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At least 379 records · Page 21Linked to original sources
PubMed2000

Lytic but not latent replication of epstein-barr virus is associated with PML and induces sequential release of nuclear domain 10 proteins.

Nuclear domains called ND10 (nuclear domain 10) are discrete nuclear protein aggregations characterized by a set of interferon-upregulated proteins including Sp100 and PML, where papova-, adeno-, and herpesviruses begin their transcription and DNA replication. Both the alpha- and betaherpesvirus subfamilies disrupt ND10 upon infection by dispersing and/or destroying ND10-associated proteins. We studied the effect of the gammaherpesvirus Epstein-Barr virus (EBV) on ND10 and its spatial distribution in the nucleus of cells during latency and lytic reactivation. In latently infected Burkitt's lymphoma, lymphoblastoid, and D98/HR1 cells, ND10 were intact, as judged by immunofluorescence localization of PML, Sp100, NDP55, and Daxx. Fluorescent in situ hybridization revealed no association between viral episomes and ND10 during latency, implying that the maintenance replication of EBV, which depends on host cell proliferation, occurs independent of ND10. As in mitosis, the EBV genomes were attached to interphase chromosomes, suggesting that they are unable to move freely within the interchromosomal space and thus unable to associate with the interchromosomally located ND10 or other nuclear domains. Upon lytic activation, ND10 became dispersed in cells expressing lytic proteins. Redistribution of ND10 proteins occurred sequentially at different stages of the lytic cycle, with Sp100, Daxx, and NDP55 dispersed before and PML dispersed after the onset of lytic replication. ND10 remnants were retained until the early stages of lytic replication, and replicating EBV genomes were frequently found beside this nuclear domain; the number of replication domains was usually lower than the average latent virus frequency. Thus, latency does not require or induce interaction of EBV with ND10 for transcription and replication, whereas lytic replication triggers dispersion of ND10 proteins and occurs in close association with PML aggregates. The required movement of chromosome-attached latent EBV episomes to ND10 after reactivation from latency might include physical release of the chromosome-bound episomes. Only episomes contacting ND10 after such a release might be able to begin lytic replication.

Cell Line↗
PubMed1995

Cellular proteins bind to the downstream component of the lytic origin of DNA replication of Epstein-Barr virus.

The lytic origin of DNA replication of Epstein-Barr virus, oriLyt, is a complex eukaryotic origin which is activated during the lytic phase of the viral life cycle. It consists of at least two independent cis-acting components, one of which plays a dual role in transcription and DNA replication. The binding of the viral factor BZLF1, a member of the AP1 family of transcription factors, to this upstream component is crucial for oriLyt function (A. Schepers, D. Pich, and W. Hammerschmidt, EMBO J. 12:3921-3929, 1993). The second cis-acting element, the downstream component of oriLyt, is equally indispensable; however, its function is unknown. In this study, the downstream component was found to be the binding target of several cellular proteins. One could be identified as Sp1 or as a related protein which binds twice to the downstream component of oriLyt. Mutational analysis indicated that Sp1 alone is not directly involved in mediating DNA replication; however, other factors which share the same binding sequence or bind closely to one of the Sp1 binding sites are likely candidates to contribute to a replication protein complex at the downstream component of oriLyt. The sequence requirements for the downstream component are remarkably stringent, indicating that at least one of the putative factors is a sequence-specific DNA-binding protein which is required for the activation of oriLyt.

Base Sequence↗
PubMed2003

Role of CCAAT/enhancer-binding protein alpha (C/EBPalpha) in activation of the Kaposi's sarcoma-associated herpesvirus (KSHV) lytic-cycle replication-associated protein (RAP) promoter in cooperation with the KSHV replication and transcription activator (RTA) and RAP.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT/enhancer binding protein alpha (C/EBPalpha) and p21(CIP-1) expression, resulting in G(0)/G(1) cell cycle arrest during the lytic cycle. RAP and C/EBPalpha are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C/EBPalpha; this analysis showed that one of these sites has a very high affinity for C/EBPalpha. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPalpha can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C/EBPalpha together resulted in a threefold increase in activity over that obtained with C/EBPalpha alone. Importantly, the introduction of exogenous Flag-tagged C/EBPalpha triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C/EBPalpha and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C/EBPalpha and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPalpha, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPalpha or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPalpha-mediated transactivation of the RAP promoter. However, the C/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C/EBPalpha and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C/EBPalpha in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C/EBPalpha was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C/EBPalpha and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPalpha occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C/EBPalpha and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPalpha. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPalpha but that full activity requires more than just the core C/EBP binding site.

