A fifth Epstein-Barr virus nuclear protein (EBNA3C) is expressed in latently infected growth-transformed lymphocytes.

Three distantly homologous neighboring long open reading frames in the Epstein-Barr virus (EBV) genome are preceded by short open reading frames. The leftmost short and long open reading frames encode EBNA3, a nuclear protein which is slightly smaller (145 kilodaltons [kDa]) than two other nuclear proteins (150 to 155 kDa) detected in Western blots (immunoblots) of latently infected cell protein (K. Hennessy, F. Wang, E. Woodland-Bushman, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5693-5697, 1986; I. Joab, D. T. Rowe, M. Bodescot, J.-C. Nicolas, P. J. Farrell, and M. Perricaudet, J. Virol. 61:3340-3344, 1987). We have demonstrated that the most rightward short (BERF3) and long (BERF4) open reading frames are spliced in frame at the 3' end of a 5-kilobase latently infected cell RNA and that this RNA begins within or upstream of the EBV long internal repeat. EBV-immune human antibodies specific for the long open reading frame translation product identified a 155-kDa protein on Western blots of latently infected cell protein and specifically reacted with large nonnucleolar nuclear granules in every latently infected cell. Expression of the cDNA in BALB/c 3T3 cells resulted in translation of full-size EBNA3C but had no effect on cell morphology, contact inhibition, or serum independence.     

Mapping of the gene coding for Epstein-Barr virus-determined nuclear antigen EBNA3 and its transient overexpression in a human cell line by using an adenovirus expression vector.

The open reading frame which lies within the Epstein-Barr virus (EBV) T2 cDNA isolated by Bodescot et al. (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:2611-2620, 1986) was inserted into a eucaryotic expression vector containing a strong adenovirus promoter. The T2 cDNA contains viral genomic sequences from the short BLRF3 open reading frame fused to the adjacent BERF1 long open reading frame. After transfection of human cells, the recombinant plasmid directed the expression of a 140-kilodalton protein. The expressed protein had the same molecular weight, subcellular localization, and immunological characteristics as the EBV-determined nuclear antigen EBNA3, which is made in lymphocytes latently infected with EBV. Immunoprecipitation of extracts of transfected cells labeled with [32P]phosphoric acid showed that the EBNA3 protein is phosphorylated.     

A bicistronic Epstein-Barr virus mRNA encodes two nuclear proteins in latently infected, growth-transformed lymphocytes.

EBNA2 is a nuclear protein expressed in all cells latently infected with and growth transformed by Epstein-Barr virus (EBV) infection (K. Hennessy and E. Kieff, Science 227:1230-1240, 1985). The nucleotide sequence of the EBNA2 mRNA (J. Sample, M. Hummel, D. Braun, M. Birkenbach, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5096-5100, 1986) revealed that it begins with a 924-base open reading frame that has an unusual potential translational initiation site (CAAATGG). This open reading frame is followed by 138 nucleotides with only one highly unlikely translational initiation site (TACATGC), which would translate a pentapeptide before the next stop codon. The last part of the mRNA is the open reading frame which encodes EBNA2. In this paper, we demonstrate that the 924-base open reading frame translates a 40-kilodalton protein in vitro or in murine cells transfected with the EBNA2 cDNA under control of the murine leukemia virus long terminal repeat. A protein of identical size was detected in EBV-transformed, latently infected human lymphocyte nuclei by using antibody specific for the leader open reading frame expressed in bacteria. Therefore, this is a rare example of a mRNA which translates two proteins from nonoverlapping open reading frames. Since the protein encoded by the leader of the EBNA mRNA is expressed in all nuclei of a latently infected cell line, it was designated EBNA-LP. EBNA-LP localizes to small intranuclear particles and differs in this respect from EBNA1, EBNA2, or EBNA3. EBNA-LP is not expressed in an EBV-transformed marmoset lymphocyte cell (B95-8) or in one EBV-infected Burkitt tumor cell line (Raji) but is expressed in three other Burkitt tumor cell lines (Namalwa, P3HR-1, and Daudi).     

Use of second-site homologous recombination to demonstrate that Epstein-Barr virus nuclear protein 3B is not important for lymphocyte infection or growth transformation in vitro.

