Biomedical subjects

Henri-Jacques Delecluse

Publications and source records attributed to Henri-Jacques Delecluse.

At least 19 recordsLinked to original sources

PubMed2026

Spontaneous lytic reactivation drives a persistent B cell-vector pathway for epithelial dissemination of the Epstein-Barr virus.

The Epstein-Barr virus (EBV) establishes lifelong B cell infection via oral transmission; however, it paradoxically drives carcinomas in anatomically distant organs with striking geographic disparities. While genomic studies frequently link specific EBV variants to these epithelial cancers, the mechanisms bridging ubiquitous infection to distant, strain-dependent malignancies remain largely unresolved. Using an induction-free primary B cell system, we identify a circulating B cell-vector pathway driving immortalized epithelial dissemination. We demonstrate that B cells infected with carcinoma-associated strains exhibit markedly higher epithelial transmission compared with those carrying lymphoid strains. This contact-dependent process requires spontaneous lytic reactivation, viral DNA replication, and de novo virion production. Crucially, those infected B cells retain their transmission capacity for months, supporting sustained epithelial seeding. Mechanistically, entry requires gH/gL engagement of EphA2/desmocollin-2 (DSC2), with actin- and PI3K-dependent endocytosis. These findings define a lytic-coupled, receptor-dependent pathway by which the EBV exploits B cells to access the epithelium, offering a mechanistic framework for understanding strain tropism and host-virus interactions.

B cell vector↗

PubMed2007

Phosphoacceptor site S173 in the regulatory domain of Epstein-Barr Virus ZEBRA protein is required for lytic DNA replication but not for activation of viral early genes.

The Epstein-Barr virus ZEBRA protein controls the viral lytic cycle. ZEBRA activates the transcription of viral genes required for replication. ZEBRA also binds to oriLyt and interacts with components of the viral replication machinery. The mechanism that differentiates the roles of ZEBRA in regulation of transcription and initiation of lytic replication is unknown. Here we show that S173, a residue in the regulatory domain, is obligatory for ZEBRA to function as an origin binding protein but is dispensable for its role as a transcriptional activator of early genes. Serine-to-alanine substitution of this residue, which prevents phosphorylation of S173, resulted in a threefold reduction in the DNA binding affinity of ZEBRA for oriLyt, as assessed by chromatin immunoprecipitation. An independent assay based on ZEBRA solubility demonstrated a marked defect in DNA binding by the Z(S173A) mutant. The phenotype of a phosphomimetic mutant, the Z(S173D) mutant, was similar to that of wild-type ZEBRA. Our findings suggest that phosphorylation of S173 promotes viral replication by enhancing ZEBRA's affinity for DNA. The results imply that stronger DNA binding is required for ZEBRA to activate replication than that required to activate transcription.

Alanine↗

PubMed2006

Amino acids in the basic domain of Epstein-Barr virus ZEBRA protein play distinct roles in DNA binding, activation of early lytic gene expression, and promotion of viral DNA replication.

The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding to DNA, in activating Rta, in stimulating early lytic gene expression, and in promoting viral DNA replication and viral late gene expression. These results are discussed in relationship to the recently solved crystal structure of ZEBRA bound to an AP-1 site.

Base Sequence↗

PubMed2006

The Epstein-Barr virus BMRF1 gene is essential for lytic virus replication.

The Epstein-Barr virus (EBV) BMRF1 protein is a DNA polymerase processivity factor. We have deleted the BMRF1 open reading frame from the EBV genome and assessed the DeltaBMRF1 EBV phenotype. DeltaBMRF1 viruses were replication deficient, but the wild-type phenotype could be restored by BMRF1 trans-complementation. The replication-deficient phenotype included impaired lytic DNA replication and late protein expression. DeltaBMRF1 and wild-type viruses were undistinguishable in terms of their ability to transform primary B cells. Our results provide genetic evidence that BMRF1 is essential for lytic replication of the EBV genome.

