Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.

The Eptstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solubilized from isolate nuclei of two EBV-transformed cell lines- Raji and AW-Ramos, by high-salt treatment. Its DNA-binding properties were studied by DNA-cellulose chromatography and a 51Cr release complement fixation assay. EBNA binds to both double-stranded and single-stranded calf thymus DNA, showing a higher affinity to double-stranded DNA. There was no detectable difference in the DNA binding of EBNA prepared from Raji and AW-Ramos cells.     

Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified 700-fold to apparent homogeneity from Raji and Namalwa cell extracts by a three-step procedure involving heat treatment, DNA-cellulose chromatography, and hydroxyapatite chromatography. Acid-fixed nuclear binding and complement fixation were used to monitor antigenic specificity. Purified EBNA was also capable of specifically inhibiting the regular anticomplement immunofluorescence reaction for EBNA against Raji target cells. The purified antigen had a molecular weight of 170,000 to 200,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it yielded a single 48,000-dalton (48K) monomer. An EBNA-associated protein was also purified from the same cell extract. It had a molecular weight of about 200,000 and yielded a single 53K protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same protein was also found in Epstein-Barr virus negative B-cell lymphoma lines. The two types of protein were characterized by amino acid composition and peptide mapping. The results showed that the 53K and 48K protein components have no long regions in common; this excludes that the smaller product arises by breakdown of the larger product. Residue distributions were different, but an excess of hydrophilic residues was found in both proteins, suggesting a certain overall similarity in properties. 53K components from different cell lines appeared to differ somewhat. Epstein-Barr virus-positive lines carry two 53K components, one of which may be a slightly modified 53K product. Immunocomplexing assay showed that the 48K, but not the 53K, protein carries EBNA specificity. In mixtures, the 53K protein is co-precipitated with the 48K protein. The data suggest that EBNA may form a complex with the 53K proten within the cell.     

Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.     

Partial purification of the Epstein-Barr virus nuclear antigen(s)

The Epstein-Barr virus nuclear antigen (EBNA) is speculated to be involved in cell transformation by the virus. Studies on the molecular properties of EBNA, however, have yielded conflicting results. In this study, three Epstein-Barr virus(EBV)-induced antigens were isolated and purified from extracts prepared from Raji cells. These antigens were able to block the anticomplement immunofluorescence reaction, indicating that all three were related to EBNA. The soluble antigen was found wholly in the cytosol fraction. An EBV-induced nuclear antigen I was found both in the cytosol and the nucleus. The EBV-induced nuclear antigen II was found associated with the chromatin. The soluble antigen and the nuclear antigen I were separated and partially purified using phosphocellulose chromatography. Each was further purified 1,400-fold with respect to the whole cell state by chromatography on CL-Sepharose 6B followed by blue dextran-Sepharose. subunit molecular weights of 70,000 were determined for each of these antigens, both in the crude and purified state, by radioimmunoelectrophoresis and gel filtration. The nuclear antigen II was purified 2,500-fold using hydroxylapatite, CL-Sepharose 6B, and blue dextran-Sepharose chromatographies. This antigen displayed two subunits by radioimmunoelectrophoresis with molecular weights of 65,000 and 70,000. Although all antigens shared similar molecular weights, the extent of their homology remains to be determined.     

Identification and characterization of a cellular protein that cross-reacts with the Epstein-Barr virus nuclear antigen.

A 62,000-dalton (62K) cell protein reacts with antisera to the 72K polypeptide of the Epstein-Barr virus nuclear antigen (EBNA) in immunoblots. This protein was initially detected in EBNA-negative as well as EBNA-positive cell lines with anti-EBNA-positive human sera. A monoclonal antibody raised against the 72K EBNA and an antiserum from a rabbit immunized with the glycine-alanine domain of EBNA also reacted with the cellular protein. The cellular protein was partially purified from Epstein-Barr virus genome-positive and -negative cell lines. Absorption experiments identified a shared antigenic determinant between the 72K EBNA and 62K cellular protein. A comparison of the 62K protein and EBNA by protease digestion did not reveal similar peptides.     