Amino Acid Sequence↗
PubMed1980

Integration of bacteriophage Mu at host chromosomal replication forks during lytic development.

The target site for bacteriophage Mu integration in a lytic cycle of infection was investigated. DNA synthesis in five Hfr strains of Escherichia coli K-12 was synchronized by amino acid starvation and was allowed to proceed for 0, 8, or 15 min before infection. The Hfr cells were then infected with Mu and were subsequently mated with nonimmune F- recipient cells. Mating was interrupted mechanically at 5-min intervals and samples were assayed for infective centers. Conjugal transfer of Mu was delayed in Hfr strains that have transfer origins 15 map units or more from the E. coli replication origin, and the delays increased as the distance between an Hfr point of origin and the replication origin increased. When a gene A mutant of Mu was used for the infection, no infective centers were generated. Infection with a gene B mutant resulted in infective center formation only after long periods of mating. These data are most consistent with a model in which infecting Mu DNA or its progeny integrate at host chromosomal replication forks.

Bacteriophage mu↗
PubMed2001

Spontaneous activation of the lytic cycle in cells infected with a recombinant Kaposi's sarcoma-associated virus.

The genetic analysis of human herpesvirus 8 (HHV8), also termed Kaposi's sarcoma-associated virus, has been hampered by severe difficulties in producing infectious viral particles and modifying the viral genome. In this article, we report the successful cloning of the HHV8 complete genome onto a prokaryotic F-plasmid replicon which allows the propagation of the recombinant viral DNA in Escherichia coli. The insertion of the F-plasmid into the HHV8 genome interrupts the ORF56 gene, whose expression product-by homology with the Epstein-Barr virus BSLF1 gene--is supposed to be necessary for lytic DNA replication. After introduction of the recombinant HHV8 DNA into 293 cells, early viral antigens are expressed, suggesting that spontaneous lytic replication is initiated. However, completion of the lytic program is prevented by the absence of the ORF56 protein, and a quasi-latent state is established. Upon reintroduction of the ORF56 viral gene, the block is overcome and infectious HHV8 virions are produced. As the recombinant HHV8 genome can be easily modified in E. coli, this experimental system opens the way to an extensive genetic analysis of other HHV8 functions.

Cell Line↗
PubMed1996

Activation of oriLyt, the lytic origin of DNA replication of Epstein-Barr virus, by BZLF1.

oriLyt, the cis-acting element of the lytic origin of DNA replication of Epstein-Barr virus, is activated by the viral transactivator BZLF1 which belongs to the extended bZIP class of transcription factors. Seven binding sites for BZLF1, so-called ZRE sites, are located within oriLyt. By mutational analysis of individual ZRE sites, we found that lytic DNA replication is dependent on only four of these sites which colocate with the promoter of the BHLF1 gene. The remaining three ZRE sites distal to the BHLF1 promoter were dispensable for DNA replication and did not contribute to long-range transcriptional activation of this promoter by BZLF1. This finding indicated that a similar set of ZRE sites is involved in DNA replication and transcriptional activation. To determine the function of BZLF1 in DNA replication, BZLF1 mutants with successive deletions in the transactivation domain were analyzed in replication assays. Unexpectedly, most BZLF1 mutants which failed to support DNA replication were found to be equally defective in transcriptional activation. Therefore, similar trans-acting domains of BZLF1 are involved both in replication and in transcription.

Binding Sites↗
PubMed2004

Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression.

