The perforin-dependent immunological synapse allows T-cell activation-dependent tumor targeting by MLV vector particles.

We have reported that retroviral particles adhered to the surface of antigen-specific T cells can be carried to metastases following adoptive transfer in vivo, a process we have called viral hitch hiking. Following antigen-driven T-cell accumulation at tumors, viral particles productively infect tumor cells via envelope/receptor dependent interactions ('hand on' of virus from the T cell to the tumor cell). We describe here a second envelope/receptor independent pathway of viral hand on from T cells, dependent on T-cell activation. We show that the endosomolytic property of perforin promotes release of viral particles from endosomes into which they are co-delivered along with cytotoxic granules from the activated T cell. Therefore, hand on of MLV particles lacking any envelope can be used for in vivo delivery of vectors, where targeting is at the extremely specific level of recognition of antigen by the T-cell receptor, thereby dispensing with the need to engineer viral envelopes. These data reveal a novel pathway by which MLV viral particles exploit a functional immunological synapse and present new opportunities both to improve the efficacy of adoptive T-cell transfer and to target vectors for systemic gene delivery.

In vivo tolerance assessment of skin after insertion of subcutaneous and cutaneous microdialysis probes in the rat.

The purpose of the study was to evaluate the trauma induced by insertion of the linear microdialysis probe in the subcutaneous and dermal tissue in the rat and to check if the microdialysis probe insertion affects transdermal drug delivery. Non-invasive bioengineering methods (TEWL, Laser Doppler Velocimeter, Chromameter) as well as histology were combined to characterize these effects. The results showed that the dermal and subcutaneous insertion of microdialysis probes did not change skin permeability, blood flow and color, confirming the safety of this technique. The probe depth did not influence the trauma. No significant physical damage after probe insertion was noticed. Thus, the present work validates the use of microdialysis in dermatopharmacokinetics studies after topical or systemic drug delivery.

A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf.

Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.

Ionizing radiation-induced MEK and Erk activation does not enhance survival of irradiated human squamous carcinoma cells.

PURPOSE: Ionizing radiation (IR) triggers several intracellular signaling cascades that have commonly been regarded as mitogenic, including the Raf-MEK-Erk kinase cascade. In addition to promoting proliferation, activated MEK and Erk may also prevent cell death induced by cytotoxic stimuli. Because Raf, MEK, and Erk are activated by IR in some tumor cell lines, this suggests that IR-induced activation of the kinase cascade may enhance the survival of irradiated cells. METHODS AND MATERIALS: IR-induced activation of MEK and Erk was assessed in irradiated UM-SCC-6 cells, a human squamous carcinoma cell line. Activation of MEK and Erk was blocked with the pharmacological inhibitor of MEK activation, PD098059. Clonogenic survival was assessed in irradiated UM-SCC-6 cells that were pretreated with nothing or with the MEK inhibitor. RESULTS: In UM-SCC-6 cells, IR doses as low as 2 Gy rapidly activated MEK and Erk. Pretreatment of the cells with the pharmacological inhibitor of MEK activation, PD098059, effectively blocked IR-induced activation of MEK and Erk. However, inhibition of the kinase cascade did not affect the clonogenic survival of irradiated cells in either early or delayed-plating experiments. CONCLUSION: Taken together, these results suggest that although MEK and Erk are rapidly activated by IR treatment, these protein kinases do not affect the clonogenic survival of irradiated UM-SCC6 cells.

Characterization and growth regulation of a rat intrahepatic bile duct epithelial cell line under hormonally defined, serum-free conditions.

Bile duct epithelial cells, or cholangiocytes, proliferate in vivo under a number of pathologic (i.e., partial hepatectomy) and pathophysiologic (i.e., bile duct ligation, malignant transformation) conditions. However, little is known about the possible growth factors that modulate these proliferative responses, in part because an in vitro model to study proliferation of nontransformed, normal cholangiocytes is not available. We report here the development of a rat cholangiocyte cell line (MMRC, minimal media-requiring rat cholangiocytes) that grows under hormonally defined, serum-free conditions on plastic and maintains a cholangiocyte phenotype. Morphologic as well as functional studies indicate that the cell line is polarized and actively transports fluid and electrolytes in an apical to basolateral direction. MMRC, when cultured for 24 mo. and passaged 80 times, have not undergone malignant transformation, because the cell line failed to grow under anchorage-independent conditions or in nude mice. Cellular proliferation is accelerated 2-8-fold by insulin, insulin-like growth factor 1, epidermal growth factor, and hepatocyte growth factor, growth factors known to stimulate tyrosine kinase receptors, and inhibited 2-10-fold by TGFbeta and IL-2. Glyco-conjugates of primary (i.e., cholic and chenodeoxycholic acid) and secondary bile acids (i.e., deoxycholic and lithocholic acid) do not alter proliferation at low concentration (1 microM), but are toxic at higher concentration (10 microM). In summary, we have developed and characterized a cholangiocyte cell line derived from normal rat liver, which grows under hormonally defined, serum-free conditions, maintains a nonmalignant, cholangiocyte phenotype, displays morphologic and functional features of polarity, and alters its proliferation rate in response to a variety of growth factors.