Antigens, Viral↗

PubMed2005

Epstein-Barr virus-induced B-cell transformation: quantitating events from virus binding to cell outgrowth.

Epstein-Barr virus (EBV) infection and growth activation of human B cells is central to virus biology and disease pathogenesis, but is poorly understood in quantitative terms. Here, using virus at defined m.o.i., the different stages of this process at the single-cell level are followed in vitro. Virus binding to the B-cell surface, assayed by quantitative PCR, is highly efficient, particularly at the low m.o.i. values that most likely reflect physiologic events in vivo. However, only 10-15 % of bound virus genomes reach the cell nucleus, as visualized by sensitive fluorescence in situ hybridization (FISH) assay; viral genomes acquired per cell nucleus range from 1 to >10, depending on the m.o.i. Thereafter, despite differences in initial genome load, almost all nuclear genome-positive cells then go on to express the virus-encoded nuclear antigen EBNA2, upregulate the cell activation antigen CD23 and transit the cell cycle. EBNA2-positive cells in the first cycle post-infection then grow out to lymphoblastoid cell lines (LCLs) just as efficiently as do cells limiting-diluted from already established LCLs. This study therefore identifies EBV genome delivery to the nucleus as a key rate-limiting step in B-cell transformation, and highlights the remarkable efficiency with which a single virus genome, having reached the nucleus, then drives the transformation programme.

B-Lymphocytes↗

PubMed2005

BZLF1 activation of the methylated form of the BRLF1 immediate-early promoter is regulated by BZLF1 residue 186.

The Epstein-Barr virus (EBV) genome is highly methylated in latently infected cells. We recently reported that the EBV immediate-early (IE) protein BZLF1 (Z) preferentially binds to and activates transcription from the methylated form of the BRLF1 IE gene promoter (Rp). We now report that serine residue 186 in the Z DNA-binding domain plays an important role in the ability of Z to bind to and activate methylated Rp. A Z mutant containing an alanine residue at position 186 [Z(S186A)] was significantly defective in binding to methylated, as well as unmethylated, ZREs (Z-responsive elements) in Rp and was unable to activate lytic EBV gene transcription from the methylated or demethylated form of the viral genome. A Z mutant containing threonine at residue 186 [Z(S186T)] bound only to the methylated form of the ZRE-2 site in Rp and induced lytic EBV gene transcription from the methylated, but not demethylated, form of the viral genome. The defect in both of these mutants was primarily due to an inability to activate the Rp in the context of the viral genome. Finally, a Z mutant containing an aspartic acid at position 186 [Z(S186D)] did not bind to either the consensus AP-1 site or to the methylated or unmethylated Rp ZRE-2 site and did not induce lytic gene transcription. These results indicate that replacement of serine with threonine at residue 186 in the Z DNA-binding domain differentially affects its ability to reactivate the unmethylated, versus methylated, viral genome.

Cell Line↗

PubMed2005

Marked variation in response of consensus binding elements for the Rta protein of Epstein-Barr virus.

The R transactivator (Rta) protein activates Epstein-Barr virus (EBV) lytic-cycle genes by several distinct mechanisms that include direct binding to viral promoters, synergy with BamHI Z EBV replication activator (ZEBRA), and activation of cellular signaling pathways. In the direct and synergistic mechanisms of action, Rta binds to specific DNA sequences that are present in the promoters of responsive genes. It has been difficult to demonstrate the capacity of Rta expressed in mammalian cells to bind DNA in vitro in order to study the relative affinities of Rta binding elements. We discovered that a short C-terminal region of Rta inhibits the ability of Rta to bind DNA in vitro. C-terminally truncated versions of Rta bind DNA efficiently and thus facilitate a comparison of consensus Rta binding elements (CRBEs) found in promoters of five Rta-responsive genes: BMLF1, BHLF1, BMRF1, BaRF1, and BLRF2. All CRBEs in the promoters of the five genes conform to the proposed recognition sequence GNCCN9GGNG, where N is any nucleotide and N9 represents a sequence of nine nucleotides. Nonetheless, CRBEs varied markedly in their abilities to bind Rta in electrophoretic mobility shift assays. Not all CRBEs bound or responded to Rta. Binding affinities of the CRBEs and the capacity to be activated by Rta in reporter assays were strongly correlated. The CRBEs from the BMLF1 and BHLF1 promoters conferred the greatest response. The response of the BMRF1, BaRF1, and BLRF2 CRBEs was less robust. By creation of chimeras, inversions, and point mutations, differences in binding affinities and transcriptional activation levels could be attributed to N9 sequence variation. The length of N9 was also critical for a maximal response. In Raji and BZLF1-knockout cells, the mRNAs of the five Rta-responsive lytic-cycle genes differed dramatically in kinetics of expression, abundance, and synergistic responses to ZEBRA and Rta. Affinities of Rta response elements for Rta are likely to play an important role in temporal regulation and the level of lytic-cycle EBV gene expression.

