A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein

We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate-induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex.     

Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells.

We have increased the copy number of Epstein-Barr virus vectors that also carry the origin of replication of simian virus 40 (SV40) by providing a transient dose of SV40 T antigen. T antigen was supplied in trans by transfection of a nonreplicating plasmid which expresses T antigen into cells carrying Epstein-Barr virus-SV40 vectors. A significant increase in vector copy number occurred over the next few days. We also observed a high frequency of intramolecular recombination when the vector carried a repeat segment in direct orientation, but not when the repeat was in inverted orientation or absent. Furthermore, by following the mutation frequency for a marker on the vector after induction of SV40 replication, it was determined that SV40 replication generates a detectable increase in the deletion frequency but no measurable increase in the frequency of point mutations.     

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.     

Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants

Summary SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are able to grow in BSC monkey kidney cells at 37° C, but give at least 10 000-fold lower yields than wild type virus in BSC cells at 32° C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product, a protein throught to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not seem to act by correcting the mutational block but rather increases the efficiency of plaque formation. This work was supported by grants CA40586 and BRSG 2S07RR07084-23 to J. M. P. and grant CA33079 to L. T., from the National Institutes of Health, Bethesda, MD.     

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.     

Expression of p40/Epstein-Barr virus nuclear antigen 1 binding protein 2

Nucleolar protein p40/EBP2 is a proliferation-associated antigen that interacts with Epstein-Barr virus nuclear antigen 1 (EBNA1) to maintain the Epstein-Barr virus (EBV) episomes. The yeast p40/EBP2 functions in the processing of 27S-A into 27S-B ribosomal RNA. The present study reports high evolutionary conservation of the cDNA-derived amino acid sequences of p40/EBP2 from frog, chicken, pig, rat, mouse, bovine, and human. p40/EBP2 is ubiquitously expressed in human tissues. It is highly expressed in myelogenous leukemia K-562 compared to other cell lines tested. The human p40/EBP2 gene is located in chromosome 1 with nine exons and eight introns. The minimal promoter region resides 300 nucleotides upstream of a putative ATG initiation codon preceded by a pyrimidine-rich region. These two regions contain eight Sp1 and four c-Ets-1 putative binding sites. Analysis of the p40/EBP2 gene and its promoter region will facilitate studies on the regulation of its expression in EBV-infected and noninfected cells.     

Presence in growth-arrested cells of cellular proteins that interact with simian virus 40 small-t antigen.

Two cellular proteins, 56K and 32K, found in association with simian virus 40 small-t antigen were not induced by viral infection. In addition, the proteins were expressed by cells in the growth arrest period, a time in which small-t function is of importance in infection and transformation.     

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.     

Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene.

Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function (hr/hf) mutant, dlA2475, delayed complementation was seen with the 6 of the 10 nonviable mutants. Of these 10 mutants, 5 formed plaques 3 to 4 days later than in control complementations, while complementation by one of the mutants, inA2827, with an insertion at amino acid 520, was delayed more than 1 week. Most mutants which showed delayed complementation replicated less well in Cos-1 cells than did a control mutant, dlA1209, which produced no T antigen. The replication of inA2827(aa520) was reduced by more than 90%. Similar interference with viral DNA replication was seen when CV-1, HeLa, or 293 cells were cotransfected with an origin-defective plasmid encoding wild-type large T antigen and with inA2827(aa520). Only one of the mutant T antigens, inA2807(aa303), was unstable. These results indicate that some of the mutant T antigens interfered with functions of wild-type T required for viral DNA replication. However, not all of the mutants which showed delayed complementation also showed interference with viral DNA replication. This indicates that mutant large T antigens may interfere trans dominantly with multiple activities of wild-type large T antigen.     

Functional interaction of nuclear transport-defective simian virus 40 large T antigen with chromatin and nuclear matrix.

We analyzed the subcellular distribution of nuclear transport-defective simian virus 40 Lys-128-mutant (cT-3 [R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984] and d10 [D. Kalderon, W. D. Richardson, A. F. Markham, and A. E. Smith, Nature (London) 311:33-38, 1984]) large T antigens in various Lys-128-mutant-transformed rodent cells and in Lys-128-mutant d10-infected TC7 cells. Small but significant amounts of the mutant large T antigens were found in association with nuclear substructures, both in mutant-transformed and in mutant-infected cells. Experiments with TC7 cells made incompetent for cell division by 60Co irradiation supported the assumption that Lys-128-mutant large T antigen did not associate with nuclear components during mitosis but most likely was transported into the nucleus because the Lys-128 mutation was leaky for nuclear transport. Low-level simian virus 40 DNA replication and production of infectious mutant virus progeny in TC7 cells indicated that the association of Lys-128-mutant large T antigen with nuclear substructures is functional.     

Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.     

Interaction of simian virus 40 small-T antigen produced in bacteria with 56K and 32K proteins of animal cells.

Small-t antigen produced in bacteria interacted with two animal cell proteins with molecular weights of 56,000 and 32,000, as did the viral antigen from infected cells. Demonstration of this specific interaction required the enrichment of native, monomeric small-t antigen from extracts in which much of the small-t antigen was highly aggregated.     

Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: studies with simian virus 40 double mutants.

Simian virus 40 mutants containing both a tsA mutation (rendering the 90,000 molecular weight [90K] T-antigen thermolabile) and a deletion between 0.54 and 0.59 map units (reducing the size and the amount of the 20K t-antigen) were used to transform Chinese hamster lung cells. The frequencies of transformation by the double mutants were comparable to that of the tsA mutant alone by both the focus and agar assays except when the cells were serum depleted before infection. Growth-arrested cells were transformed (using the agar assay) by the deletion mutants at less than 2% the frequency found when the 20K t-antigen was normal. Growth arrest had very little effect on the temperature sensitivity of the resultant transformed cell lines whether or not the deletion was present.     

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.     

Deletion mutations in the small t antigen gene alter the tissue specificity of tumors induced by simian virus 40.

Subcutaneous injection of wild-type simian virus 40 into Syrian hamsters normally induces fibrosarcomas at the injection site. We showed that subcutaneous injection of three different small t deletion mutants (dl884, dl883, and dl890) led to the formation of abdominal reticulum cell sarcomas (lymphomas) in about 15% of the animals bearing tumors. The remainder of the tumors were fibrosarcomas occurring with prolonged latencies at the site of injection. We postulated that, in the absence of an active small t protein, which is thought to have cell growth-promoting properties, the mutant virus preferentially transforms rapidly proliferating lymphoid cells.     

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.