Analysis of structural proteins of purified murine cytomegalovirus.

Murine cytomegalovirus propagated in mouse embryo fibroblasts was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 x g for 90 min. (ii) The concentrated virus was passed through a Bio-Rad Bio-Gel A-15m column to eliminate contaminating materials smaller than 15 x 10(6) daltons. Most of the virus was recovered in the void volume of the column. (iii) Two consecutive centrifugations through 20 to 50% potassium tartrate gradients were performed. After the second tartrate gradient centrifugation, symmetrical, coinciding peaks of plaque titer, protein, and radioactivity were found at a density between 1.20 g/cm3 and 1.21 g/cm3. To establish purification criteria, virus was purified from two different mixtures: [35S]methionine-labeled extracellular virus, mixed with an equal volume of unlabeled normal culture fluid, and unlabeled extracellular virus mixed with an equal volume of [35S]methionine-labeled normal culture fluid. At the end of the procedure, the extent of purification, as judged by the ratio of cellular to viral radioactivity was at least 70-fold. Virus proteins were analyzed by electrophoresis on a 5 to 20% gradient polyacrylamide gel slab. After gel electrophoresis,, Coomassie brilliant blue staining profiles and autoradiograms of the purified virus preparations were compared. At least 33 virus structural protein bands were present. The molecular weights of these proteins ranged from 11,500 to 255,000. The sum of the molecular weights of the virus structural proteins was 2,462,000. Autoradiograms obtained from electrophoresis of purified [14C]glucosamine-labeled virus showed that at lease 6 of the 33 viral structural proteins were glycoproteins.     

Herpesvirus sylvilagus I. Polypeptides of virions and nucleocapsids.

Herpesvirus sylvilagus was propagated in juvenile cotton tail rabbit kidney cells and purified from the cytoplasmic fraction of the infected cells. The purification procedure included zonal centrifugation through a 5 to 30% dextran t-10 gradient, followed by equilibrium centrifugation in a 5 to 50% potassium tartrate gradient. H. sylvilagus formed one band after centrifugation through the tartrate gradient at a density of 1.22 g/cm3. Contamination of the purified virus preparation by cellular proteins was less than 0.2% as determined by the removal of radioactivity from an artificially mixed sample containing [35S]methionine-labeled control cells and nonlabeled infected cells. H. sylvilagus nucleocapsids were isolated from infected cell nuclei and purified by sedimentation through a 36% sucrose cushion, followed by equilibrium centrifugation in 5 to 50% tartrate gradient. Forty-four polypeptides ranging in molecular weight from 18,000 to 230,00 were resolved when [35S]methionine-labeled enveloped H. sylvilagus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Seventeen polypeptides found within the enveloped virus were also identified with the nucleocapsid. Six additional nucleocapsid polypeptides han no counterparts within the enveloped virus. The major polypeptide within both the virus and the nucleocapsid had a molecular weight of 150,000.     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.     

Location of an F-pilin pool in the inner membrane.

Polyacrylamide gel analysis of [35S]methionine-labeled membrane preparations from Escherichia coli has revealed the presence of five polypeptides present only in the membranes of cells containing the conjugative plasmid F. In addition to the previously reported product of traT, polypeptides migrating with apparent molecular weights of 100,000, 23,500, 12,000, and 7,000 were resolved. Membrane preparations from F traJ mutants lacked these polypeptides, indicating that all of these proteins are tra gene products. The 7,000-molecular-weight polypeptide comigrated with unlabeled purified F-pilin protein. About 4 to 5% of the total radioactive label in whole membrane preparations was present in this polypeptide, indicating the existence of a substantial pool of membrane-associated F-pilin. The polypeptide could be extracted from whole membrane preparations with Triton X-100 and was found in the inner membrane fraction of membranes separated by sucrose density centrifugation.     

Substructures and Polypeptides of Visna Virus

The protein of Visna virus, disrupted by 8 M guanidine hydrochloride and heating, was resolved into 10 polypeptides by agarose gel column chromatography in 6 M guanidine hydrochloride. Two of the peaks contained glycopolypeptides. Nonidet-disrupted virions were resolved into two fractions by potassium tartrate gradient centrifugation, with densities of 1.08 and 1.24 g/ml, respectively. About 70% of the viral DNA polymerase directed by added template was released into the light fraction, in which very little endogenous enzyme activity was detected. Also released into the light fraction were all of the glycopolypeptides, 50% of the viral RNA, and a part of each of the other viral protein components. The data indicate that extensive degradation of subviral structures occurred, even under mild conditions for virion disruption. The 1.24-g/ml fraction was composed of 50% of the viral RNA, most of the endogenous DNA polymerase activity (80%), and a major internal polypeptide (GuHCl6) with an estimated mol wt of 28,000. Two other polypeptides were also consistently detected in the heavy fraction, but they constituted less than 25% of the ribonucleoprotein complex, compared with 75% for GuHCl6.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. I. Electrophoretic and immunochemical analyses

We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.     

