Expression and analysis of the Epstein-Barr virus BARF1-encoded protein from a tetracycline-regulatable adenovirus system.

Epstein-Barr virus (EBV) has been associated with human cancers of lymphocytic or epithelial origin. Potential functions of the BARF1 early gene in EBV oncogenesis emerged from our observations showing expression of BARF1-encoded protein in nasopharyngeal carcinoma biopsies, and induction of either malignant transformation (in rodent fibroblast and human B cell lines) or immortalization (in monkey primary epithelial cells) following BARF1 transfection. We previously reported expression of the BARF1 product as a cytoplasm/membrane-associated protein from 293-tTA cells infected with a BARF1-recombinant adenovirus. Since constitutive expression of BARF1 from this heterologous system became inefficient, we developed a tetracycline-regulatable recombinant vector expressing BARF1 and green fluorescent protein from a dicistronic message. As here reported, stable and efficient expression of BARF1 from this vector in either permissive or non-permissive cell lines, allowed the first sequencing identification and further molecular characterization of BARF1-encoded protein.

Expression of BARF1 gene encoded by Epstein-Barr virus in nasopharyngeal carcinoma biopsies.

We reported previously that the EBV BARF1 open reading frame encodes a Mr 31,000-33,000 protein (p31) with potential transforming and oncogenic properties. This gene was found capable of transforming both: (a) the rodent fibroblast lines Balbc/3T3 and NIH3T3 into cells producing aggressive tumors in newborn rats; and (b) the human EBV-negative B-cell line Louckes into cells leading to small tumors, which disappeared 3 weeks after injection. Our recent study showed that BARF1 ORF expression may confer the property of immortalization to primary kidney epithelial cells (M. X. Wei et al., Oncogene, 14: 3073-3081, 1997). Because this suggested that BARF1 could be involved in epithelial malignancy, we investigated its transcriptional and translational expressions in Algerian nasopharyngeal carcinoma (NPC) biopsies by reverse transcription-PCR and immunoblotting using rabbit polyclonal antisera prepared against two synthetic peptides corresponding to distinct, predicted epitopes of the BARF1 protein (NGGVMKEKD, amino acids 172-180, and GKNDKEE, amino acids 203-209). The BARF1 ORF was found to be transcribed and translated in >85% of our NPC biopsies, with high p31 protein level detected in several NPC patient biopsies as well as in NPC-derived xenografts. Our observation of BARF1 expression in a large proportion of NPC epithelial cells suggests that this EBV gene might play an important role in the malignant transformation of human epithelial cells in vivo.

Phenotypic and genetic characterization of thymidine kinase from clinical strains of varicella-zoster virus resistant to acyclovir.

Varicella-zoster virus (VZV) is a common herpesvirus responsible for disseminated or chronic infections in immunocompromised patients. Effective drugs such as acyclovir (ACV), famciclovir (prodrug of penciclovir), and foscarnet are available to treat these infections. Here we report the phenotypic and genetic characterization of four ACV-resistant VZV strains isolated from AIDS patients and transplant recipients. Sensitivity to six antiviral drugs was determined by an enzyme-linked immunosorbent assay, viral thymidine kinase (TK) activity was measured by comparing [(3)H]thymidine and 1-beta-D-arabinofuranosyl-[(3)H]thymine as substrates, and the TK gene open reading frame was sequenced. Three strains were found to be TK deficient, and the fourth was a mixed population composed of TK-positive and TK-deficient viruses. Each strain presented a unique TK gene mutation that could account for ACV resistance. In one strain, the deletion of two nucleotides at codon 215 induced a premature stop signal at codon 217. In another strain, a single nucleotide addition at codon 167 resulted in a premature stop signal at codon 206. In both other strains, we identified amino acid substitutions already described in other ACV-resistant VZV strains: either Glu-->Gly at residue 48 or Arg-->Gly at residue 143. According to our work and data previously reported on resistant VZV strains, there are three areas in the TK gene where 71% of the mutations described to date are located. These areas are putative candidates for a genotypic diagnosis of ACV resistance.

Establishment of a monkey kidney epithelial cell line with the BARF1 open reading frame from Epstein-Barr virus.