Binding Sites↗

PubMed2005

Mutation of a single amino acid residue in the basic region of the Epstein-Barr virus (EBV) lytic cycle switch protein Zta (BZLF1) prevents reactivation of EBV from latency.

Zta, the product of the BZLF1 gene carried by Epstein-Barr virus (EBV), is crucial for reactivation of EBV from latency. Zta is a member of the bZIP family of transcription factors, and in common with many of these, Zta possesses a conserved cysteine residue in its basic region (C189) and a further cysteine residue in its ZIP region (C222). We demonstrate that C189 is required to reactivate EBV from latency but C222 is not and that this single amino acid affects two independent functions of Zta, (i) binding to a Zta-responsive site and (ii) manipulating the cell cycle.

Cell Cycle↗

PubMed2005

Epstein-Barr virus lytic infection is required for efficient production of the angiogenesis factor vascular endothelial growth factor in lymphoblastoid cell lines.

Although Epstein-Barr virus (EBV)-associated malignancies are primarily composed of cells with one of the latent forms of EBV infection, a small subset of tumor cells containing the lytic form of infection is often observed. Whether the rare lytically infected tumor cells contribute to the growth of the latently infected tumor cells is unclear. Here we have investigated whether the lytically infected subset of early-passage lymphoblastoid cell lines (LCLs) could potentially contribute to tumor growth through the production of angiogenesis factors. We demonstrate that supernatants from early-passage LCLs infected with BZLF1-deleted virus (Z-KO LCLs) are highly impaired in promoting endothelial cell tube formation in vitro compared to wild-type (WT) LCL supernatants. Furthermore, expression of the BZLF1 gene product in trans in Z-KO LCLs restored angiogenic capacity. The supernatants of Z-KO LCLs, as well as supernatants from LCLs derived with a BRLF1-deleted virus (R-KO LCLs), contained much less vascular endothelial growth factor (VEGF) in comparison to WT LCLs. BZLF1 gene expression in Z-KO LCLs restored the VEGF level in the supernatant. However, the cellular level of VEGF mRNA was similar in Z-KO, R-KO, and WT LCLs, suggesting that lytic infection may enhance VEGF translation or secretion. Interestingly, a portion of the vasculature in LCL tumors in SCID mice was derived from the human LCLs. These results suggest that lytically infected cells may contribute to the growth of EBV-associated malignancies by enhancing angiogenesis. In addition, as VEGF is a pleiotropic factor with effects other than angiogenesis, lytically induced VEGF secretion may potentially contribute to viral pathogenesis.

Cell Line↗

PubMed2005

Epstein-Barr virus lytic infection contributes to lymphoproliferative disease in a SCID mouse model.

