RESUMEN

Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.

Asunto(s)

Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/genética , Proteínas Inmediatas-Precoces/metabolismo , Transactivadores/metabolismo , ADN Viral/metabolismo , Desoxirribonucleasas , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/genética , Linfoma , Ácido Fosfonoacético/farmacología , ARN Mensajero , Inhibidores de la Transcriptasa Inversa/farmacología , Transactivadores/genética , Células Tumorales Cultivadas

RESUMEN

The ZEBRA protein mediates the switch between the latent and lytic life cycles of Epstein-Barr virus. Z(S186A), a point mutant in ZEBRA's basic domain in which serine 186 is changed to alanine, is unable to induce expression of lytic cycle mRNAs or proteins from the latent EBV genome even though it retains the ability to activate transcription from reporters bearing known ZEBRA-responsive promoters (A. L. Francis et al., J. Virol. 71:3054-3061, 1997). We now describe three distinct phenotypes of ZEBRA mutants bearing different amino acid substitutions at S186. These phenotypes are based on the capacity of the mutants to activate expression of the BRLF1 and BMRF1 genes, which are targets of ZEBRA's action, and to synergize with the BRLF1 gene product Rta (R transactivator) in activating expression of downstream genes. One mutant class, represented by Z(S186T), was similar to the wild type, although reduced in the capacity to activate BRLF1 and BMRF1 early lytic cycle genes from the latent virus. A second class, represented by Z(S186C) and Z(S186G), was impaired in transcriptional activation, unable to activate early lytic cycle products from the latent virus, and not rescued by overexpression of Rta. A third class, Z(S186A), although unable by itself to activate BRLF1 or other lytic cycle genes, synergized with Rta. Rta rescued the capacity of Z(S186A) to activate the BMRF1 early lytic cycle gene from the latent virus. All mutant classes bound to DNA in vitro, although their capacity to bind to different ZEBRA response elements varied. Serine 186 of ZEBRA is a critical residue that is required for the distinct activities of induction of BRLF1 expression and for synergy with Rta. Since only Z(S186T) among the mutants behaved similarly to the wild type, activation of BRLF1 likely requires phosphorylation of S186. However, since Z(S186A) could synergize with Rta, synergy with Rta does not appear to be dependent on phosphorylation of S186. S186 likely mediates DNA recognition on the BRLF1 promoter in the context of the latent virus, protein-protein interactions, or both. The Z(S186) mutants define the amino acid side chains required for these functions.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Proteínas Inmediatas-Precoces/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Proteínas Virales , Antígenos Virales/genética , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/química , Humanos , Proteínas Inmediatas-Precoces/genética , Fosforilación , Mutación Puntual , Regiones Promotoras Genéticas , Serina , Relación Estructura-Actividad , Transactivadores/química , Factores de Transcripción/genética

RESUMEN

Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.

Asunto(s)

Genes Virales , Herpesvirus Humano 8/genética , Quimiocinas/genética , Expresión Génica , Genes Inmediatos-Precoces , Humanos , Cinética , ARN Viral/análisis , Sarcoma de Kaposi/virología , Células Tumorales Cultivadas

RESUMEN

Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein-Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein-Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.

Asunto(s)

Genes Virales , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/virología , Activación Viral/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Herpesvirus Humano 8/fisiología , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Transcripción Genética , Células Tumorales Cultivadas

RESUMEN

The ZEBRA protein from Epstein-Barr virus (EBV) activates a switch from the latent to the lytic expression program of the virus. ZEBRA, a member of the bZIP family of DNA-binding proteins, is a transcriptional activator capable of inducing expression from viral lytic cycle promoters. It had previously been thought that ZEBRA's capacity to disrupt EBV latency resided primarily in its ability to activate transcription of genes that encode products required for lytic replication. We generated a point mutant of ZEBRA, Z(S186A), that was not impaired in its ability to activate transcription; however, this mutation abolished its ability to initiate the viral lytic cascade. The mutant, containing a serine-to-alanine substitution in the DNA-binding domain of the protein, bound to several known ZEBRA-binding sites and activated transcription from reporters bearing known ZEBRA-responsive promoters but did not disrupt latency in EBV-infected cell lines. Therefore, initiation of the EBV lytic cycle by the ZEBRA protein requires a function in addition to transcriptional activation; a change of serine 186 to alanine in the DNA-binding domain of ZEBRA abolished this additional function and uncovered a new role for the ZEBRA protein in disruption of EBV latency. The additional function that is required for initiation of the lytic viral life cycle is likely to require phosphorylation of serine 186 of the ZEBRA protein, which may influence either DNA recognition or transcriptional activation of lytic viral promoters in a chromatinized viral episome.