The binding of simian virus 40 large T antigen to the polyphosphate backbone of nucleic acids

Simian virus 40 (SV40) large tumor antigen (T antigen), a phosphoprotein found in nuclei of SV40-infected and -transformed cells, binds nonspecifically to DNA. To study this mechanism the binding properties of T antigen to double-stranded (ds) and single-stranded (ss) DNA-cellulose as well as to phosphocellulose were compared. After incubation of [35S] methionine or [3H] leucine/[32 P] phosphate radioactively-labeled cell extracts at different pH values (6.0, 7.3, 9.0) with DNA- or phosphocellulose, bound and unbound species of T antigen were purified and analyzed by SDS-polyacrylamide gel electrophoresis for both the yield and the possible correlation with protein phosphorylation. T antigens bound with comparable affinities to ds- and ss-DNA-cellulose and phosphocellulose. These results suggest the binding of T antigen to the polyphosphate backbone of DNA as a molecular mechanism for its nonspecific binding. The evidence for this observation was supported by blocking the binding of T antigen to DNA-cellulose by divalent cations (Ca2+, Mg2+). 3H/32P ratios of T antigen obtained by double-labeling cells for various times imply that higher phosphorylated forms of T antigen bound more strongly to ds- and ss-DNA as well as to phosphocellulose. Thus, in the presence of cellular proteins and other components the binding activity of T antigen to the polyphosphate backbone of DNA seems to be positively correlated with its phosphorylation. These observations are consistent with the hypothesis that the binding affinities of SV40 T antigen to host cell DNA may be regulated by its phosphorylation.     

Epstein-Barr virus (EBV) antigenic determinants on subunits of the EBV determined nuclear antigen (EBNA)

In order to characterize the substructure of the Epstein-Barr virus determined nuclear antigen (EBNA) which is considered to have a molecular weight of 180 K in its native form, we have examined the antigenic specificity of the polypeptides obtained after denaturation of this molecule. Two procedures were employed; treatment by sodium dodecyl sulfate (SDS) and heat followed by gel electrophoresis, or denaturation by guanidine hydrochloride followed by gel filtration, which allowed us to detect a specific antigenic activity in the 50 K region, following dialysis. The denatured molecules could be reassociated into larger molecules (50 to 180 K) which retain the property of binding to fixed nuclei, as does native EBNA. These results indicate that EBNA has a polymeric structure and that 50 K subunits carry the antigenic determinants.     

Mitochondria contain a distinct DNA topoisomerase.

A topoisomerase (nicking-closing enzyme) has been isolated from rat liver mitochondria. It has purified by double-stranded DNA-cellulose chromatography approximately 50,000-fold, based on the crude mitochondrial extract. It possesses a minimum specific activity of 1.9 x 10(5) units/mg. The enzyme has been shown to be distinctly mitochondrial, differentiated from the nuclear topoisomerase by its sensitivity to the intercalating drug, ethidium bromide, and to the non-intercalating trypanocidal drug, Berenil.     

Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.     

Biochemical characterization of Epstein-Barr virus nuclear antigen 2A and an associated ATPase activity.

A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5'-[p-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 microM and 11 microM, respectively, and the Vmax for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 microM, respectively. Phosphotyrosine had a Ki of 480 microM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.     

On the DNA polymerase III of mouse myeloma: partial purification and characterization.

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...     

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.     