A hallmark of all herpesvirus is the ability to exist in either a latent, or lytic, state of replication, enabling the lifelong infection of its host. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) can efficiently establish a latent infection in a variety of cell types in vitro, making it a valuable model for the study of latency and reactivation. To facilitate the identification of KSHV lytic replication, and allow subsequent experiments with live cells, a recombinant virus, rKSHV.219, was constructed using JSC-1 cells that expresses the red fluorescent protein (RFP) from the KSHV lytic PAN promoter, the green fluorescent protein (GFP) from the EF-1alpha promoter, and with the gene for puromycin resistance as a selectable marker. rKSHV.219 from JSC-1 cells was used to infect Vero cells for purification of the recombinant virus. Vero cells were also used for the production of rKSHV.219 at levels of 10(5)-10(6) infectious units (IU) of virus per milliliter using a combination of KSHV/RTA expressed from a baculovirus vector, BacK50, and butyrate. Virus produced from Vero cells was used to infect human fibroblasts (HF), 293, DU145, T24, HaCaT, and HEp-2 cells, and in all cells except 293 cells, only a latent infection was established with GFP expression, but no RFP expression. In 293 cells, 10-15% of cells showed lytic gene expression. Both primary and immortalized microvascular endothelial cells (MVEC) were also infected with rKSHV.219, and reduced spontaneous lytic replication was found in immortalized cells. In all cells used in this study, rKSHV.219 efficiently established latent infections from which the virus could be reactivated to productive lytic replication. This work also demonstrated strong synergy between KSHV/RTA and butyrate for the activation of KSHV lytic replication and the production of infectious virus.

Animals↗
PubMed1998

The position of the ZEBRA activation domain does not influence its biological activity.

Epstein-Barr virus (EBV) is a human herpesvirus which latently infects B lymphocytes. EBV encodes a unique transcriptional activator, known as ZEBRA, which can disrupt viral latency in B cells and induce lytic viral replication. Furthermore, ZEBRA has been shown to bind at the EBV origin of lytic replication, and is necessary for viral DNA replication to occur. Previously we demonstrated that heterologous activation domains can fully substitute for the ZEBRA activation domain. Here we extend those results by showing that the position of the ZEBRA activation domain or a heterologous replacement domain does not influence its ability to function in the disruption of EBV latency. In this study three novel clones were constructed in which the ZEBRA activation region was repositioned to the carboxy terminus of the protein. These mutants were used to demonstrate that the ability of ZEBRA's wild type domain to function in the complex biological process of virus activation is not compromised by altering its position within the protein.

B-Lymphocytes↗
PubMed2004

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: cis-acting requirements for replication and ori-Lyt-associated RNA transcription.

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors such as virally encoded origin-binding protein and DNA replication enzymes. Recently, the origins of lytic DNA replication (ori-Lyt) in Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified and a virally encoded bZip protein, K8, has been shown to specifically bind to the origin. To map cis-acting elements within KSHV ori-Lyt that are required for DNA replication function and to define the nature of K8 bZip protein binding to the origin, we constructed consecutive internal deletion mutations across the core domain of a KSHV ori-Lyt and tested them for DNA replication function in a transient replication assay. This mutagenesis study allowed the identification of four components within the ori-Lyt, and all were indispensable for ori-Lyt function. The first component contains eight CCAAT/enhancer binding protein (C/EBP) binding motifs that organize as four spaced C/EBP palindromes. Each palindrome contains two head-to-head CCAAT consensus motifs that are separated by a 13- or 12-bp space sequence. Substitution mutagenesis of these C/EBP motifs showed that these C/EBP palindromes are required for both K8 binding and ori-Lyt-dependent DNA replication. The second component is an 18-bp AT palindrome, which is essential for ori-Lyt function. The third component was determined to be a 32-bp previously unidentified sequence and is required for DNA replication. The last component consists of an open reading frame 50 (ORF50)/Rta responsive element (RRE) and a TATA box. We showed that the binding of an ORF50/Rta protein to the RRE was essential for ori-Lyt-dependent DNA replication. The presence of a functional RRE and a downstream TATA box suggested that this region serves as an ORF50/Rta-dependent promoter and a transcription event may be necessary for ori-Lyt-dependent DNA replication. Using a luciferase reporter system, we demonstrated that the region of the RRE and TATA box constitutes an ORF50/Rta-dependent promoter. Furthermore, a polyadenylated RNA of 1.4 kb was identified downstream of the promoter.

Amino Acid Sequence↗
PubMed2006

Contribution of C/EBP proteins to Epstein-Barr virus lytic gene expression and replication in epithelial cells.

The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPbeta and had limited C/EBPalpha expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPalpha and C/EBPbeta. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPbeta expression and a prolonged increase in C/EBPalpha expression. In AGS/BX1 cells, endogenous C/EBPalpha and C/EBPbeta proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPalpha and C/EBPbeta proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPbeta and C/EBPalpha expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.