Kinetic and molecular identification of sodium-dependent glucose transporter in normal rat cholangiocytes.

While previous work has demonstrated that monosaccharides can be absorbed from bile, studies of sugar transport by the biliary, epithelia (i.e., cholangiocytes) are lacking. Using a novel model of polarized rat cholangiocytes in primary culture, designated normal rat cholangiocytes (NRC), we examined directly the uptake and transcellular transport of a nonmetabolizable monosaccharide, methyl alpha-D-glucopyranoside (AMG). When the apical or basolateral domain of cholangiocytes was exposed to radiolabeled AMG or sucrose (control), only apical absorption of AMG was evident. This apical uptake was time dependent, saturable, and significantly inhibited (> or = 90%) by removal of Na+ or in the presence of phlorizin (0.1 mM), a competitive inhibitor of the Na(+)-glucose cotransporter. The transcellular flux of AMG was also polar (i.e., apical to basolateral). Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of the transcript for the specific Na(+)-glucose cotransporter SGLT1 in NRC and in freshly isolated cholangiocytes but not in purified hepatocytes; in contrast, the transcript for SGLT2 was absent in all liver samples. In situ RT-PCR on frozen sections of normal rat liver showed that SGLT1 was expressed exclusively in cholangiocytes. Immunoblot analysis using a specific polyclonal antibody for the facilitative glucose transporter GLUT1 demonstrated it to be present in vesicles derived from NRC enriched in basolateral plasma membrane domains. Our data are consistent with the concept that SGLT1 is present on the apical domain of biliary epithelia and, in conjunction with GLUT1 on the basolateral domain, accounts for glucose absorption from bile.

Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.

The study of intrahepatic bile duct epithelial cells (i.e., cholangiocytes) has been limited by the lack of a polarized in vitro model that allows easy access to both apical and basolateral cell surfaces. Therefore, we developed a cell line of polarized normal rat cholangiocytes (NRCs) and established conditions that produced a confluent monolayer of cells grown on collagen-coated filters of tissue culture inserts. We passaged NRCs at high density to collagen-coated, tissue-culture inserts and measured transepithelial electrical resistance. We evaluated ultrastructural features by transmission and scanning electron microscopy. gamma-glutamyl-transpeptidase (gamma GT) was visualized in cultured cells by enzyme histochemistry, and cytokeratin (CK)-7, CK-19, vimentin, and desmin staining was done by immunohistochemistry. We studied the biologic responsiveness and functional polarity of NRCs by measuring their levels of cyclic AMP after addition of forskolin with or without somatostatin to either the apical or basolateral chambers. When seeded with approximately 1 x 10(5) cells/cm2, the NRCs formed a confluent monolayer in 72 hr. Transepithelial electrical resistance increased over time, achieving a maximum of 625 (+- 25) ohms.cm2 by 1 week after confluence. Transmission and electron microscopy scanning showed the apical cell surface to be tightly packed with microvilli with a heterogeneous display of cilia ranging from none to 20 to 30 cilia/cell. On transmission, apically positioned tight junctions and vesicles were apparent; nuclei were oriented basally and the basolateral surface was characterized by membrane interdigitations. NRCs stained positively for the cholangiocyte marker proteins, gamma-glutamyl-transpeptidase, CK-7, and CK-19, and negative for the mesenchymal markers, vimentin, and desmin. Exposure of the basolateral (but not the apical) cell surface to somatostatin caused a 60% inhibition of forskolin-induced increases in intracellular levels of cyclic AMP, suggesting the presence of somatostatin receptors exclusively on the basolateral plasma membrane domain. We have developed a unique model of primary cultures of normal rat cholangiocytes in which the apical and basolateral surfaces are easily accessible; the cells develop intermediate-strength tight junctions, retain their cholangiocyte phenotype, display morphologic and functional polarity, and are responsive to hormones. This model should be useful for the assessment of vectorial transport of solutes and other constituents of blood and bile, as well as for studying growth regulation of cholangiocytes.