ATP phosphohydrolase (ATPase) activity of a polyoma virus T antigen

Among the various polyoma virus T antigens which have so far been identified, only the large-T and a 63 000-Mr polypeptide were found to bind to double-stranded calf thymus DNA. The proteins were not retained on single-stranded DNA-cellulose columns, and a purification procedure was designed on the basis of this observation. Purified fractions (approx. 1000-fold) exhibited an enzymatic activity which converts ATP into ADP and Pi. This activity was quantitatively inhibited after preincubation in the presence of anti-(polyoma T antigen) immunoglobulins and was shown to be dependent on a virus-coded gene product (alpha gene) on the basis of the following observations: (a) ATPase activity from cells infected with tsa mutants of polyoma was reduced after a shift to the restrictive temperature; (b) the enzyme purified from tsa-infected cells maintained at the permissive temperature was more thermolabile in vitro than that prepared in parallel from cells infected with wild-type virus.     

Large-scale Epstein-Barr virus EBNA1 protein purification

The protein–DNA and protein–protein interactions of Epstein–Barr virus nuclear antigen 1 (EBNA1) are known to play an important role in the many functions of this viral protein. Large quantities of pure EBNA1 protein would be useful in biochemical assays to elucidate such interactions. In particular, the crystal structure of the full-length protein would be important to show possible regions of interaction and/or post-translational modification. Recently, we described a novel approach to overexpress and purify EBNA1 from Escherichia coli; however, it is not ideal for large-scale production of EBNA1. We were able to optimize this protocol by (1) adding a polyethyleneimine precipitation step prior to Ni–NTA chromatography to reduce complexity of the sample and remove nucleic acid, (2) optimizing the Ni–NTA gradient to further separate EBNA1 from impurities, and (3) concluding with a MonoS cation-exchange chromatography step to further purify and concentrate EBNA1. We were able to recover 10-mg quantities of pure EBNA1 protein.     

The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion

Background

Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known.

Methodology/Principal Findings

Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing ∼120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation.

Significance

Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore, production of EBNA1-derived DRiPs is down-regulated in cells, possibly limiting the antigenicity of EBNA1.     

Antigenic structure of double-stranded RNA analogues having varying activity in interferon induction.

Antibodies were induced by immunization of rabbits with methylated bovine serum albumin complexes of: poly(I).poly(BC), an effective interferon inducer; poly(c7A).poly(rT), a noninducer that can block induction by active poly(A).poly(rT); and poly(A).poly(Um), which has neither inducing nor blocking activity. Similar complexes of f2 phage RNA or tRNA did not induce anti-nucleic acid antibodies. Each anti-polynucleotide serum contained some antibodies specific for double-stranded structure. Antibodies were immunospecifically purified from precipitates made with each serum and homologous or cross-reacting double-stranded polynucleotides. The purified antibodies distinguished among varying helices bearing base or ribose modifications. Antipoly(I).poly(BC) specificity paralleled that of the interferon induction system. Anti-poly(A).poly(Um) specificity favored the 2'-modified polymers. Anti-poly(c7A).poly(rT) antibodies were the least discriminating. Cross-reaction results indicated that some antibodies reacted with determinants that included both sugar-phosphate backbones. In far antibody excess, antigen:antibody ratios in precipitating complexes reached a minimum of 7 to 12 base pairs per bivalent IgG molecule. Single antigenic determinants may span about 4 base pairs, with primary contact sites including the phosphate groups and the furanose.     

Antigens and autophagy: the path less travelled?

The Epstein-Barr virus (EBV)-coded nuclear antigen (EBNA) 1, a latent cycle protein endogenously expressed in EBV-transformed B lymphoblastoid cell lines (LCLs), is reported to be processed for CD4(+) T cell recognition by an intracellular route involving antigen delivery to the endosome/lyosome (MHC class II loading) compartment via macroautophagy. In contrast we find that, in the same cell type, two other virus-coded nuclear proteins of the latent cycle, EBNA2 and EBNA3C, are processed by a different route that is unaffected by autophagy inhibition. This involves the intercellular transfer of an antigenic moiety, detectable in cell-free culture supernatants, and its uptake and processing as exogenous antigen by neighboring cells. The process is cumulative and leads over several days of LCL culture to high levels of CD4+ T cell epitope display. The presentation of certain EBV lytic cycle proteins to CD4+ T cells has also recently been found to involve a similar intercellular antigen transfer. It becomes important to know why, even in the same cell type, some antigens but not others appear to access the MHC class II presentation pathway by autophagy.     