Immunological and Chemical Identification of Intracellular Forms of Adenovirus Type 2 Terminal Protein

Highly purified adenovirus type 2 terminal protein (TP) with an apparent M_r of 55,000 (55K) was prepared in quantities of 10 to 30 μg from guanidine hydrochloride- or sodium dodecyl sulfate-disrupted virions (60 to 120 mg). Guinea pigs were immunized with 14 to 20 injections of TP in amounts of 1 to 2 μg. Antiserum to TP was used to study the intracellular polypeptides related to adenovirus type 2 TP. By immunoprecipitation with anti-TP serum, we identified 80K and 76K polypeptides in the nucleoplasmic and cytoplasmic S100 fractions of [³⁵S]methionine-labeled cells early and late after infection with Ad2. By immunoautoradiographic analysis which eliminates coprecipitation of unrelated proteins, we identified an 80K polypeptide (probably an 80K-76K doublet) in unlabeled, late infected cells, using anti-TP serum and ¹²⁵I-labeled staphylococcal protein A. About two- to threefold-higher levels of the 80K and 76K polypeptides were present in the nucleoplasm than in the S100 fraction, and two- to threefold-higher levels were found in late infected cells than in early infected cells (cycloheximide enhanced, arabinofuranosylcytosine treated). We did not detect the 80K or 76K polypeptide in uninfected cells, indicating that these polypeptides are virus coded. Tryptic peptide map analysis showed that the 80K and 76K polypeptides are very closely related and that they share peptides with the DNA-bound 55K TP. Our data provide the first direct demonstration of intracellular 80K and 76K forms of TP. The intracellular 80K and 76K polypeptides are closely related or identical to the 80K polypeptide that Challberg and co-workers (Proc. Natl. Acad. Sci. U.S.A. 77:5105-5109, 1980) detected at the termini of adenovirus DNA synthesized in vitro and to the 87K polypeptide that Stillman and co-workers (Cell 23:497-508, 1981) translated in vitro. We did not detect the 55K TP in early or late infected cells, consistent with the proposal by Challberg and co-workers that the 80K polypeptide is a precursor to the virion-bound TP and that the conversion of the 80K polypeptide to the 55K TP occurs during virus maturation. The 80K and 76K polypeptides have many more methionine-containing tryptic peptides than does the 55K TP, and most of the tryptic peptides unique to the 80K and 76K polypeptides are very hydrophobic. Thus, the conversion of the 80K and 76K polypeptides to the 55K TP may involve the removal of a specific hydrophobic protein region.     

Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates.

To determine whether certain outer membrane proteins are associated with growth of Bacteroides thetaiotaomicron on polysaccharides, we developed a procedure for separating outer membranes from inner membranes by sucrose density centrifugation. Cell extracts in 10% (wt/vol) sucrose-10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) were separated into two fractions on a two-step (37 and 70% [wt/vol]) sucrose gradient. These fractions were further resolved into outer membranes (p = 1.21 g/cm3) and inner membranes (p = 1.14 g/cm3) on sucrose gradients. About 20 to 26% of the total 3-hydroxy fatty acids from lipopolysaccharide and 2 to 3% of the total cellular succinate dehydrogenase activity were recovered in the outer membrane preparation. The inner membrane preparation contained 22 to 49% of the total succinate dehydrogenase activity and 2 to 3% of the total 3-hydroxy fatty acids from lipopolysaccharide. Outer membranes contained a lower concentration of protein (0.34 mg/mg [dry weight]) than did the inner membranes (0.68 mg/mg [dry weight]). Molecular weights of inner membrane polypeptides ranged from 11,000 to 133,000. The most prominent polypeptides had molecular weights ranging from 11,000 to 26,000. In contrast, the molecular weights of outer membrane polypeptides ranged from 17,000 to 117,000. The most prominent polypeptides had molecular weights ranging from 42,000 to 117,000. There were several polypeptides in the outer membranes of bacteria grown on polysaccharides (chondroitin sulfate, arabinogalactan, or polygalacturonic acid) which were not detected or were not as prominent in outer membranes of bacteria grown on monosaccharide components of these polysaccharides.     