Recent advances in the isolation of liver cells.

The development of new and refined separation techniques--including FACS, FFE, CFE and isopyknic gradients--has had a profound impact on the ability of investigators to isolate specific cell types from the liver. Although some of these techniques, such as FFE, may be of limited preparative value, they are nonetheless important analytical tools that detect subtle differences among cell subpopulations. The isolation of highly purified preparations of liver cells in large yields requires the use of more conventional purification methods such as CFE and isopyknic centrifugation. Immunological approaches represent a key development for the isolation of specific liver cell types, especially when they are used in combination with other techniques. Excellent, reliable and relatively simple techniques now exist to isolate highly purified preparations of hepatocytes, cholangiocytes, KCs, SCs, FSC, myofibroblasts and pit cells. Additional work is necessary to refine techniques for the isolation of dendritic cells and lymphocytes.

Biological characteristics of primary cultures of human gallbladder epithelial cells.

Epithelial cells of the gallbladder have potential to represent an important model for studies of ductal epithelial in normal and pathological states. We therefore initiated studies to establish human gallbladder epithelial cells (GBEC) in culture. GBEC were isolated by trypsinization of small tissue fragments from human gallbladders obtained at cholecystectomy; cells were plated on tissue culture dishes and grown in defined MCDB 153 medium containing added growth factors. In this medium, GBEC showed a plating efficiency of approximately 1%; those GBEC that attached formed colonies and proliferated, as demonstrated by autoradiographic analysis of [3H]thymidine incorporation into DNA. Cultured GBEC expressed two markers found on GBEC in situ, i.e., gamma-glutamyl transpeptidase and cytokeratin 19. By using various attachment substrates, with and without added serum, increased plating efficiency and better growth were achieved. When type IV collagen was used as substrate and 10% fetal bovine serum was added to MCDB 153, passage of GBEC was possible, and cells proliferated through five to six population doublings. GBEC in culture under all conditions eventually enlarged, showed vacuolization, and demonstrated irreversible growth arrest. Nonetheless, the culture conditions described here allow for preparation of large quantities of highly enriched human GBEC.

The ins and outs of membrane movement in biliary epithelia.

We have developed a novel technique for the isolation from normal rat liver of morphologically polar, intrahepatic bile duct epithelial cells which exhibit clathrin-coated pits. Using electron microscopic cytochemistry, we demonstrated receptor-mediated endocytosis of EGF by cultured IBDEC. Also, using freshly isolated polar couplets of IBDEC, we demonstrated that these cells participate in fluid-phase endocytosis. Finally, using a novel fluorescence unquenching assay and our isolated bile duct epithelial cell model, we showed that secretin stimulates exocytosis in IBDEC, a finding compatible with the possibility that secretin-induced changes in ductular bile flow may occur by an exocytic process. The availability of a reproducible and reliable technique to prepare liver cell fractions highly enriched in intrahepatic bile duct epithelial cells with morphologic polarity has made it possible to do direct experiments on the functions of intrahepatic bile duct epithelial cells, including the study of plasma membrane movement (i.e., endocytosis and exocytosis). With the availability of this technique, other studies previously impossible to carry out in IBDEC are now feasible. Such studies are too numerous to mention, but would include experiments on ligand binding, transport of macromolecules, assessment of metabolic activities and toxicity studies, to name just a few. Indeed, virtually any question that has been asked about hepatocytes and addressed using isolated hepatocytes can now be directed toward isolated intrahepatic bile duct epithelial cells. Finally, the methodology described here is theoretically applicable to human liver. Indeed, intrahepatic bile duct epithelial cells are considered to be involved in the pathogenesis of several kinds of drug and immunologically induced liver diseases, including allograft rejection, primary biliary cirrhosis, and primary sclerosing cholangitis. The availability of the technology described here should make feasible direct experimental approaches to questions in all of these areas.

Morphologic demonstration of receptor-mediated endocytosis of epidermal growth factor by isolated bile duct epithelial cells.