Asunto(s)

Alanina/fisiología , Proteínas de Unión al ADN/fisiología , Herpesvirus Humano 4/fisiología , Serina/fisiología , Transactivadores/fisiología , Activación Transcripcional , Proteínas Virales , Latencia del Virus , Linfocitos B/citología , Sitios de Unión , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/genética , Expresión Génica , Genes Reporteros , Herpesvirus Humano 4/genética , Humanos , Mutación Puntual , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Transactivadores/genética , Células Tumorales Cultivadas

RESUMEN

We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/virología , Antígenos Virales/inmunología , Proteínas de la Cápside , Cápside/inmunología , Herpesvirus Humano 8/inmunología , Sarcoma de Kaposi/virología , Infecciones Oportunistas Relacionadas con el SIDA/sangre , Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/análisis , Antígenos Virales/genética , Secuencia de Bases , Células COS , Cápside/análisis , Cápside/genética , Línea Celular , Clonación Molecular , Reacciones Cruzadas , ADN Viral , Escherichia coli , Expresión Génica , Herpesvirus Humano 8/genética , Humanos , Datos de Secuencia Molecular , ARN Viral/análisis , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Sarcoma de Kaposi/sangre , Sarcoma de Kaposi/inmunología , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas

RESUMEN

The BC-1 cell line, derived from a body cavity-based, B-cell lymphoma, is dually infected with Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In these studies, the relationships between these two gammaherpesviruses and BC-1 cells were characterized and compared. Single-cell cloning experiments suggested that all BC-1 cells contain both genomes. In more than 98% of cells, both viruses were latent. The two viruses could be differentially induced into their lytic cycles by chemicals. EBV was activated into DNA replication and late-gene expression by the phorbol ester tetradecanoyl phorbol acetate (TPA). KSHV was induced into DNA replication and late-gene expression by n-butyrate. Amplification of both EBV and KSHV DNAs was inhibited by phosphonoacetic acid. Induction of the KSHV lytic cycle by n-butyrate was accompanied by the disappearance of host-cell beta-actin mRNA. Induction of EBV by TPA was not accompanied by such an effect on host-cell gene expression. Induction of the KSHV lytic cycle by n-butyrate was associated with the expression of several novel polypeptides. Recognition of one of these, p40, served as the basis of development of an assay for antibodies to KSHV in the sera of infected patients. BC-1 cells released infectious EBV; however, there was no evidence for the release of encapsidated KSHV genomes by BC-1 cells, even though n-butyrate-treated cells contained numerous intranuclear nucleocapsids. The differential inducibility of these two herpesviruses in the same cell line points to the importance of viral factors in the switch from latency to lytic cycle.

Asunto(s)

Replicación del ADN , Herpesvirus Humano 4/fisiología , Herpesvirus Humano 8/fisiología , Linfoma de Células B/virología , Sarcoma de Kaposi/virología , Latencia del Virus , Replicación Viral , Animales , Antígenos Virales , Butiratos/farmacología , Ácido Butírico , ADN Viral/análisis , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/aislamiento & purificación , Humanos , Linfoma de Células B/patología , Microscopía Electrónica , Biosíntesis de Péptidos , Ácido Fosfonoacético/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero , ARN Viral/análisis , Conejos , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Virión

