not on smooth areas of the cell surface where virus was not budding. The cell surface associated gp36 was found not to be incorporated into budding virions. A few cells in all three cell lines were found to produce only a few or no MuMTV particles and in these cells, unlike in the high virus-producing cells, considerable quantities of gp52 were expressed on the surface membrane. All three cell lines were found to shed large amounts of the MuMTV env precursor polyprotein as well as the mature non-virion-associated glycoprotein, gp52, into the culture medium. The envelope precursor protein (P75^env) that was shed into the culture medium was found to differ from the predominant form of the cellular env precursor Pr70^env in that (1) P75^env migrated with an apparent higher molecular weight than Pr70^env in SDS gels; (2) Pr70^env contained only the core oligosaccharide, whereas P75^env contained fucose in addition to the core sugars; (3) two-dimensional gel electrophoretic analysis showed that Pr70^env could be resolved into three to four components migrating in the basic region of the isoelectric focussing gel (pH 7–8), whereas P75^env was resolved into 9–13 components migrating in a more acidic region of the gel (pH 5–7). The molecular structure of the exfoliated gp52 was found to be similar to that of the gp52 that was incorporated into the virions although the virion-associated gp52 was not the source of the gp52 in the medium. Our quantitative pulse-chase studies suggest that of the two populations of MuMTV env precursors that are present in MuMTV-producing cells, only Pr70^env is processed intracellularly to give rise to the mature MuMTV envelope proteins gp52 and gp36.     

Diabetic sera react with the glutamic acid decarboxylase molecule in a dimeric-oligomeric form

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). This was initially identified as a 64–65 kD molecule according to migration in gels after immunoprecipitation from pancreatic islets. We studied the antigenicity of two different radiolabelled preparations of GAD, derived either by affinity purification from porcine brain and known to contain GAD 65 and GAD 67, or by expression from a cDNA for human GAD 65 by rabbit reticulocyte lysate (RRL). Radiolabelled immunoprecipitated pellets from the reaction of potent antisera to GAD from patients with IDDM were examined by autoradiography after SDS–PAGE under reducing or non-reducing conditions. Also, preparations of porcine brain GAD were ‘depleted’ of GAD by exposure to antisera, and then similarly re-examined. Autoradiography of radiolabelled GAD either affinity purified from porcine brain, or expressed by RRL, showed that the immunoprecipitated protein migrated under non-reducing conditions according to a M_r of ~110–130 kD, corresponding to dimeric forms of monomeric GAD of ~55–65 kD. Depletion by immunoprecipitation of this minor higher M_r component from preparations of GAD left, in the supernatant, an abundance of GAD of M_r 64–65 kD corresponding to monomer that was completely non-reactive with potent IDDM sera. We conclude that IDDM sera react with the GAD molecule in a dimeric (or oligomeric) form. Our findings have general connotations for self-tolerance and autoimmunity.