Antigens, Viral↗
PubMed2002

De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells.

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is central to the pathogenesis of the endothelial neoplasm Kaposi's sarcoma (KS) and is also linked to the rare B-cell tumor known as primary effusion lymphoma (PEL). Latently infected PEL cell lines can be induced to enter the lytic cycle and produce KSHV virions. However, such cells do not support de novo infection or serial propagation of KSHV. These limitations have prevented the development of systems for the genetic analysis of KSHV and have impeded a deeper understanding of KS pathogenesis. Here we show that human dermal microvascular endothelial cells immortalized by expression of telomerase can be readily infected by KSHV virions produced by PEL cells. Infection is predominantly latent, but a small subpopulation enters the lytic cycle spontaneously. Phorbol ester (tetradecanoyl phorbol acetate [TPA]) treatment of latently infected cells leads to enhanced induction of lytic KSHV replication, resulting in foci of cytopathic effect. There is no cytopathic effect or viral DNA expansion when infected TIME cells (telomerase-immortalized microvascular endothelial cells) are TPA induced in the presence of phosphonoacetic acid (PAA), an inhibitor of herpesvirus replication. Supernatants from phorbol-induced cultures transfer latent KSHV infection to uninfected cells, which can likewise be induced to undergo lytic replication by TPA treatment, and the virus can be further serially transmitted. Serial passage of the virus in TIME cells is completely inhibited when TPA treatment is done in the presence of PAA. Latently infected endothelial cells do not undergo major morphological changes or growth transformation, and infection is lost from the culture upon serial passage. This behavior faithfully recapitulates the behavior of spindle cells explanted from primary KS biopsies, strongly supporting the biological relevance of this culture system. These findings suggest that either the stability or the growth-deregulatory potential of the KSHV latency program in endothelial cells is more limited than might be predicted by analogy with other oncogenic viruses.

Caenorhabditis elegans Proteins↗
PubMed1996

Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma.

Kaposi sarcoma (KS) is the leading neoplasm of HIV-infected patients and is also found in several HIV-negative populations. Recently, DNA sequences from a novel herpesvirus, termed KS-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8) have been identified within KS tissue from both HIV-positive and HIV-negative cases; infection with this agent has been proposed as a possible factor in the etiology or pathogenesis of the tumor. Here we have examined the pattern of KSHV/HHV-8 gene expression in KS and find it to be highly restricted. We identify and characterize two small transcripts that represent the bulk of the virus-specific RNA transcribed from over 120 kb of the KSHV genome in infected cells. One transcript is predicted to encode a small membrane protein; the other is an unusual polyadenylylated RNA that accumulates in the nucleus to high copy number. This pattern of viral gene expression suggests that most infected cells in KS are latently infected, with lytic viral replication likely restricted to a much smaller subpopulation of cells. These findings have implications for the therapeutic utility of currently available antiviral drugs targeted against the lytic replication cycle.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) contains two functional lytic origins of DNA replication.

We used a transient-transfection replication assay to identify two functional copies of the human herpesvirus 8 (HHV8) lytic origin of DNA replication (oriLyt). BCLB-1 cells were transfected with HHV8 subgenomic fragments containing the putative lytic origin along with a plasmid expressing viral transactivator open reading frame (ORF) 50. The HHV8 left-end oriLyt (oriLyt-L) lies between ORFs K4.2 and K5 and is composed of a region encoding various transcription factor binding sites and an A+T-rich region and a G+C repeat region. The right-end oriLyt (oriLyt-R) maps between ORF 69 and vFLIP, a region similar to the RRV oriLyt, and is an inverted duplication of oriLyt-L.

Cell Line↗
PubMed1999

Redox control of Epstein-Barr virus replication by human thioredoxin/ATL-derived factor: differential regulation of lytic and latent infection.

