Analysis of early and late Epstein-Barr virus associated polypeptides by immunoprecipitation.

The Epstein-Barr virus-producing cell lines P3HR-1 and B95-8 and the nonproducer cell lines Raji clone No. 7 and NC37 were induced to viral antigen synthesis by the tumor promoter TPA and then analyzed by immunoprecipitation with human sera for early and late virus-associated polypeptides. After labeling of producer cells for a 4-day period with [³⁵S]methionine, two polypeptides with molecular weights of 140,000 and 150,000 were identified reacting predominately with virus capsid antigen (VCA⁺) sera. Analysis of purified Epstein-Barr virus demonstrated that the 140,000 polypeptide presumably represents an envelope protein while the 150,000 polypeptide is a nucleocapsid protein. In 4-hr radioactively labeled producer cells an additional polypeptide with a molecular weight of 130,000 was found to be immunoreactive with VCA⁺ sera. Immunoprecipitation of [³⁵S]methionine-labeled cell extracts from nonproducer cells resulted in the specific precipitation of two polypeptides with molecular weights of 85,000 and 35,000 which most likely represent early EBV-associated proteins. Producer cells exhibit three additional apparently early EBV-associated polypeptides with molecular weights of 120,000, 18,000, and 16,000. None of these polypeptides could be detected in EBV genome-negative Ramos cells after TPA treatment.     

A transfection assay for transformation by feline sarcoma virus proviral DNA

We report that fibroblast lines derived from mouse (NIH 3T3) and mink (CCL-64) can be transformed via transfection with the total cellular DNA extracted from cells transformed with feline sarcoma virus. Replication of helper virus is not required in the recipient cell. Transformation of recipient cells is observed when the donor DNA is extracted from either virus producer or nonproducer transformed cell lines. A transforming virus can be rescued from nonproducer transformed recipients upon superinfection with a replication competent helper virus.     

Necessity for two human chromosomes for human chorionic gonadotropin production in human-mouse hybrids

Through a series of human-mouse hybrids we have identified that two human chromosomes, 10 and 18, must be present for production of the pregnancy protein hormone human chorionic gonadotropin (hCG). Human choriocarcinoma cells producing hCG were hybridized to mouse cells. From 49 independent clones three hybrid clones continued to produce whole hCG. Chromosomal analysis was done on the 3 producer clones and 5 nonproducer clones. The additional 41 nonproducer clones were genetically characterized by isozymes. Only when chromosomes 10 and 18 were present in a clone would the whole hCG molecule be produced. Clones with only 10 or only 18 did not produce hormone. Nine subclones of a producer clone confirmed this observation. Three subclones retaining both 10 and 18 continued to produce hCG. This study demonstrated the need to use cellular chromosome data and population enzyme data to identify two chromosomes necessary for hCG production in heterogeneous human-mouse hybrids.     

Effect of n-butyrate on cellular and viral DNA synthesis in cells latently infected with Epstein-Barr virus

Cellular DNA synthesis and Epstein-Barr virus (EBV)-specific DNA synthesis was examined after n-butyrate treatment of the producer line P3HR-1. Butyrate was found to effectively inhibit cellular DNA synthesis. This was followed by activation of the viral cycle as seen by marked increase in the expression of the EBV early antigen (EA), viral capsid antigen, and EBV-specific DNA synthesis. In spite of efficient induction of EA in the nonproducer Raji line, there was no increase in EBV-specific DNA synthesis above the level of the untreated control. It is likely that the cellular inhibitory effects of n-butyrate are responsible for the activation of EBV in producer lines and that different cellular controls, not affected by n-butyrate, are responsible for keeping the virus latent in nonproducer cells.     

Identification of a 39,000-dalton protein in cells transformed by the FBJ murine osteosarcoma virus

Two components of the FBJ murine osteosarcoma virus complex have been isolated separately in tissue culture; the FBJ murine leukemia virus (FBJ-MLV) by dilution and the FBJ murine sarcoma virus (FBJ-MSV) by the establishment of nonproducer transformed rat cells. Analysis of these cells using MLV antisera indicated that there were no new proteins related to viral structural proteins specifically associated with the presence of the FBJ-MSV genome. The FBJ-MSV nonproducer cells were used to induce tumors in syngeneic and allogeneic F₁ rats. Sera from tumor-bearing rats were examined for activity against FBJ-MSV-specific antigens. A number of sera were found to precipitate a 39,000-dalton protein, p39, from several producer and nonproducer FBJ-MSV transformed rodent cells, but not from cells transformed by other strains of MSV or cells infected with MLV. Precipitation of p39 was not blocked by the presence of excess viral proteins, indicating that p39 is not related to the viral structural proteins. This conclusion was confirmed by methionine tryptic peptide analysis which showed that the fingerprint of p39 was distinct from those of the viral gag or env gene proteins. The data demonstrate the presence of a unique antigenic protein, unrelated to the MLV proteins, in FBJ-MSV transformed cells.     