Reactivity of human lipoproteins with purified lecithin: cholesterol acyltransferase during incubations in vitro

Studies have been performed to determine the proportion of the esterified cholesterol in high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) that is attributable to a direct action of lecithin: cholesterol acyltransferase on each lipoprotein fraction. Esterification of [3H]cholesterol was examined in 37 degrees C incubations of either: (a) unseparated whole plasma, (b) plasma reconstituted after prior ultracentrifugation to separate the 1.21 g/ml supernatant, (c) a mixture comprising the 1.21 g/ml supernatant of plasma and purified lecithin: cholesterol acyltransferase or (d) the same mixture as (c) after supplementation with a preparation of partially purified lipid transfer protein. Each of these incubations was performed using samples collected from four different subjects, two of whom had normal and two of whom had elevated concentrations of plasma triacylglycerol. At the completion of 3-h incubations, the lipoproteins were separated into multiple fractions by gel filtration to obtain a continuous profile of esterified [3H]cholesterol across the whole spectrum of lipoproteins. There was an appearance of esterified [3H]cholesterol in each of the major lipoprotein fractions in all incubations. In unseparated plasma, 56% of the total (mean of four experiments) was in HDL, 33% in LDL and 11% in VLDL. A comparable distribution was observed in the incubations of reconstituted plasma and in the samples to which partially purified lipid transfer protein had been added. In the absence of lipid transfer protein activity in incubations containing purified lecithin: cholesterol acyltransferase, 73% of the esterified [3H]cholesterol was in HDL, 25% in LDL and only 1% in VLDL. It has been concluded that at physiological concentrations of lipoproteins, 70-80% of the cholesterol esterifying action of lecithin: cholesterol acyltransferase is confined to the HDL fraction, with most of the remainder involving the LDL fraction. Of the newly formed esterified cholesterol incorporated into LDL during incubations of unseparated plasma, it was apparent that more than 70% was independent of activity of the lipid transfer protein. Of that incorporated into VLDL in unseparated plasma, in contrast, almost 90% was derived as a transfer from other fractions as a consequence of activity of the lipid transfer protein.     

Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-2-infected cells.

The virus-coded 72000-Mr DNA-binding protein from adenovirus-type-2-infected cells has been purified to homogeneity by DEAE-cellulose chromatography, selective precipitation and gel filtration. The 72000-Mr DNA-binding protein is phosphorylated and the phosphate is covalently linked predominantly to serine. Analysis of tryptic digests of the 32P-labeled 72000-Mr protein showed that the phosphate residue(s) is present in only one peptide. The DNA-binding fraction contains an additional non-phosphorylated protein with an approximate molecular weight of 45000. Tryptic peptide maps of [35S]methionine-labeled 72000-Mr and 45000-Mr polypeptides are indistinguishable. The amino acid compositions of the 72000-Mr and 45000-Mr polypeptides show closely related distributions. An antiserum produced against the purified 72000-Mr DNA-binding protein precipitates both the 72000-Mr and the 45000-Mr protein from extracts of adenovirus-infected cells. Immunofluorescence studies revealed DNA-binding protein to be accumulated in characteristic structures in nuclei of the infected cells.     

Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.

Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.     

Purification and buoyant density of infectious bovine rhinotracheitis virus.

A purification scheme for infectious bovine rhinotracheitis virus utilizing rate-zonal centrifugation in a 10-40% potassium tartrate gradient was described. The density of IBRV in the potassium tartrate gradient was found to be 1.22 g/cm3. Electron microscopic examination of purified virus preparations revealed homogeneous populations of enveloped virions with minute projections on the envelope surface.     

Genetic analysis of adenovirus type 2 III. Temperature sensitivity of processing viral proteins.

Using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis of [35S]methionine-labeled adenovirus type 2-infected KB cell extracts, a total of 23 virus-induced polypeptides was detected. This technique was applied to the analysis of the temperature-sensitive mutant, ts 1, which has previously been shown to be defective in a late function. By means of pulse-chase experiments, ts 1 was shown to be defective in the processing of the precursor polypeptide (Pre VII) to the major core protein VII. Two other putative precursor polypeptides, Va (27K) and Vb (24K), were also not processed. Thus, the ts 1 mutation blocked the appearance of six post-translational clevage products, i. e., polypeptides VI, VII, VIII, X, XI, and XII. All of these polypeptides are virion components. Processing was temperature sensitive in a shift-up experiment, whereas it was normal in a shift-down experiment. The kinetics of the temperature-shift experiments suggested that infectious virus could be recovered if enough time is provided for processing to take place. Processing was not inhibited by cycloheximide. The analysis of purified virus particles and empty shells (TCs) revealed the presence of the precursor and putative precursor polypeptides Pre-VII, Va and Vb, instead of their cleavage products, in both types of particles. Based on these results we propose that the ts 1 gene codes for or regulates an endoprotease which is responsible for the completion of the last step in virus maturation, that is, the conversion of "young virions" into mature infectious virions by a series of maturation cleavages.     