Most Epstein-Barr virus (EBV)-positive tumor cells contain one of the latent forms of viral infection. The role of lytic viral gene expression in EBV-associated malignancies is unknown. Here we show that EBV mutants that cannot undergo lytic viral replication are defective in promoting EBV-mediated lymphoproliferative disease (LPD). Early-passage lymphoblastoid cell lines (LCLs) derived from EBV mutants with a deletion of either viral immediate-early gene grew similarly to wild-type (WT) virus LCLs in vitro but were deficient in producing LPD when inoculated into SCID mice. Restoration of lytic EBV gene expression enhanced growth in SCID mice. Acyclovir, which prevents lytic viral replication but not expression of early lytic viral genes, did not inhibit the growth of WT LCLs in SCID mice. Early-passage LCLs derived from the lytic-defective viruses had substantially decreased expression of the cytokine interleukin-6 (IL-6), and restoration of lytic gene expression reversed this defect. Expression of cellular IL-10 and viral IL-10 was also diminished in lytic-defective LCLs. These results suggest that lytic EBV gene expression contributes to EBV-associated lymphoproliferative disease, potentially through induction of paracrine B-cell growth factors.

Animals↗

PubMed2005

BFRF1 of Epstein-Barr virus is essential for efficient primary viral envelopment and egress.

The molecular mechanisms that underlie maturation and egress of Epstein-Barr virus (EBV) virions are only partially characterized. We have recently shown that the BFRF1 gene, the EBV positional homolog of herpes simplex virus type 1 and pseudorabies virus UL34, is expressed early during EBV lytic replication and that it is found predominantly on the nuclear membrane (A. Farina, R. Santarelli, R. Gonnella, R. Bei, R. Muraro, G. Cardinali, S. Uccini, G. Ragona, L. Frati, A. Faggioni, and A. Angeloni, J. Virol. 74:3235-3244, 2000). These data suggest that the BFRF1 protein might be involved in viral primary envelopment. To precisely determine the function of this protein, we have constructed an EBV mutant devoid of the BFRF1 gene (BFRF1-KO). 293 cells carrying BFRF1-KO showed no differences in comparison with wild-type EBV in terms of DNA lytic replication or expression of late viral proteins upon induction of the lytic cycle. However, binding assays and infection experiments using cell lines or human cord blood lymphocytes showed a clear reduction in the viral mutant titers. Complementation experiments with BFRF1-KO and a BFRF1 expression vector restored viral titers to levels similar to those for the wild-type control, showing that the modifications that we introduced were limited to the BFRF1 gene. Electron microscopic observations showed that the reduction in viral titers was due to sequestration of EBV nucleocapsids in the nuclei of lytically induced cells. This suggests that BFRF1 is involved in transport of the maturing virion across the nuclear membrane. This hypothesis was further supported by the observation that BFRF1 is present in maturing intracellular virions but not in their extracellular counterparts. This implies that BFRF1 is a key protein for EBV maturation.

Cell Line↗

PubMed2005

Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: interactions with BFRF1 and with the nuclear lamina.

We have reported in the accompanying paper that the BFRF1 protein of Epstein-Barr virus (EBV) is important for efficient primary viral envelopment and egress (A. Farina, R. Feederle, S. Raffa, R. Gonnella, R. Santarelli, L. Frati, A. Angeloni, M. R. Torrisi, A. Faggioni, and H.-J. Delecluse, J. Virol. 79:3703-3712). Here we describe the characterization of the product of the EBV BFLF2 gene, which belongs to a family of conserved herpesviral genes which include the UL31 genes of herpes simplex virus and of pseudorabies virus and whose products are known to interact with UL34, the positional homolog of BFRF1. BFLF2 is an early transcript and is expressed in a variety of cell lines upon EBV lytic cycle activation. Western blotting of purified virion preparations showed that BFLF2 is a component of intracellular virions but is absent from mature extracellular virions. Coimmunoprecipitation experiments indicated that BFLF2 interacts with BFRF1, which was confirmed by immunofluorescence confocal microscopy showing that the two proteins colocalize on the nuclear membrane not only upon cotransfection in epithelial cells but also during viral replication. In cells carrying an EBV mutant with the BFRF1 gene deleted (293-BFRF1-KO cells) BFLF2 expression was low, and it was restored to wild-type levels upon treatment of the cells with the proteasome inhibitor MG132. Furthermore, recomplementing the 293-BFRF1-KO cells by BFRF1 transfection restored BFLF2 expression to the wild-type level. In addition, when expressed alone BFLF2 was localized diffusely inside the nucleus, whereas in the presence of BFRF1 the two proteins colocalized at the nuclear rim. Finally, 293 epithelial cells transfected with either protein or cotransfected were analyzed by electron microscopy to investigate potential alterations in the morphology of the nuclear membrane. The ultrastructural analysis revealed that (i) BFRF1 caused duplications of the nuclear membrane, similar to those reported to occur during the course of herpesviral replication, and (ii) while BFLF2 alone did not cause any apparent alteration, coexpression of the two proteins dramatically induced profound convolutions of the duplicated nuclear membrane. Both biochemical and morphological analysis showed association of the BFRF1-BFLF2 complex with a component of the nuclear lamina, lamin B. Taken together, these results and those of the accompanying paper (Farina et al., J. Virol. 79:3703-3712) indicate an important role of BFRF1 and BFLF2 in the early steps of EBV maturation at the nuclear membrane.