Recombinant Epstein-Barr viruses with a stop codon inserted into the nuclear protein 3B (EBNA 3B) open reading frame were generated by second-site homologous recombination. These mutant viruses infected and growth transformed primary B lymphocytes, resulting in the establishment of lymphoblastoid cell lines (LCLs). Polymerase chain reaction analysis and Southern hybridizations with infected cell DNA demonstrated the presence of the mutant EBNA 3B and the absence of wild-type EBNA 3B. Immunoblot analysis of the LCLs with affinity-purified EBNA 3B antibodies confirmed the absence of EBNA 3B cross-reactive protein. Virus was reactivated from two of these infected LCLs and serially passaged through primary B lymphocytes. The newly infected cells contained only the mutant recombinant virus. No difference was noted between mutant and wild-type recombinants, derived in parallel, in latent (other than EBNA 3B) or lytic cycle-infected cell virus protein expression or in the growth of the latently infected transformed cell lines. These data indicate that the EBNA 3B protein is not critical for primary B-lymphocyte infection, growth transformation, or lytic virus infection in vitro.     

Prediction and demonstration of a novel Epstein-Barr virus nuclear antigen.

The protein sequence predicted by the Epstein Barr virus (EBV) BERF4 open reading frame includes a tetrapeptide, Lys-Arg-Pro-Arg (KRPR), shown for other proteins to be a component of a signal for rapid nuclear localization. A subgenomic fragment of EBV DNA containing BERF4 has been incorporated into an expression vector, transfected onto primate cells and the nuclear distribution of the resulting protein established by immunofluorescence using EBV positive human sera. These sera contained high titres of antibodies to a fusion protein, produced in E. coli, consisting of beta-galactosidase and the C-terminal 167 amino acids of BERF4. Immunoaffinity purified antibodies reactive with the EBV component of the fusion show the molecular weight of this antigen in EBV immortalized B-cell lines to be about 160 kD. The demonstration that BERF4 contains an exon encoding a nuclear protein identifies a new EBNA gene (EBNA-6) and suggests that KRPR is a signal sequence common to a number of viral and cellular nuclear polypeptides which bind to nucleic acids and may therefore be of predictive value in identifying karyophilic proteins.     

Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA.

We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DNA sequences cloned into a prokaryotic F-factor plasmid. These mini-EBVs, when introduced into an EBV-containing lymphoblastoid cell, can be packaged by the endogenous helper virus. One such mini-EBV was found to have a single C residue deleted from its EBNA3a open reading frame. When packaged, this mini-EBV initiates proliferation of infected primary human B lymphocytes only in conjunction with a complementing helper virus. Proliferation of the infected cells, however, was maintained either alone by the mini-EBV containing the mutated EBNA3a open reading frame or alone by its derivative in which the EBNA3a open reading frame had been healed of its lesion by recombination with the helper virus. The mini-EBV with a wild-type EBNA3a open reading frame when packaged alone can both initiate and maintain proliferation upon infection of primary human B lymphocytes. These findings identify 41% of EBV DNA which is sufficient to immortalize primary human B lymphocytes and provide an assay to distinguish virus contributions to initiation or maintenance of cell proliferation or both. They also identify EBNA3a as a transforming gene, which contributes primarily to the initiation of cell proliferation.     

Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment

We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes.     

Identification of the human papillomavirus E2 protein in genital tract tissues.

A 27-kilodalton protein representing approximately 60% of the E2 open reading frame of human papillomavirus type 6 (HPV-6) was synthesized in a bacterial expression system. Affinity-purified polyclonal antibody to this protein detected the probable E2 gene product as a 50-kilodalton protein in most condylomas by Western blot (immunoblot) analysis. The E2-positive condylomas were associated with HPV-6, HPV-11, HPV-16, or unidentified HPVs.     

Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor.

The nucleotide sequence of black beetle virion (BBV) RNA2 has been determined. RNA2 is 1399 b long. Its 5' terminus is capped. Its 3' terminus has an unidentified moiety that renders the RNA resistent to polyadenylation and ligation. The first AUG codon at base 23 is followed by an open reading frame for a protein 407 amino acids long, the predicted size of coat protein precursor. A second open reading frame for a putative protein 72 amino acid residues long begins at base 1110. No other large open reading frames exist. The 5' half of the RNA can be folded into a long, imperfect hairpin of high predicted stability. The 3' half of the RNA can fold into a complex set of multiply bifurcated stem and loop regions.     

Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide.

We have constructed a cDNA library from the cytoplasmic RNAs of Raji cells, a Burkitt's lymphoma cell line latently infected with Epstein-Barr virus. We report here the characterization of a cDNA representing a spliced RNA transcribed from the IR1-U2 region of the viral genome. The cDNA is 1007 bp long. The 5' region contains three tandem repeats of two exons, 66 and 132 bp, which are transcribed from the IR1 repeats. The 3' region is formed from four exons transcribed from U2. An open reading frame extends from the 5' end to position 784, and includes the repeats. This reading frame presumably corresponds to the carboxy-terminal 261 amino acids of a polypeptide containing several repeats of a 66 amino acid sequence. Since it would be encoded by the IR1-U2 region of the viral genome, the putative polypeptide might be involved in the process of growth-transformation of B-lymphocytes.