Growth of B95-8 cells and expression of Epstein-Barr virus lytic phase in serum-free medium.

Epstein-Barr virus (EBV)-transformed tamarin (Saguinus oedipus) cells (B95-8) were selected for growth in medium with reduced serum and then transferred to serum-free medium which consisted of RPMI 1640 supplemented with insulin, transferrin, and selenium. Serum-free cells in continuous passage for 1 year had a morphology, growth rate, and culture density which approached those of B95-8 cells grown with serum. The cells expressed virus-induced antigens, including the EBV-associated DNA polymerase. Cells exposed to EBV-inducing agents, n-butyric acid and phorbol 12-myristate-13-acetate, produced transforming virus with titers comparable to those of cultures grown with serum. These findings demonstrate that serum is neither required for the growth of B95-8 cells nor necessary for induction or full expression of the EBV lytic phase in these cells.     

Activation of the alternative complement pathway by EBV and the viral envelope glycoprotein, gp350

The EBV-producing B lymphoblastoid cell line B95-8 was found to efficiently activate the alternative C pathway whether assessed with Mg-EGTA-treated human serum or with mixtures of the purified proteins of the pathway (PAP). The ability of the cells to activate was markedly increased after stimulation of EBV replication by treatment of the cells with a phorbol ester, and decreased by treatment of the cells with a viral polymerase inhibitor. Alternative pathway activation was dependent on the presence of either properdin or EBV-immune IgG; the addition of either alone to the PAP led to the deposition of 200,000 C3 molecules/cell. The addition of both properdin and immune IgG to the PAP markedly increased C3 binding to a level of 800,000 molecules/cell. Several lines of evidence indicate that the major external glycoprotein of EBV, gp350, mediates alternative pathway activation by B95-8 cells. First, the ability to activate C positively correlated with gp350 expression on the surface of the EBV-producing cells and gp350- cells failed to activate; second, the anti-EBV antibody in immune human sera which enhanced activation specifically immunoprecipitated gp350 from membranes of B95-8 cells; third, a significant proportion of the C3 which became bound to the cells during activation was attached either to gp350 or to the anti-gp350 antibody found in immune human sera; and fourth, purified gp350, as well as EBV, efficiently activated the alternative pathway. These results indicate that gp350, an EBV envelope glycoprotein, is an efficient alternative pathway activator and its expression on cell membranes is associated with the ability to activate C.     

Inhibition of Epstein-Barr virus DNA synthesis and late gene expression by phosphonoacetic acid.

Growth of lymphoblastoid cells (B95-8, Raji) is not inhibited by the presence of 0.4 mM phosphonoacetic acid. The synthesis of Epstein-Barr virus (EBV) in the producer line B95-8 is completely inhibited, as shown by the total inhibition of viral capsid antigen synthesis. Early viral antigens are made normally in the presence of phosphonoacetic acid, but EBV DNA synthesis is blocked in cells entering the productive cycle. Nonproducer cells in the population replicate the resident EBV DNA by a mechanism that is resistant to phosphonoacetic acid. These results are consistant with the hypotheses that EBV DNA is replicated by two mechanisms, one in the noninduced cell and a different mechanism in the producer cell, and that prior replication of EBV DNA, probably by the second mode, is a prerequisite for late gene expression.     

Epstein-Barr virus-specific DNase activity in nonproducer Raji cells after treatment with 12-o-tetradecanoylphorbol-13-acetate and sodium butyrate.

An Epstein-Barr virus (EBV)-specific DNase was induced in EBV nonproducer Raji cells after treatment with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate. The increase in EBV DNase activity was related to the appearance of early antigen-positive cells. The enzyme had a sedimentation coefficient of 4S and was resistant to 300 mM KCl, and its induction did not depend on viral DNA synthesis. The EBV-specific DNase activity was specifically inhibited by sera from patients who had nasopharyngeal carcinoma with high early antigen activities but not by sera from normal, healthy individuals. There was a correlation between the degree of anti-EBV DNase activity and the titers of early antigen antibody.     

EB病毒胸苷激酶基因的扩增

ＥＢ病毒胸苷激酶基因的扩增陈尚武,陈瑞君,黄迪,朱振宇,马涧泉（中山医科大学生化教研室,广州５１００８９）（中山医科大学肿瘤研究所,广州５１００６０）关键词Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒,胸苷激酶,基因,聚合酶链反应鼻咽癌（ｎａｓｏｐｈａｒｙｎｇｅａｌ...     