Human thioredoxin (hTRX)/adult T-cell leukemia (ATL)-derived factor (ADF) was originally reported as an interleukin-2 (IL-2) receptor-alpha-inducing factor produced by human T-cell lymphotropic virus-1-positive (HTLV-1+) cell lines. Growing evidence indicates that hTRX/ADF plays important roles in cellular responses against oxidative stress and is involved in a variety of cellular functions. A high level of hTRX/ADF expression is also observed in other human virus-infected cell lines including those of Epstein-Barr virus (EBV) and human papillomavirus. In this report, we analyzed the effect of hTRX/ADF on lytic amplification and persistent replication of EBV as a model for lytic versus latent phase of viral replication in host cells. Addition of hTRX/ADF clearly suppressed lytic replication of EBV in Raji cells and B95-8 cells induced to the lytic phase of 12-O-tetradecanoylphorbol-13-acetate (TPA), and it prevented the death of these cells evoked by the lytic induction. In contrast, hTRX/ADF did not have any effect on persistent replication in the latent phase. These data indicated that hTRX/ADF prevents EBV-transformed cells from proceeding into the lytic phase and regulates cohabitation of EBV and its host cells.

Carcinogens↗
PubMed1991

Molecular basis of latency in pathogenic human viruses.

Several human viruses are able to latently infect specific target cell populations in vivo. Analysis of the replication cycles of herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus suggests that the latent infections established by these human pathogens primarily result from a lack of host factors critical for the expression of viral early gene products. The subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of these viral regulatory proteins and lead to a burst of lytic viral replication. Latency in these eukaryotic viruses therefore contrasts with latency in bacteriophage, which is maintained primarily by the expression of virally encoded repressors of lytic replication.

HIV-1↗
PubMed1990

Identification of the lytic origin of DNA replication in human cytomegalovirus by a novel approach utilizing ganciclovir-induced chain termination.

Infection with human cytomegalovirus in the presence of the antiviral nucleotide analog ganciclovir results in continuing low-level viral DNA synthesis and the accumulation of relatively small fragments of double-stranded progency DNA. These fragments consistently proved to represent amplification of sequences from only one small section of the viral genome (EcoRI-V) lying near the center of the unique L segment. Further mapping revealed that the viral sequences represented in these fragments occurred in gradients of abundance that decreased in both directions from a point near 0.35 to 0.4 map unit. The proportion of amplified sequences increased with both time after infection and dosage of ganciclovir used. We conclude that the primary lytic cycle replication origin of human cytomegalovirus lies within a 3- to 4-kb region immediately upstream and to the right of the promoter for the single-stranded DNA-binding protein (DB140). The amplified origin-containing DNA molecules appeared to arise by continuing rounds of bidirectional initiation on truncated fragments of the genome that were generated as a result of chain termination effects induced by the incorporation of ganciclovir into the viral DNA. Inspection of the DNA sequence in the vicinity of ori-Lyt revealed a large complex upstream region that may be a noncoding intergenic domain and that bears no homology to any previously described herpesvirus origin. This 2.5-kb region includes many duplicated and inverted sequences, together with consensus CRE/ATF and other transcription factor-binding sites, and an interesting set of 23 copies of an interspersed decamer consensus element AAAACACCGT that is also conserved at the equivalent locus in simian cytomegalovirus. This work represents the first identification of an origin domain in a cytomegalovirus genome and is the first demonstration of a bidirectional mechanism for any herpesvirus lytic cycle origin.

Base Sequence↗
PubMed2001

Epstein-Barr virus immediate-early protein BRLF1 induces the lytic form of viral replication through a mechanism involving phosphatidylinositol-3 kinase activation.

Expression of the Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 induces the lytic form of viral replication in most EBV-positive cell lines. BRLF1 is a transcriptional activator that binds directly to a GC-rich motif present in some EBV lytic gene promoters. However, BRLF1 activates transcription of the other IE protein, BZLF1, through an indirect mechanism which we previously showed to require activation of the stress mitogen-activated protein kinases. Here we demonstrate that BRLF1 activates phosphatidylinositol-3 (PI3) kinase signaling in host cells. We show that the specific PI3 kinase inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus vector to induce the lytic form of EBV infection, while not affecting lytic infection induced by a BZLF1 adenovirus vector. Furthermore, we demonstrate that the requirement for PI3 kinase activation in BRLF1-induced transcriptional activation is promoter dependent. BRLF1 activation of the SM early promoter (which occurs through a direct binding mechanism) does not require PI3 kinase activation, whereas activation of the IE BZLF1 and early BMRF1 promoters requires PI3 kinase activation. Thus, there are clearly two separate mechanisms by which BRLF1 induces transcriptional activation.

DNA-Binding Proteins↗