Identification of herpesvirus sylvilagus-induced polypeptides in productively infected cells.

Polypeptides synthesized in cell cultures infected with high multiplicities of herpesvirus sylvilagus were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts. Initiation of polypeptide synthesis was detected by 6 h after infection. The maximum intensity of many [35S]methionine-labeled viral bands was observed at 45 h after infection. Production of detectable infectious virus began between 18 and 24 h and reached a plateau at 48 h after infection. Immunoprecipitation of cell extracts identified a minimum of 45 virus-induced polypeptides ranging in molecular weight from 230,000 to 27,000. The major polypeptide appeared to have a molecular weight of 150,000. The pattern of these extracts suggested that the synthesis of host polypeptides is stimulated during the first 12 h and thereafter reduced, but not completely inhibited, during the remaining course of infection.     

Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.     

Complex of DNA with chromatin proteins investigated by isopycnic centrifugation in metrizamide.

Complexes of mouse main band DNA with a fraction of non-histone proteins (NHP), having a high affinity for DNA, in the absence or presence of histones have been investigated by gradient centrifugation in metrizamide. Two types of complexes were formed at an input ratio of NHP to DNA between 1 and 2.5. In metrizamide gradients a majority of DNA was found in the light complex (at the density of 1.14-1.16 g/cm3) even at the very high NHP to DNA ratio. When histones were present in the reaction mixture, most of the DNA was found in the heavy complex (1.19-1.21 g/cm3). The electrophoretic profiles of the proteins recovered from the heavy and light complexes were different; some fractions of nonhistone proteins were present only in the heavy component.     

Association of newly synthesized poly(A) polymerase with four distinct polypeptides

Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.     

Biophysical Properties of Frog Virus and Its Deoxyribonucleic Acid: Fate of Radioactive Virus in the Early Stage of Infection

Frog virus (FV-3) was banded by isopycnic centrifugation in cesium chloride, sucrose, or potassium tartrate. Two bands of infectivity were regularly found at positions in cesium chloride corresponding to densities of 1.26 and 1.30 g/cm(3), respectively. Deoxyribonucleic acid from either band had the following characteristics: double-stranded; a T(m) of 76.3 C in 0.1 SSC (0.015 m NaCl plus 0.015 m sodium citrate) and a buoyant density of 1.720 g/cm(3) in cesium chloride, corresponding to a guanine plus cytosine content of 56 to 58% and a molecular weight of 130 x 10(6) daltons, determined by velocity sedimentation. These data, together with electron micrographs of sections of cells infected with material from either band suggest that two types of infectious frog virus particles exists, rather than a second virus in the frog virus stocks. The composition of frog virus was determined. It was found that highly purified preparations of frog virus were composed of 55.8% protein, 30.1% deoxyribonucleic acid, and 14.2% lipid. The kinetics of adsorption and uncoating of FV-3 was studied with radioactive virus. Uncoating is comparatively rapid and in contrast to poxvirus is unaffected by inhibitors of protein synthesis.     

Polypeptides of the Epstein-Barr virus membrane antigen complex.

Epstein-Barr virus (EBV)-associated membrane antigens have been purified from the plasma membranes of the producer cell line P3HR-1 NONO. The antigens were assayed with a specific rabbit anti-ebv antiserum using an 125I-labeled staphylococcal protein A binding assay. The antigens have been shown to be present on purified plasma membranes. Treatment of the plasma membranes with Triton X-100 allows the separation of two antigenically distinct classes of antigens, one soluble and one insoluble in the detergent. Immunoprecipitates of [125I5- and [35S]methionine-labeled, detergent-soluble antigens contained three major polypeptides of molecular weights of 350,000, 140,000, and 75,003 (on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and several minor components. These polypeptides were all specifically precipitated from four EBV-producer cell lines, P3HR-1, P3HR-1 NONO, B95-8, and 7744. They could not be precipitated from producer cell lines treated with phosphonoacetic acid, which inhibits late viral functions, nor could they be precipitated from nonproducer cell lines. The 350,000 and 75,000 molecular weight polypeptides bound to Ricin and lentil lectin columns; however, most of the 140,000 molecular weight material did not. A component of molecular weight 220,000 (prominent only in P3HR-1 NONO) was probably a degradation product of the 350,000 molecular weight polypeptide.