Blotting, Western↗

PubMed2005

Molecular genetics of DNA viruses: recombinant virus technology.

Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecular genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All available techniques are based on homologous recombination between a targeting vector carrying the mutated version of the gene of interest and the recombinant virus. After modification, the mutant viral genome is stably introduced into eukaryotic cells permissive for viral lytic replication. In these cells, mutant viral genomes can be packaged into infectious particles to evaluate the effect of these mutations in the context of the complete genome.

Bacteriophage lambda↗

PubMed2004

Efficient somatic gene targeting in the lymphoid human cell line DG75.

Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy. Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest. Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines. Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin. The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells. The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles. The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination. Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells.

Burkitt Lymphoma↗

PubMed2004

Reactivation of latent Epstein-Barr virus by methotrexate: a potential contributor to methotrexate-associated lymphomas.

BACKGROUND: Patients with rheumatoid arthritis or polymyositis treated with methotrexate (MTX) develop Epstein-Barr virus (EBV)-positive lymphomas more frequently than patients treated with other, equally immunosuppressive regimens. Here we determined whether MTX, in contrast to other commonly used medications for rheumatoid arthritis or polymyositis, is unique in its ability to induce the release of infectious EBV from latently infected cells. METHODS: The effect of MTX and other immunosuppressant drugs on EBV replication in vitro was assessed using latently infected EBV-positive lymphoblastoid and gastric carcinoma cell lines. Inhibitors of signal transduction pathways were used to define requirements for induction of lytic infection. Drug effects on transcription of the two EBV immediate-early promoters (BRLF1 and BZLF1) and on promoter constructs lacking cis-acting sequences required for activation by other effectors was examined using reporter gene assays. EBV viral load in rheumatoid arthritis and polymyositis patients receiving MTX was compared with that in patients receiving other immunosuppressive medications. Statistical tests were two-sided. RESULTS: MTX activated the release of infectious EBV from latently infected cell lines in vitro, and MTX treatment was associated with activation of the two viral immediate-early promoters in reporter gene assays. Induction of lytic EBV infection by MTX required the p38 MAP kinase, PI3 kinase, and MEK pathways and specific cis-acting motifs in the two viral immediate-early promoters. Patients treated with MTX-containing regimens had statistically significantly higher mean EBV loads in their blood than patients treated with immunosuppressing regimens that did not include MTX (40 EBV copies per 10(6) cellular genomes versus 5.1 copies; geometric mean fold difference in copies = 10.8, 95%, confidence interval = 3.0 to 38; P = .011). CONCLUSION: MTX may promote EBV-positive lymphomas in rheumatoid arthritis and polymyositis patients by its immunosuppressive properties as well as by reactivating latent EBV.

Arthritis, Rheumatoid↗

PubMed2004

The EBV lytic switch protein, Z, preferentially binds to and activates the methylated viral genome.