A non-virion surface antigen on Moloney sarcoma virus-transformed non-producer and producer cells

Sera from STU mice bearing sarcomas induced by cells producing the Moloney sarcoma-helper-virus (M-MSV/MLV) complex were cytotoxic for these cells as well as for M-MSV non-producer and M-MLV producer cells. Analysis by polyacrylamide gel electrophoresis of 125I-labelled surface antigens immunoprecipitated with such sera revealed the virus envelope glycoprotein gp71 on the producer cells and an additional antigen of mol. wt 55 K on the M-MSV-transformed producer and non-producer cells. This antigen was not found on non-transformed M-MLV-producing cells and was neither related serologically to structural polypeptides of murine C-type viruses nor to components of embryonal STU mouse fibroblasts and foetal bovine serum.     

Epstein-Barr virus-induced proteins. III. Analysis of polypeptides from P3HR-I-EBV-superinfected NC37 cells by immunoprecipitation

P3HR-I-EBV induces in superinfected nonproducer NC37 cells at least 13 [³⁵S]methionine-labeled polypeptides with molecular weights between 18,000 and 150,000, which were identified by immunoprecipitation using human sera. Application of Ara-C inhibited the synthesis of the two structural polypeptides p150 and p140 while the synthesis of the other polypeptides was not affected.     

Effect of the diterpene ester TPA on Epstein-Barr virus antigen- and DNA synthesis in producer and nonproducer cell lines

Diterpene esters like TPA (12-O-tetradecanoylphorbol-13-acetate) which are effective tumor promoters in mice, induce the synthesis of Epstein-Barr virus capsid antigen (VCA) in producer lines and of the early antigen complex (EA) in nonproducer lines. In three lymphoblastoid lines which produce nontransforming (P3HR-1) or transforming virus (B95-8, QIMR-WIL) and in two nonproducer lines (Raji, NC 37) the role of TPA on viral DNA synthesis was studied. At different times after addition of TPA the percentage of VCA- and EA-positive cells was determined and the amount of viral DNA per cell estimated by measuring the reassociation kinetics of labeled EV DNA in the presence of cellular DNA. In virus-producing lines the number of VCA-positive cells increased about 3- to 5-fold from 7–10% to 30–40%, whereas the amount of viral DNA per cell increased 10- to 20-fold. Thus, TPA not only increases the number of viral antigen-producing cells, but also enhances viral DNA replication. In nonproducer lines the amount of EBV DNA per cell was constant despite efficient EA induction by TPA. This indicates that the induction of EA is not linked to the replication of viral DNA.     

Differential effect of phosphonoacetic acid on early antigen synthesis in two Epstein-Barr virus producer cell lines.

The effect of phosphonoacetic acid (PAA) on the expression of Epstein-Barr virus (EBV)-determined antigens in producer (P3HR-1 and B95-8) lymphoblastoid cell lines (LCLs) and iododeoxyuridine (IUDR)-treated nonproducer cell lines (Raji and F137) was investigated. PAA effectively inhibited spontaneous early antigen (EA) synthesis in the P3HR-1 LCL only, while it had no effect on EA production in the B95-8 cell line nor on the IUDR-induced EA in nonproducer LCLs. The inhibition of spontaneous EA in P3HR-1 cell was dose and time dependant, and removal of PAA from cultures and subsequent propagation of these cells in PAA-free medium did not restore spontaneous EA production. The data reported show that EA is synthesized by at least two mechanisms in P3HR-1 cells, one of which is sensitive to PAA, thus suggesting that viral DNA synthesis is required for spontaneous EA synthesis only in the P3HR-1 cells; however, in these same cells, viral DNA synthesis would not be required for the IUDR-induced EA production.     