We previously reported that the BARF1 (BamH1-A right frame 1) gene product from Epstein-Barr Virus (EBV) may have oncogenic properties since injection into new-born rats of transfected cell lines resulted in the development of BARF1 expressing tumors, which were aggressive in the case of murine fibroblasts and transient in that of human B lymphocytes. As EBV has been associated with nasopharyngeal carcinoma (NPC) and evidence of BARF1 transcription in this cancer was emerging from our biopsy analyses, we examined the effects of BARF1 transfection into primate primary epithelial cells. The expression of the BARF1 open reading frame in primary monkey kidney epithelial cells led us to the establishment of continuously dividing lines. The BARF1 transfectants showed the major characteristics of immortalized cells: morphological change, short cell doubling time, ability to divide at low cell density and continuous growth over 50 passages. Injection of BARF1 transfectants into nude mice did not induce any tumor. Established subclones were shown to be epithelial cells expressing known keratins as well as the BARF1 coded mRNA and protein. This is the first report indicating that expression of the BARF1 gene product in primary epithelial cells may contribute to the establishment of cell lines.

Expression of the protein encoded by Epstein-Barr virus (EBV) BARF1 open reading frame from a recombinant adenovirus system.

Epstein-Barr virus (EBV) has been associated with human cancers of lymphocytic or epithelial origin, but viral functions implied in oncogenesis are not yet clear. We previously reported the oncogenic transformation of rodent fibroblast and human B lymphocyte cell lines by the BARF1 coding sequence from EBV. We more recently observed immortalizing effects of this gene on monkey kidney primary epithelial cells. Here we describe an efficient recombinant adenovirus expression system which allowed us to characterize BARF1 translation products, with the help of rabbit polyclonal antibodies raised to the entire protein. The present data demonstrate that BARF1 encodes a 31-33 kDa hydrophobic protein, linked to cell membranes though also recovered in the cytosol, and recognized by human sera from patients with various EBV-related pathologies.

Relationship between nasopharyngeal carcinoma and high antibody titers to Epstein-Barr virus-specific thymidine kinase.

Epstein-Barr virus (EBV) has long been implicated in nasopharyngeal carcinoma (NPC). Recent studies in our and other laboratories have shown a correlation between the disease and high antibody titers to EBV-specific DNase. These data led us to also examine serial sera from healthy adults and patients with infectious mononucleosis or NPC, for their capacity to neutralize the EBV-specific thymidine kinase (TK) activity from chemically induced EBV-carrying human lymphoblastoid cells. Our results were the following: (i) sera were found that efficiently blocked the EBV-specific TK activity of induced-Raji TK- cell extracts, but not the host-cell TK activity from EBV-negative BJAB cells; (ii) a relationship appeared between high levels of EBV-specific TK-neutralizing activity in sera and NPC pathology, even though in this preliminary study the degrees of EBV-induced TK-blocking activity detected in sera were not significantly correlated with EBV-specific antibody titers; (iii) the EBV-induced TK-neutralizing activity was found in the main IgG fraction derived from NPC sera. These data must be compared with other known antibody responses to EBV for their clinical interest in NPC control.

Identification of an Epstein-Barr virus-specific desoxyribonuclease gene using complementary DNA.

We have recently obtained 18 distinct cDNA clones representing different genes expressed in the early phase of EBV infection. One of them, c37, which is situated at the position 12907-122451 in the B95-8 viral genome, is shown here to code for a viral desoxyribonuclease [DNase]. Cell free translation of c37-selected messenger RNA yielded a protein of about 52 KDa which was immunoprecipitated by a high EA titer serum from nasopharyngeal carcinoma patient. This protein showed a DNase activity which was resistant to high salt concentrations (150 to 300 mM KCl) and was specifically neutralized by EA positive serum. These properties are typical of the EBV-specific DNase activity that we recently described in chemically induced EBV-transformed lymphoid cells. The same results were obtained on cell-free translation of the native RNA synthesized in vitro from pGEM-37 plasmid containing the entire c37 cDNA sequence (1.53 Kb). These data indicate that the BGLF5 open reading frame contained in c37 encodes for the EBV-specific DNase.

Characterization of an Epstein-Barr virus-induced thymidine kinase.

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.