RESUMEN

The viral capsid antigen complex of Epstein-Barr virus (EBV), an important serodiagnostic marker of infection with the virus, consists of at least four components, with molecular masses of 150, 110, 40, and 21 kDa. Here we show that the 21-kDa component of the viral capsid antigen consists of products of two EBV genes, BFRF3 and BLRF2. Both products were expressed from late transcripts, were recognized by human antisera, and were present in virions. The BFRF3 product, but not that of BLRF2, fulfilled the definition of ZEBRA-associated protein p21 (ZAP21). In cells in which EBV was lytically replicating, BFRF3 protein was coimmunoprecipitated together with ZEBRA by a rabbit antiserum directed against amino acids 197 to 245 of BZLF1. In EBV-negative cells cotransfected with BZLF1 and BFRF3 expression vectors, BFRF3 was also coimmunoprecipitated with this antiserum. Although this antiserum could not detect BFRF3 on an immunoblot, it was able to immunoprecipitate BFRF3 in the absence of ZEBRA expression. The rabbit antiserum to amino acids 197 to 245 of BZLF1 was found to detect the same epitope at the carboxy end of BFRF3 as was recognized by rabbit antiserum to BFRF3 itself. Thus, coimmunoprecipitation of BFRF3 p21 with ZEBRA appeared to be due to cross-reactivity of the immunoprecipitating antiserum rather than to direct association of ZEBRA and BFRF3 p21.

Asunto(s)

Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Cápside/inmunología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/inmunología , Transactivadores/inmunología , Infecciones Tumorales por Virus/inmunología , Proteínas Virales , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Cápside/genética , Proteínas de Unión al ADN/metabolismo , Epítopos/inmunología , Infecciones por Herpesviridae/sangre , Herpesvirus Humano 4/genética , Humanos , Immunoblotting , Pruebas de Precipitina , Conejos , Transactivadores/metabolismo , Células Tumorales Cultivadas , Infecciones Tumorales por Virus/sangre , Virión/metabolismo

RESUMEN

A newly recognized gamma herpesvirus known as Kaposi sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is present in Kaposi sarcomas and body-cavity-based lymphomas. Here we identify a novel abundant 1.2-kb RNA, polyadenylated nuclear RNA (PAN RNA), encoded by the virus. The majority of cDNAs produced from poly(A)-selected RNA isolated from a human body cavity lymphoma cell line 48 hr after butyrate induction of KSHV lytic replication represented PAN RNA. Within PAN RNA were two 9 and 16 nt stretches with 89% and 94% identity to U1 RNA. A third stretch of 14 nt was 93% complementary to U1. The 5' upstream region of PAN RNA contained both proximal and distal sequence elements characteristic of regulatory regions of U snRNAs, whereas the 3' end was polyadenylylated. PAN RNA was transcribed by RNA polymerase II, lacked a trimethylguanosine cap, and did not associate with polyribosomes. PAN RNA formed a speckled pattern in the nucleus typical of U snRNAs and colocalized with Sm protein. Therefore, PAN represents a new type of RNA, possessing features of both U snRNA and mRNA.

Asunto(s)

Herpesvirus Humano 8/genética , ARN Viral/biosíntesis , Sarcoma de Kaposi/virología , Secuencia de Bases , Linfoma de Burkitt , Butiratos/farmacología , Ácido Butírico , Línea Celular , ADN Complementario , Guanosina/análogos & derivados , Herpesvirus Humano 8/aislamiento & purificación , Herpesvirus Humano 8/fisiología , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genética , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Replicación Viral/efectos de los fármacos