It was recently shown that intrahepatic bile duct epithelial cells in situ or after isolation from rat liver have coated pits and vesicles, suggesting that they participate in receptor-mediated endocytosis. Therefore, using a morphologic approach and epidermal growth factor coupled to horseradish peroxidase or colloidal gold as probes, we studied freshly isolated or short-term cultured intrahepatic bile duct epithelial cells prepared from normal rat liver to determine if they participate in receptor-mediated endocytosis. Immunoelectron microscopy using a monoclonal antibody against the epidermal growth factor receptor was also used to examine for the presence of the growth factor receptor on the cells. Immediately after isolation, the cells did not internalize either epidermal growth factor-horseradish peroxidase or epidermal growth factor-colloidal gold; no growth factor receptor could be shown on these cells by immunocytochemistry, either. In contrast, cells cultured for 24 h bound and internalized both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold at 37 degrees C and showed growth factor receptors diffusely distributed on the plasma membrane. When cultured cells exposed to epidermal growth factor-colloidal gold were fixed with glutaraldehyde containing saponin and tannic acid, colloidal gold particles were observed in coated pits and in coated and uncoated vesicles. Preincubation of cultured cells with native epidermal growth factor completely blocked the internalization of both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold. When rat liver was stained in situ for epidermal growth factor receptor, reaction product was observed by immunoelectron microscopy exclusively on the basal surface of the plasma membrane of the intrahepatic bile duct epithelial cells. These results indicate that bile duct epithelial cells internalize epidermal growth factor by endocytosis via coated pits containing receptors localized in situ exclusively to the basal domain of their plasma membranes. The data demonstrate for the first time that intrahepatic bile duct epithelial cells participate in receptor-mediated endocytosis and raise the possibility that they are a target for epidermal growth factor.

Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver.

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.

Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver.

To study directly the functions of the cells that line the bile ducts inside the liver, we developed a new technique for isolating intrahepatic bile duct epithelial cells (IBDECs) from normal rat liver. Parenchymal and nonparenchymal cells were separated from whole liver by enzymatic digestion and mechanical disruption; subpopulations of individual nonparenchymal cells then were isolated by serial counter-flow elutriation, isopycnic centrifugation, and immunoaffinity separation with a specific monoclonal antibody against an antigen on the plasma membrane of IBDECs. Using this approach, we isolated 1.2 +/- 0.2 x 10(6) (mean +/- SE) viable (greater than 95% trypan blue exclusion) cells, greater than 95% of which were identified as IBDECs by morphologic appearance and specific cytochemical markers. The IBDECs averaged 7.4 +/- 0.16 microM in diameter and retained their in situ appearance, including morphologic polarity. They appeared as single cells or as cell doublets attached by tight junctions that excluded ruthenium red. Microvilli were abundant and were restricted to the apical (i.e., luminal) domain of the plasma membrane. Coated pits were observed on both apical and basolateral cell surfaces. Internally, IBDECs contained a well-developed system of organelles, including mitochondria, Golgi, and discrete types of vesicles, such as coated vesicles, multivesicular bodies, and lysosomes. These results indicate that a highly purified suspension of viable, morphologically intact, and polar IBDECs can be prepared from normal rat liver using a novel approach that separates liver cells on the basis of size, density, and specific membrane components. The availability of such a model will allow experimental studies to be performed directly on IBDECs, an approach that has not previously been possible.

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species.

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.

Isolation of steroid receptor binding protein from chicken oviduct and production of monoclonal antibodies.

Previous studies have shown that the molybdate-stabilized progesterone receptor from the chick oviduct contains a nonhormone binding component with a molecular weight of 90 000. This protein has also been shown to be associated with some other molybdate-stabilized steroid receptors of the oviduct. In order to access this larger pool of the receptor binding protein, we have developed an isolation procedure based on the observation that the protein is selectively shed from proteins adsorbed to heparin-agarose when molybdate is removed. The protein obtained by this procedure is shown to be the same as that isolated from affinity-purified progesterone receptor as compared by protease digestion and one-dimensional peptide mapping. Four immunoglobulin G secreting hybridoma cell lines were generated against the 90 000-dalton antigen. All of the antibodies recognize the 90 000-dalton protein obtained by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. In addition, two of the antibodies complex the molybdate-stabilized progesterone receptor as demonstrated by sedimentation analysis on sucrose gradients. One of these antibodies was used to show the presence of the 90 000-dalton component in molybdate-stabilized glucocorticoid and androgen receptors and also to show its presence in brain, liver, and skeletal muscle, but not in serum.

Characterization of a major protein with a molecular weight of 160,000 associated with the viral capsid of Epstein-Barr virus.