Purification and characterization of the DNA-binding activity of the Epstein-Barr virus DNA polymerase accessory protein BMRF1 gene products, as expressed in insect cells by using the baculovirus system.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) BMRF1 gene product, the EBV DNA polymerase accessory protein, under the control of the polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced two phosphoproteins of 52 and 50 kDa and one unphosphorylated protein of 48 kDa, recognized by anti-BMRF1 protein-specific monoclonal antibody. The major protein bands were 50 and 48 kDa. The expressed BMRF1 gene products were purified to near homogeneity from the nuclear extract of the recombinant baculovirus-infected insect cells by double-stranded DNA-cellulose column chromatography followed by heparin-agarose column chromatography. The purified BMRF1 gene products exhibited higher binding affinity for double-stranded DNA than for single-stranded DNA without ATP hydrolysis. The protein-DNA interaction did not necessarily require a primer terminus. The present system will open the way for the biochemical characterization of the EBV DNA polymerase accessory protein.     

Identification of an Epstein-Barr virus nuclear antigen by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis.

A 65,000-dalton (65K) antigen found in Raji cells by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis has been identified as an Epstein-Barr virus nuclear antigen (EBNA). This identification is based on the following evidence. The 65K antigen is detected in Raji cells but not in three Epstein-Barr virus (-) human B cell lines. It is not detected with EBNA (-) sera. The 65K antigen is found predominantly in the nucleus and co-elutes with EBNA during partial purification by DNA-Sepharose and Blue Dextran-Sepharose chromatography. Finally, the partially purified 65K antigen is an effective absorbant of EBNA antibody as measured in an anticomplement immunofluorescence assay. Antigens with molecular weights of 72, 70, and 73K have been detected in B95-8, P3HR-1, and Namalwa cells, respectively. These antigens are the likely homologues of the 65K Raji EBNA. In addition, an Epstein-Barr virus-associated, 81K DNA-binding antigen has been detected in both B95-8 and Raji cells.     

Bioreactor-scale production and one-step purification of Epstein-Barr nuclear antigen 1 expressed in baculovirus-infected insect cells

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated malignancies and is essential for EBV-genome maintenance. Antibodies to EBNA1 are abundantly detected in serum of most EBV carriers but EBNA1 escapes recognition by effector T-lymphocytes. To further study the functional and immunological characteristics of EBNA1 it is important to have sufficient quantities of purified EBNA1 available. This paper describes a simple, reproducible method for the production and purification of EBV-encoded EBNA1 expressed in insect cells (bEBNA1). For quantification of EBNA1 expression levels in cell lines and for monitoring bEBNA1 purification and overall yields we developed a quantitative and EBNA1-specific capture ELISA. We observed that EBV-positive cell lines express EBNA1 at different levels, with the B cell lymphoblastoid cell line X50/7 having the highest production. However, much larger quantities (380-fold) were obtained by expressing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells. Scaling-up experiments revealed that bEBNA1 expression kinetics and protein stability are identical in 1-liter stirred bioreactors when compared to expression in stationary culture flasks. Optimal expression was reached after 72 h following inoculation at 1 pfu/cell, when insect cell viability was about 50%. For purification the nuclear fraction containing most of the bEBNA1 (>95%) was isolated. Solubilized bEBNA1 was purified by a one-step oriP DNA-Sepharose affinity purification procedure, using biotinylated PCR-amplified family of repeats (FR)-domain products immobilized onto streptavidin agarose. A >200-fold specific enrichment was reached and yields of bEBNA1 with an estimated purity of >95%.