Programming of events in Epstein-Barr virus-activated cells induced by 5-iododeoxyuridine

Programming of the Epstein-Barr (EB) virus productive cycle was studied in synchronized nonproducer Raji cells and producer EB-3 cells after activation by 5-iododeoxy uridine (IdU) added for 60 min during the S-1 period of the cell cycle. The activated Raji cells synthesized only the virus-associated early antigen (EA) complex, while some activated EB-3 cells also synthesized virus structural antigens (VCA).Synthesis of EA occurred at a time which corresponded to the early G-2 period of the cell cycle. DNA synthesis after removal of the IdU was not required for subsequent synthesis of EA. The time of EA synthesis, including associated RNA and protein requirements, was the same in cells allowed to complete the S phase as in cells where DNA synthesis was inhibited by 1-B-d-arabino furanosylcytosine (ara-C) or hydroxyurea (HU) added at the time of removal of the IdU. The findings indicated that synthesis of EA was independent of the cells' S phase and suggested a possible association between EA synthesis and synthesis of cell G-2 proteins.Synthesis of EA in abortively infected Raji cells was followed by low levels of deoxycytidine (dCyt) incorporation which persisted for several hours. At least some EA-positive cells were also able to reach mitosis, indicating that synthesis of EA did not result in immediate cell death.Synthesis of VCA occurred 3–4 hr after synthesis of EA in approximately 50% of the activated EB-3 cells. Synthesis of VCA required a short period of DNA synthesis (approximately 60 min) after EA synthesis. The inability of approximately 50% of the activated EB-3 cells to synthesize VCA was ascribed to toxic effects of the incorporated IdU which precluded the cells from carrying out events required for initiation of the short period of DNA synthesis subsequent to completion of the S phase and EA synthesis. Those EB-3 cells which did progress to VCA synthesis showed productive replication of virus DNA which continued for several hours after the initial appearance of VCA.     

Biochemical characterization of two Epstein-Barr virus early antigen-associated phosphopolypeptides

Epstein-Barr virus (EBV) nonproducer cells NC37 induced to viral early antigen (EA) synthesis by the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) were labeled at Day 4 after induction with 32P, and were analyzed by immunoprecipitation with human EA-positive sera. By employing this method the appearance of two phosphopolypeptides of 50 and 58K (pp50 and pp58) was well correlated with EA complex. Partial V8 protease digestion and two-dimensional peptide analysis revealed that the polypeptides pp50 and pp58 are related. The analysis of phosphoamino acids indicated that pp58 contains phosphoserine and phosphothreonine to the same percentage, whereas in pp50 only phosphoserine was found. The analysis of the subcellular distribution revealed that pp50 and pp58 are located in the chromatin. Both phosphopolypeptides exhibit DNA-binding activity, and are recognized by two monoclonal antibodies described recently (R3 and 1108-1).     

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.     

Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate.

n-Butyrate was found to increase the number of virus producer cells to a dramatic extent in the Epstein-Barr virus (EBV)-carrying P3HR-1 and B95-8 lines. Induction was also seen in the nonproducer Raji and the low producer Daudi lines, but at a mucch lower level. The virus containing supernatant of the butyrate treated P3HR-1 cells induced preferentially EBNA in EBV-negative Ramos target cells, whereas the spontaneously produced virus induced predominantly EA in Raji indicator cells. This suggests a possible difference in the biological properties of the butyrate induced vs the prototype virus. In addition to providing a convenient method to obtain a high yield of viral-DNA and virus antigen-producing cells in the severely restricted EBV system, the findings raise interesting questions on the mechanism of EBV induction, and its possible relationship to the known differentiation inducing ability of n-butyrate.     

Hepatitis B virus core antigen (HBc Ag) accumulation in an HBV nonproducer clone of HepG2-transfected cells is associated with cytopathic effect

Two clones of the hepatoblastoma HepG2 cell line transfected with complete hepatitis B virus deoxyribonucleic acid (HBV DNA) were studied. The kinetics and cytopathic effect of HBV Ag production in these two clones (one of which was an HBV producer) were compared to those of the parent HepG2 cell line. The presence of hepatitis B surface antigen (HBs Ag) and hepatitis B e antigen (HBe Ag) was determined by commercial enzyme-linked immunosorbent assay (ELISA). A hepatitis B core antigen (HBc Ag)-specific ELISA assay was developed, using monoclonal anti-HBc to detect HBc Ag. Amounts of HBs, HBe, and HBC Ags were partially quantified in both intracellular and extracellular compartments. The HBV producer clone excreted high levels of HBc, HBe, and HBs Ags from the beginning of the growth phase, and no cytopathic effect was observed. The HBV nonproducer clone produced high levels of HBs and HBe Ags, but there was no detectable HBc Ag in the supernatant; instead, HBc Ag accumulated in the intracellular compartment. In this clone, significant cell death was observed 4 days after cell confluency, corresponding with notable HBc Ag release into the supernatant. These results suggest a cytopathic effect associated with HBc Ag accumulation in the HBV nonproducer clone, but no cytopathic effect in the HBV producer clone. This suggests that virological factors as well as the host's immune response may be considered in explaining liver injury occurring in hepatitis B.     