RESUMEN

The lytic cycle of Epstein-Barr virus (EBV) can be activated by transfection of the gene for ZEBRA, a viral basic-zipper (bZip) transcriptional activator. ZEBRA and cellular AP-1 bZip activators, such as c-Fos, have homologous DNA-binding domains, and their DNA-binding specificities overlap. Moreover, EBV latency can also be disrupted by phorbol esters, which act, in part, through AP-1 activators. It is not known whether ZEBRA and AP-1 factors play equivalent roles in the initial stages of reactivation. Here the contribution of ZEBRA's basic DNA recognition domain to disruption of latency was analyzed by comparing ZEBRA with chimeric mutants in which the DNA recognition domain of ZEBRA was replaced with the analogous domain of c-Fos. Chimeric ZEBRA/c-Fos proteins overexpressed in Escherichia coli bound DNA with the specificity of c-Fos; they bound a heptamer AP-1 site and an octamer TPA response element (TRE). ZEBRA bound the AP-1 site and an array of ZEBRA response elements (ZREs). In assays with reporter genes, both ZEBRA and ZEBRA/c-Fos chimeric mutants activated transcription from Zp, a promoter of the ZEBRA gene (BZLF1) that contains the TRE and multiple ZREs. However, despite their capacity to activate reporters bearing Zp, neither ZEBRA nor the c-Fos chimeras activated transcription from Zp in the context of the intact latent viral genome. In contrast, ZEBRA but not ZEBRA/c-Fos chimeras activated Rp, a second viral promoter that controls ZEBRA expression. Hence, transcriptional autostimulation by transfected ZEBRA occurred preferentially at Rp. Both ZEBRA and the ZEBRA/c-Fos chimeras activated transcription from reporters with multimerized AP-1 sites. However, in the context of the virus, only ZEBRA activated the promoters of two early lytic cycle genes, BMRF1 and BMLF1, that contain an AP-1 site. Thus, overexpression of an activator that recognized AP-1 and TRE sites was not sufficient to activate EBV early lytic cycle genes.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Proteínas Virales , Linfocitos B/citología , Linfocitos B/metabolismo , Secuencia de Bases , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética , Latencia del Virus

RESUMEN

ZEBRA has been shown to activate model reporter genes consisting of synthetic oligomerized ZEBRA response elements upstream of a minimal CYC1 promoter fused to beta-galactosidase in the yeast Saccharomyces cerevisiae. Here it is shown that in S. cerevisiae ZEBRA activates transcription of natural Epstein-Barr virus promoters. Two Epstein-Barr virus promoters were shown to be activated by ZEBRA in S. cerevisiae: Zp, the promoter that regulates expression of BZLF1, which encodes ZEBRA; and EAp, the promoter controlling expression of BMRF1, which encodes diffuse early antigen, EA-D. These observations indicate that neither mammalian-specific nor virally encoded coactivators are obligatory for ZEBRA to stimulate expression from these two promoters. Zp was also strongly activated by endogenous yeast factors. EAp was not activated by yeast factors. The results show that in S. cerevisiae and in B cells, ZEBRA dominates the response of EAp; ZEBRA plus endogenous cell factors activate Zp.

Asunto(s)

Antígenos Virales/genética , Proteínas de Unión al ADN/fisiología , Herpesvirus Humano 4/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transactivadores/fisiología , Activación Transcripcional , Proteínas Virales/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ARN/fisiología , Transactivadores/genética

RESUMEN

The ZEBRA protein activates expression of Epstein-Barr virus early-lytic-cycle genes in human B lymphocytes. Here it is shown that ZEBRA also behaves as a sequence-specific transcriptional activator in Saccharomyces cerevisiae. Deletional mutagenesis defined three regions of ZEBRA that participate in activation in S. cerevisiae. These regions are designated YI (amino acids [aa] 1 to 25), YII (aa 51 to 102), and YIII (aa 228 to 245). Two of the three regions of the native ZEBRA protein act together to mediate activation when assayed on ZEBRA binding sites. However, when fused to the DNA binding domain of GAL4 and assayed on GAL4 binding sites, regions YII and YIII were each sufficient to confer activation in S. cerevisiae. Regions of ZEBRA which affected activation in S. cerevisiae were also required in human B lymphocytes. The amino-terminal region of ZEBRA (aa 1 to 98) was required for activation both in S. cerevisiae and in human B cells; deletion of the carboxy-terminal 18 aa also significantly reduced activation in both cell types. Thus, the behavior of ZEBRA in human B cells and S. cerevisiae suggests that the protein contains universal activation motifs that interact with conserved components of the transcription machinery. However, certain deletion mutants of ZEBRA containing mutations in the N-terminal region exhibited discordant behaviors in S. cerevisiae and in B cells. For example, deletion of ZEBRA aa 26 to 51 impaired activation to a great extent in B cells but had little or no effect in S. cerevisiae. The discordant mutants may reflect interactions with a variable domain of a conserved component or unique interactions with specialized components of the basal transcription apparatus in different cells.