A monoclonal antibody designated V3 was produced against a late protein associated with the Epstein-Barr virus-induced viral capsid antigen complex. The antibody reacted with discrete patches in the nuclei of infected cells as well as with virus particles, as shown by immunofluorescence and ultrastructural immunoperoxidase staining. The molecular weight of the protein precipitated by this monoclonal antibody was ca. 160,000. All anti-viral capsid antigen antibody-positive sera tested in an enzyme-linked immunosorbent assay reacted with this purified protein. The synthesis of the antigen was inhibited by phosphonoacetic acid but was not affected by tunicamycin, indicating that this was a late nonglycosylated viral protein. No differences were noted between the protein isolated from the P3HR-1 and B-95-8 cell lines as determined by immunoprecipitation and peptide mapping. By isoelectric focusing, this protein had a pI on the basic side ranging from 7.5 to 9.0.

Production of monoclonal antibody to a late intracellular Epstein-Barr virus-induced antigen.

A monoclonal antibody designated L2 was produced against a late intracellular protein induced by Epstein-Barr virus (EBV). This protein was expressed in cells producing virus but not in EBV genome-positive nonproducer cell lines, EBV genome-negative cell lines, or producer cultures cultivated in the presence of phosphonoacetic acid as determined by immunofluorescence. In addition, the antibody did not react with the membranes of infected cells indicating that it was not directed against an EBV-induced membrane antigen component. The monoclonal antibody was shown to recognize a glycoprotein with a molecular weight of approximately 125K by SDS-polyacrylamide gel electrophoresis. This glycoprotein was consistently found to be slightly larger when isolated from the P3HR-1 cell line as opposed to the B-95-8 cell line. A similar difference was also noted by comparison of peptide maps of this protein isolated by immunoaffinity chromatography from the two cell lines. Serological studies indicated that this 125K glycoprotein was a major component of the viral capsid-antigen (VCA) complex as defined by immunofluorescence.

Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies.

Three monoclonal antibodies were produced against the Epstein-Barr virus-induced early antigen complex. These antibodies were shown to be specific for the early antigen complex by the fact that they only reacted with cells supporting a permissive or abortive Epstein-Barr virus infection and their synthesis was not affected by inhibitors of viral DNA synthesis. One monoclonal antibody, designated R3, was directed against a diffuse component of the early antigen complex since it reacted by immunofluorescence with cells fixed in acetone or methanol. The other two monoclonal antibodies, designated K8 and K9, reacted with a methanol-sensitive restricted component of this complex. The appearance of the R3 antigen in P3HR-1 superinfected Raji cells occurred approximately 4 h earlier than the antigen detected by K8. By both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoelectrophoresis, it was determined that the R3 monoclonal antibody recognized two major polypeptides with molecular weights of approximately 50,000 to 52,000, whereas K8 and K9 precipitated a protein of approximately 85,000. The R3 monoclonal antibody also immunoprecipitated an in vitro primary translation product. It was, therefore, possible to map this product to the Epstein-Barr virus DNA BamH1 M fragment. These in vitro products were slightly smaller than the in vivo proteins, suggesting that these proteins probably undergo posttranslational modification during the virus replication cycle.

Identification of Epstein-Barr virus strain differences with monoclonal antibody to a membrane glycoprotein.

Two monoclonal antibodies directed against Epstein--Barr virus (EBV)-induced membrane antigens (MA) were isolated in this study. On of the monoclonal antibodies, designated 2F5.6, was an IgG2 which, as detected by membrane and fixed cell immunofluorescence, reacted with MA-positive lymphoblastoid cell lines that produced transforming EBV but not with the MA-positive P3HR-1 cell line that produced the lytic, nontransforming strain of this virus. This antibody precipitated the Mr 320,000/350,000 glycoprotein from B-95 virus infected cultures and the Mr 300,000 and 220,000/250,000 glycoproteins from Raji cells superinfected with P3HR-1 virus but did not precipitate any of these EBV-specific glycoproteins from the P3HR-1 cell line. In contrast, the second monoclonal antibody, IgM designated B10.3, reacted with all virus-producing cell lines including the P3HR-1 cell line. The identity of the glycoprotein that serves as the target for this antibody is still unknown. Neither antibody had neutralizing activity against the B-95 or P3HR-1 strain of EBV. These results indicated that the 2F5.6 monoclonal antibody was directed against an antigenic determinant on the major membrane glycoprotein which is common to transforming strains of EBV but absent from the lytic P3HR-1 stain whereas the B10.3 monoclonal antibody was directed against a group-specific EBV-induced membrane determinant.