DNA methylation promotes gene silencing, yet the Epstein-Barr virus immediate-early protein, BZLF1 (Z), converts the virus from the latent to the lytic form of infection even when the viral genome is highly methylated. Here we show that methylation of CpG motifs in Z-responsive elements of the viral BRLF1 immediate-early promoter enhances Z binding to, and activation of, this promoter. Demethylation of the viral genome impairs Z activation of lytic viral genes. Z is the first transcription factor that preferentially binds to, and activates, a methylated promoter. These results identify an unexpected mechanism by which Epstein-Barr virus circumvents the inhibitory effects of viral genome methylation.

Binding Sites↗

PubMed2004

Functional analysis of Epstein-Barr virus SM protein: identification of amino acids essential for structure, transactivation, splicing inhibition, and virion production.

The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of cellular and viral gene expression that binds and stabilizes target mRNAs and shuttles from nucleus to cytoplasm. SM enhances expression of several EBV genes required for lytic replication and is essential for virion production. SM increases accumulation of specific mRNAs but also inhibits expression of several intron-containing transcripts. The mechanism by which SM inhibits gene expression is poorly understood. The experiments described here had several aims: to determine whether specific domains of SM were responsible for activation or inhibition function; whether these functions could be separated; and whether one or more of these functions were essential for virion production. A mutational analysis of SM was performed, focusing on amino acids in SM that are evolutionarily conserved among SM homologs in other herpesviruses. Mutation of the carboxy-terminal region of SM revealed a region that is likely to be structurally important for SM protein conformation. In addition, several amino acids were identified that are critical for activation and inhibition function. A specific mutation of a highly conserved cysteine residue revealed that it was essential for gene inhibition but not for transactivation, indicating that these two functions operate through independent mechanisms. Furthermore, the ability of wild-type SM and the inability of the mutant to inhibit gene expression were shown to correlate with the ability to inhibit splicing of a human target gene and thereby prevent accumulation of its processed mRNA. Surprisingly, some mutations which preserved both activation and inhibition functions in vitro nevertheless abolished virion production, suggesting that other SM functions or protein-protein interactions are also required for lytic replication.

Amino Acid Sequence↗

PubMed2004

The BRRF1 early gene of Epstein-Barr virus encodes a transcription factor that enhances induction of lytic infection by BRLF1.

The switch from the latent to the lytic form of Epstein-Barr virus (EBV) infection is mediated by expression of the viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1 (R). An EBV early protein, BRRF1 (Na), is encoded by the opposite strand of the BRLF1 intron, but the function of this nuclear protein in the viral life cycle is unknown. Here we demonstrate that Na enhances the R-mediated induction of lytic EBV infection in 293 cells latently infected with a recombinant EBV (R-KO) defective for the expression of both R and Na. Na also enhances R-induced lytic infections in a gastric carcinoma line (AGS) carrying the R-KO virus, although it has no effect in a Burkitt lymphoma line (BL-30) stably infected with the same mutant virus. We show that Na is a transcription factor that increases the ability of R to activate Z expression from the R-KO viral genome in 293 cells and that Na by itself activates the Z promoter (Zp) in EBV-negative cells. Na activation of Zp requires a CRE motif (ZII), and a consensus CRE motif is sufficient to transfer Na responsiveness to the heterologous E1b promoter. Furthermore, we show that Na enhances the transactivator function of a Gal4-c-Jun fusion protein but does not increase the transactivator function of other transcription factors (including ATF-1, ATF-2, and CREB) known to bind CRE motifs. Na expression in cells results in increased levels of a hyperphosphorylated form of c-Jun, suggesting a mechanism by which Na activates c-Jun. Our results indicate that Na is a transcription factor that activates the EBV Zp IE promoter through its effects on c-Jun and suggest that Na cooperates with BRLF1 to induce the lytic form of EBV infection in certain cell types.

Genes, Immediate-Early↗