Asunto(s)

Linfocitos B , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Herpesvirus Humano 4/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transactivadores/genética , Factores de Transcripción , Transcripción Genética , Proteínas Virales , Secuencia de Bases , Análisis Mutacional de ADN , Proteínas Fúngicas , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión , Eliminación de Secuencia , Especificidad de la Especie

RESUMEN

ZEBRA is an Epstein-Barr virus (EBV) transcriptional activator that mediates a genetic switch between the latent and lytic states of the virus by binding to the promoters of genes involved in lytic DNA replication and activating their transcription. A computer survey revealed that 9 of 23 potential or known ZEBRA-responsive EBV genes contained two or more upstream binding sites; this suggested that ZEBRA can stimulate transcription synergistically. By using a series of synthetic promoters bearing one, two, three, five, and seven upstream recognition sites, we showed that ZEBRA activates transcription synergistically when templates bearing multiple sites were compared with a template bearing a single site. This phenomenon was observed in both uninfected and EBV-infected B-lymphoid cells and in vitro in a HeLa cell nuclear extract. DNase I footprinting was used to show that the synergy was not due to cooperative DNA binding mediated by direct contact between ZEBRA dimers. The in vitro experiments revealed two manifestations of synergy. One was seen when the levels of transcription observed with the same amounts of ZEBRA added to templates bearing different numbers of sites were compared. The other was observed when the two lowest concentrations of ZEBRA that stimulated measurable transcription from any given template were compared. On the basis of both the number of sites and the calculated Kd of ZEBRA for a single site, we estimated that the critical concentration of ZEBRA needed to elicit transcriptional synergy corresponds to a site occupancy of two or three bound ZEBRA dimers. Our results have biologic implications for both the EBV lytic cycle and other processes in which the concentration of an activator changes either temporally or spatially.

Asunto(s)

ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Virales , Herpesvirus Humano 4/genética , Regiones Promotoras Genéticas , TATA Box , Transactivadores/metabolismo , Transcripción Genética , Activación Transcripcional , Proteínas Virales , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/fisiología , Clonación Molecular , ADN Viral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I , Escherichia coli/genética , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Cinética , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Moldes Genéticos

RESUMEN

Lymphoid cell lines were established which stably carry the Epstein-Barr viral (EBV) BZLF1 gene on an extrachromosomal plasmid. These lines, which spontaneously synthesize the BZLF1 gene product, ZEBRA, were examined for expression of EBV genes which were activated by ZEBRA. Cell lines which acquired oriP plasmids without BZLF1 served as controls. The extent of activation differed among derivatives of four cell lines. X50-7 cells, which harbor a standard latent EBV, could be induced by ZEBRA to produce transforming virus; a cellular subclone of this line was induced to express EBV late antigens but did not release transforming virus. In two other cell lines, Raji and ER, ZEBRA activated only a group of early antigens. Using immunofluorescence and immunoblotting with monoclonal antibodies and Northern analysis five EBV early genes were shown to be induced in cells stably transformed by oriP/BZLF1 plasmids. ZEBRA itself was activated; thus BZLF1 is autostimulatory. Four other activated genes were components of the diffuse (EA-D) and restricted (EA-R) early antigens (BMRF1, BMLF1, BHRF1, and BORF2). Stable cell lines with extrachromosomal BZLF1 expression vectors will ultimately be useful in a variety of experiments designed to study regulation of this gene, to analyze the effects of mutations on ZEBRA protein function, and to define the full spectrum of viral and cellular genes which are activated by and interact with the ZEBRA protein.