Studies on human parainfluenza virus 3: characterization of the structural proteins and in vitro synthesized proteins coded by mRNAs isolated from infected cells

The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein-synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease.     

Preparation of autologous antiserum against SSV nonproducer cells and its partial characterization

In order to prepare an autologous antiserum against SSV nonproducer cells (SSV-NP cells), goat fibroblasts from a biopsy specimen were infected with SSV (SSAV) and transformed nonproducer clones were isolated. One of these clones was propagated in autologous goat serum and inoculated into the same goat. Characterization of the resulting antiserum by a surface specific binding assay showed specific reactivity with NRK-SSV nonproducer cells, which cannot be adsorbed by an SSAV virus resin. The data suggest that the serum recognizes cell surface molecules, which might be specific for transformation by SSV.     

Epstein-Barr virus-induced polypeptides: a comparative study with superinfected Raji, IUdR-Treated, and N-butyrate-treated P3HR-1 cells

Three Epstein-Barr virus (EBV)-induced in vitro systems, EBV-superinfected Raji, IUdR-, and n-butyrate-treated P3HR-1 cells, were studied using a radioimmunoprecipitation method, and the EBV-specific polypeptide profiles were analyzed with anti-EBV human sera. In the three cell systems, 16, 20, and 22 polypeptides were identified, respectively. All 16 polypeptides detectable in the EBV-infected Raji cells belonged to the early products insensitive to phosphonoacetic acid (PAA) and were also shared by the other two systems. Among these common polypeptides, two with molecular weights of 140,000 (140K) and 120K were identified both in the cytoplasm and in the nucleus. However, these are not the major components of EA induced in the EBV-infected Raji cells. All other 14 polypeptides were found exclusively in the cytoplasm and were precipitated by all VCA(+)EA(+) sera, indicating that all of these polypeptides are EA components and that 90K, 54K, and 38K in particular are the major constituents of the EA. The six polypeptides (150K, 145K, 75K, 46K, 34K, 18K), which were detectable in n-butyrate-treated P3HR-1 cells, and four of which are common to those in IUdR-treated P3HR-1 cells, are the late virus-specific products sensitive to PAA. In contrast to the early polypeptides, these late products were found both exclusively in the cytoplasm and the nucleus. Among these products the 150K polypeptide is probably the main component of VCA.     

Genetic analysis of the defectiveness in strain MC29 avian leukosis virus.

Avian myelocytomatosis virus MC29 is defective in replication. The extent of this defectiveness was analyzed in complementation experiments. Continuous nonproducer quail cell lines transformed by MC29 were superinfected with different helper viruses. Infectious MC29 pseudotypes were formed only with helper viruses which belonged to the avian leukosis-sarcoma virus complex, e.g., Rous-associated virus type 1 (RAV-1) or ring-necked pheasant virus (RPV). Helper viruses of other retroviral species, such as reticuloendotheliosis virus (REV), golden pheasant virus (GPV), or an amphotrophic murine leukemia virus (MuLV-1313), could not rescue MC29 from these nonproducer cells but complemented the envelope-defective Bryan high titer strain of Rous sarcoma virus. Host range and interference patterns of MC29 rescued from nonproducer cells indicated that the envelope specificity of the pseudotypes was determined exclusively by the activating helper virus. Cocultivation or Sendai virus-induced fusion of MC29 nonproducer cells with quail or chicken cells transformed by the defective Bryan high titer strain of Rous sarcoma virus did not result in complementation of the defects in either of the two viruses. Fluorescent antibody staining failed to detect virus-specific antigens at the surface of MC29 nonproducer cells. Temperature-sensitive mutants of Rous sarcoma virus with lesions in the genes coding for the RNA-directed DNA polymerase (pol gene) or the nonglycosylated structural proteins (gag gene) did not acquire wild-type characteristics when grown in MC29 nonproducer cells. It is concluded that MC29 is defective in all three genes essential for the replication of retroviruses, namely, env, pol, and gag.