Purification and properties of a DNA primase from Nicotianatabacum

A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase α-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase. Received: 8 May 1997 / Accepted: 5 June 1997     

A 3' to 5' exonuclease activity is associated with phage 029 DNA polymerase

Bacteriophage 029 produces its own DNA polymerase which is encoded by gene 2 [Watabe, K. and Ito, J. (1983) Nucleic Acid Res. 11, 8333]. This 029 DNA polymerase has been purified by phospho-cellulose, DEAE-cellulose, double-stranded DNA cellulose chromatography and glycerol gradient centrifugation. An exonuclease activity associated with the DNA polymerase was found through all the steps of the purification. This nuclease preferably degrades single-stranded DNA from the 3' to the 5' terminus direction, suggesting that the enzyme plays a role for proofreading during DNA replication. While DNA polymerase activity isolated from cells infected with temperature sensitive mutant of gene 2 is thermolabile, the nuclease activity is not significantly reduced at the restrictive temperature.     

Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity.

The role of Epstein-Barr virus (EBV) early antigen diffuse component (EA-D) and its relationship with EBV DNA polymerase in EBV genome-carrying cells are unclear, EBV-specified DNA polymerase was purified in a sequential manner from Raji cells treated with phorbol-12,13-dibutyrate and n-butyrate by phosphocellulose, DEAE-cellulose, double-stranded DNA-cellulose, and blue Sepharose column chromatography. Four polypeptides with molecular masses of 110,000, 100,000, 55,000, and 49,000 daltons were found to be associated with EBV-specified DNA polymerase activity. A monoclonal antibody which could neutralize the EBV DNA polymerase activity was prepared and found to recognize 55,000- and 49,000-dalton polypeptides. An EA-D monoclonal antibody, R3 (G. R. Pearson, V. Vorman, B. Chase, T. Sculley, M. Hummel, and E. Kieff, J. Virol. 47:183-201, 1983), was also able to recognize these same two polypeptides associated with EBV DNA polymerase activity. It was concluded that EBV EA-D polypeptides, as identified by R3 monoclonal antibody, are critical components of EBV DNA polymerase.     

Inhibition of Epstein-Barr virus (EBV) release from P3HR-1 and B95-8 cell lines by monoclonal antibodies to EBV membrane antigen gp350/220.

Antibody-mediated inhibition of Epstein-Barr virus (EBV) release from the EBV-productive cell lines P3HR-1 and B95-8 was probed with two monoclonal antibodies (MAbs), 72A1 and 2L10, which immunoprecipitated the same EBV membrane antigen (MA) gp350/220 found with the 1B6 MAb with which inhibition of EBV release from P3HR-1 cells was first described. These three MAbs were not equivalent in either MA reactivities or functional effects, reflecting the variable expression of different epitopes of gp350/220. 1B6 recognized MA on P3HR-1 cells, which expressed predominately the gp220 form of MA. 1B6 did not recognize (or barely recognized) a determinant on B95-8 cells. MAbs 2L10 and 72A1 reacted as well with B95-8 cells as they did with P3HR-1 cells. MAbs 1B6 and 2L10 neutralized neither P3HR-1 nor B95-8 virus, but 72A1 neutralized both viruses. MAbs 1B6 and 72A1 inhibited P3HR-1 virus release, as measured by the assay for infectious virus and by DNA hybridization analysis of released virus, but 2L10 had no such activity. 72A1 (but not 1B6) inhibited release of EBV from B95-8 cells. These experiments pointed to the presence of three different epitopes on gp350/220, identified with the respective MAbs and having varying involvement in virus neutralization and virus release inhibition.     

Incorporation of the purified Epstein Barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions

The 145-kDa molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into 14C-phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti-CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3d. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

Immunoaffinity purification and properties of Drosophila melanogaster DNA polymerase alpha-primase complex

Two hybrid cell lines (DM88-5E12 and DM88-4C9) secreting monoclonal antibodies against DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells were established by immunizing mice with the complex partially purified by a conventional method. The IgG subclasses of both antibodies were IgG1. Both antibodies immunoprecipitated the DNA polymerase alpha-primase complex from D. melanogaster Kc cells. The DNA-polymerizing activity was neutralized by 4C9 antibody, but not by 5E12 antibody. The DNA priming activity was not neutralized by either antibody. These antibodies did not cross-react to HeLa DNA polymerase alpha-primase complex. A rapid, two-step purification of DNA polymerase alpha-primase complex from D. melanogaster Kc cell was carried out by 5E12 antibody column chromatography followed by single-stranded DNA cellulose column chromatography. The immunoaffinity-purified enzyme had both DNA-polymerizing and DNA-priming activities with the specific activities of 50,000 and 2,000 units/mg, respectively. The effects of aphidicolin, NEM, ddTTP, BuPdGTP, and DMSO on the enzyme activity showed that the purified enzyme was DNA polymerase alpha, but not DNA polymerase beta, gamma, or delta. The purified enzyme consisted of polypeptides with apparent molecular weights of 180 (and 145, 140, 130 kDa), 72, 63, 51, and 49 kDa. The 5E12 antibody was shown to bind to all the high-molecular-weight polypeptides, 180, 145, 140, and 130 kDa, by immuno-Western blotting analysis.     