Asunto(s)

Replicación del ADN , ADN Viral/biosíntesis , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Transactivadores/genética , Replicación Viral/genética , Línea Celular , Transformación Celular Viral , Farmacorresistencia Microbiana/genética , Prueba de Complementación Genética , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Mutación , Plásmidos/genética , ARN Mensajero/metabolismo , Transcripción Genética

RESUMEN

An Epstein-Barr viral gene (ZEBRA) is identified that, in human lymphoblastoid cells, activates a switch causing the virus to shift from the latent to the replicative phase of its life cycle. We have shown that a 2.7-kilobase-pair rearranged Epstein-Barr virus DNA fragment of this gene (BamHI fragment WZhet) induced transient expression of viral replicative antigens and polypeptides when it was transfected into a somatic cell hybrid, which was derived from the fusion of an epithelial line cell with a Burkitt lymphoma cell. We now show that this rearranged WZhet fragment, when introduced stably into lymphoblastoid cells, will activate expression of the complete viral replicative cycle in 1-10% of the lymphoblastoid cells, leading to production of biologically active virions that can immortalize primary lymphocytes. The transfected plasmid appears to be regulated in a manner analogous to the complete Epstein-Barr virus genome.

Asunto(s)

Transformación Celular Viral , Replicación del ADN , Genes Virales , Herpesvirus Humano 4/genética , Transfección , Antígenos Virales/análisis , Línea Celular , Humanos , Linfocitos , Plásmidos , Replicación Viral

RESUMEN

Cloned fragments of the Epstein-Barr virus (EBV) genome were used to examine tissues from 145 patients for the presence of EBV DNA by two techniques: (1) nucleic acid hybridization of cell spots from which the DNA had been extracted in situ and (2) hybridization of DNA that had been transferred to nitrocellulose by Southern blotting. EBV DNA was found in tissues from four adults and five children with American Burkitt's lymphoma, infectious mononucleosis, lymphoma following bone marrow transplant, central nervous system lymphoma, nasopharyngeal carcinoma, and fatal polyclonal B-cell lymphoma following mononucleosis; two patients also had chronic pneumonitis, failure to thrive, and abnormal immune function. Six of the nine patients whose tissues contained EBV DNA had a demonstrable or presumed associated immunologic disorder. EBV DNA was not found in normal tissues or in a variety of hematologic neoplasms and other disorders. Nucleic acid hybridization methods can be used for the routine examination of the association of EBV with lymphomas and other lymphoproliferative syndromes occurring in immunodeficient individuals.

Asunto(s)

ADN Viral/análisis , Herpesvirus Humano 4/genética , Síndromes de Inmunodeficiencia/complicaciones , Linfoma/microbiología , Trastornos Linfoproliferativos/microbiología , Neoplasias Nasofaríngeas/microbiología , Síndrome de Inmunodeficiencia Adquirida/microbiología , Adolescente , Adulto , Neoplasias Encefálicas/microbiología , Linfoma de Burkitt/microbiología , Preescolar , Enzimas de Restricción del ADN , Insuficiencia de Crecimiento/microbiología , Femenino , Genes Virales , Humanos , Lactante , Mononucleosis Infecciosa/microbiología , Trastornos Linfoproliferativos/complicaciones , Masculino , Persona de Mediana Edad , Hibridación de Ácido Nucleico , Neumonía/microbiología

RESUMEN

We recently identified, by means of cotransformation of LTK- cells, a region of the Epstein-Barr virus (EBV) genome (the BamHI K fragment) that encodes or induces an EBV nuclear neoantigen (EBNA) serologically related to the EBNA found in lymphoid cells carrying the entire EBV genome. We now find that a second EBV DNA fragment, BamHI M, is also able to give rise to cotransformed LTK- cells with stable expression of a nuclear antigen. The BamHI K and M fragments have no apparent DNA homology. Many human sera that are reactive to EBNA in Raji cells detect both antigens; however, certain anti-EBNA-positive human sera are discordant and react only with the BamHI M or only with the BamHI K nuclear antigen. Every Raji cell appears to express both "M" and "K" antigens; D98 Raji cells, a somatic cell hybrid, express only "K" antigen. The K antigen is found on metaphase chromosomes of LTK cells and Raji cells. The M-induced antigen is not located on chromosomes when the cells are in metaphase but is present as granules within the nucleus.