DNA of Epstein-Barr Virus. II. Comparison of the Molecular Weights of Restriction Endonuclease Fragments of the DNA of Epstein-Barr Virus Strains and Identification of End Fragments of the B95-8 Strain

Incubation of the DNA of the B95-8 strain of Epstein-Barr virus [EBV (B95-8) DNA] with EcoRI, Hsu I, Sal I, or Kpn I restriction endonuclease yielded 8 to 15 fragments separable on 0.4% agarose gels and ranging in molecular weight from less than 1 to more than 30 x 10(6). Bam I and Bgl II yielded fragments smaller than 11 x 10(6). Preincubation of EBV (B95-8) DNA with lambda exonuclease resulted in a decrease in the Hsu I A and Sal I A and D fragments, indicating that these fragments are positioned near termini. The electrophoretic profiles of the fragments produced by cleavage of the DNA of the B95-8, HR-1, and Jijoye strains of EBV were each distinctive. The molecular weights of some EcoRI, Hsu I, and Sal I fragments from the DNA of the HR-1 strain of EBV [EBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were identical to that of fragments produced by cleavage of EBV (B95-8) DNA with the same enzyme, whereas others were unique to each strain. Some Hsu I, EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV (B95-8) DNA were present in half-molar abundance relative to the majority of the fragments. In these instances, the sum of the molecular weights of the fragments was in excess of 10(8), the known molecular weight of EBV (HR-1) and (B95-8) DNA. The simplest interpretation of this finding is that each EBV (HR-1), and possibly also (B95-8), DNA preparation contains two populations of DNA molecules that differ in the arrangement of DNA sequences about a single point, such as has been described for herpes simplex virus DNA. Minor fragments could also be observed if there were more than one difference in primary structure of the DNAs. The data do not exclude more extensive heterogeneity in primary structure of the DNA of the HR-1 strain. However, the observation that the relative molar abundance of major and minor fragments of EBV (HR-1) DNA did not vary between preparations from cultures that had been maintained separately for several years favors the former hypothesis over the latter.     

Identification and characterization of a DNase induced by Epstein-Barr virus.

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoyl-phorbol-13-acetate, induced a DNase activity in the Epstein-Barr virus-producer cell line P3HR-1. The elution patterns of the enzyme from DEAE-cellulose, phosphocellulose, and DNA-cellulose columns were different from virus-associated DNA polymerase activity. The partially purified activity could be neutralized to the extent of 90% by sera of patients with nasopharyngeal carcinoma. Purified immunoglobulin G from sera of nasopharyngeal carcinoma patients inhibited this enzyme and that obtained from superinfected Raji cells to the same extent. The partially purified enzyme preferred native DNA as a substrate over denatured DNA and 3'-terminally labeled activated calf thymus DNA. The activity was inhibited by high ionic strength. Phosphonoformic acid did not have any effect on this enzyme activity.     

Rapid purification of overexpressed T4 DNA polymerase

A method for purifying T4 DNA polymerase from cells harboring overexpression plasmids is described. T4 DNA polymerase is precipitated from induced, lysed cells with polyethyleneimine, then extracted and fractionated further with (NH4)2SO4 before chromatography on a column of single-stranded DNA cellulose. This procedure can be completed in three days and consistently provides enzyme preparations which are at least 98% pure. When necessary, one further chromatography step provides T4 DNA polymerase suitable for recombinant DNA applications.     

DNA Polymerase in the Core of the Human Hepatitis B Virus Candidate

Experiments were done to show that the human hepatitis B antigen (HBAg)-associated DNA polymerase is a component of Dane particles and their antigenically distinct cores prepared by Nonidet P-40 detergent treatment of Dane particles. Before detergent treatment, the DNA polymerase was precipitated by serum containing anti-HB surface antigen (anti-HB(s)) but not with serum containing anti-HB core antigen (anti-HB(c)). After detergent treatment, the enzyme was precipitated by anti-HB(c)- and not by anti-HB(s)-containing serum. Highly purified 16- to 25-nm HBAg particles blocked only the precipitation of DNA polymerase in untreated HBAg preparations. The 110S structure with which the DNA reaction product remains associated in Nonidet P-40-treated preparations was identified as Dane particle core by immunoprecipitation with serum containing anti-HB(c). The DNA polymerase and the radioactive DNA reaction product were used as markers for core in immunoprecipitation tests for anticore. In such assays, 8 of 11 human sera with anti-HB(s) activity and all of 10 sera from chronic HBAg carriers were found to contain anti-HB(c) activity.