Asunto(s)

Antígenos Virales/genética , Núcleo Celular/inmunología , Transformación Celular Neoplásica , Herpesvirus Humano 4/inmunología , Animales , Linfoma de Burkitt , Línea Celular , Cromosomas/inmunología , Enzimas de Restricción del ADN , Técnica del Anticuerpo Fluorescente , Herpesvirus Humano 4/genética , Humanos , Células L/fisiología , Ratones , Hibridación de Ácido Nucleico

RESUMEN

The P3J-HR-1 strain of Epstein-Barr virus (EBV) fails to immortalize human lymphocytes. We wished to understand the nature of the genomic alterations which correlated with the loss of this ability. As a first step, the heterogeneity of DNA molecules in the P3J-HR-1 line was eliminated by cell cloning. Then a physical map was prepared of virion DNA from one cell clone, designated FF452-3. By comparison with the genomes of two EBVs, B95-8 and FF41, which are competent to immortalize lymphocytes, we identified a total of eight modifications of BamHI and EcoRI restriction endonuclease fragments of EBV (FF452-3) DNA consisting of insertions, deletions, or loss of a restriction endonuclease recognition site. To determine which of these alterations might be responsible for the loss of transforming phenotype, we examined homologous DNA fragments of the Jijoye strain of EBV, the progenitor of the HR-1 strain which still retains the ability to immortalize lymphocytes. We also studied viral DNA in lymphocytes transformed in vitro by Jijoye virus. Six of the eight alterations were found both in Jijoye and in clonal HR-1 DNA and were presumably genomic traits characteristic of this lineage of EBV. A small deletion in the BamHI-K fragment of HR-1 DNA was not found in Jijoye virion DNA, but this deletion was present in intracellular Jijoye DNA. Thus only one major genomic lesion in HR-1 DNA, a deletion of at least 2.4 x 10(6) molecular weight of DNA from a fused BamHI-H-Y fragment, consistently distinguished Jijoye DNA from its non-immortalizing P3J-HR-1 derivative. This deletion is likely to affect EBV genes which are directly or indirectly involved in immortalizing lymphocytes.

Asunto(s)

Transformación Celular Viral , Deleción Cromosómica , Herpesvirus Humano 4/genética , Mutación , Secuencia de Bases , Línea Celular , Enzimas de Restricción del ADN , ADN Viral/genética , Genes Virales , Humanos , Linfocitos , Hibridación de Ácido Nucleico

RESUMEN

We wished to learn whether the genomes of strains of EMB isolated from patients with infectious mononucleosis are consistently distinguishable from those of strains from Burkitt's lymphoma. The genome of a new transforming strains (FF41) of EBV isolated from saliva of a patient with uncomplicated infectious mononucleosis was compared with the DNA of B95-8, the only other available virus from mononucleosis. It had been found previously that B95-8 has a deletion of about 8 Md in the region of the physical map represented by the Eco RI C, Hind III D, and Bam HI I fragments. The W91 and HR-1 isolates for Burkitt's lymphoma are not deleted in this region and it had been proposed that additional information was characteristic of EBV isolates from Burkitt's lymphoma. By means of restriction enzyme analysis, blot hybridization experiments and molecular cloning of FF41 DNA we demonstrate that the deletion found in B95-8 is not present in the new mononucleosis isolate. The FF41 genome contains an extra 8 Md of DNA, represented by Bam HI fragments B', W' and I', which are located in a larger Eco RI C fragment. Thus the genome of this salivary isolate contains DNA that had previously been regarded to be unique to strains from Burkitt's lymphoma. It is therefore unlikely that major insertions or deletions in the EBV genome account for differences in disease manifestation following EBV infection.

Asunto(s)

Linfoma de Burkitt/microbiología , ADN Viral/genética , Genes Virales , Herpesvirus Humano 4/genética , Mononucleosis Infecciosa/microbiología , Enzimas de Restricción del ADN/metabolismo , Saliva/microbiología , Especificidad de la Especie