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J S Pagano

Publications and source records attributed to J S Pagano.

At least 19 recordsLinked to original sources
PubMed2001

Interferon regulatory factor 7 mediates activation of Tap-2 by Epstein-Barr virus latent membrane protein 1.

Transporter associated with antigen processing 2 (Tap-2) is responsible for ATP-dependent transport of peptides from the cytosol to the endoplasmic reticulum, where peptides bind to newly synthesized human leukocyte antigen (HLA) class I molecules, which are essential for cellular immune responses. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) has been shown to induce the expression of Tap-2. In this study, the induction of endogenous Tap-2 by LMP-1 is shown to be associated with and requires the expression of interferon regulatory factor 7 (IRF-7). In DG75 Burkitt's lymphoma (BL) cells, in which LMP-1 induces the expression of IRF-7, LMP-1 induced endogenous Tap-2, and ectopic expression of IRF-7 could enhance the induction. In Akata BL cells, in which LMP-1 could not induce IRF-7, LMP-1 could not induce Tap-2. Addition of IRF-7, which complements the defect in Akata cells, could stimulate the expression of Tap-2. Furthermore, LMP-1 and IRF-7A but not other IRF-7 splicing variants could activate endogenous Tap-2. A Tap-2 promoter reporter construct could be activated by the overexpression of IRF-7A. The activation could be specifically enhanced by LMP-1 and was dependent on an intact interferon-stimulated response element (ISRE) present in the Tap-2 promoter. Also, IRF-7 can bind to the Tap-2 promoter under physiological conditions in vivo, as shown by formaldehyde cross-linking, as well as to the Tap-2 ISRE in vitro, as shown by gel mobility shift assays. Furthermore, LMP-1 facilitates the phosphorylation and nuclear translocation of IRF-7. These data point to the role of IRF-7 as a secondary mediator of LMP-1-activated signal transduction for Tap-2 as follows: LMP-1 stimulates the expression of IRF-7 and facilitates its phosphorylation and nuclear translocation, and then the activated IRF-7 mediates the activation of the cellular Tap-2 gene. The induction of Tap-2 by IRF-7 and LMP-1 may have an important implication for the immune response to EBV and its persistence in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Aspirin inhibits tumor cell invasiveness induced by Epstein-Barr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression.

Matrix metalloproteinases (MMPs) are thought to play crucial roles in tumor invasion and metastasis. Because we have shown that EBV latent membrane protein 1 (LMP1) enhances MMP-9 expression by activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 (T. Yoshizaki, et al., Proc. Natl. Acad. Sci. USA, 95: 3621-3626, 1998), we therefore tested whether up-regulation of MMP-9 by LMP1 could be correlated with enhanced invasiveness of tumor cells in vitro. Whether aspirin and sodium salicylate could reduce invasiveness and whether LMP1 could enhance MMP-9 expression in tumors grown in nude mice were also tested. C33A cells stably expressing LMP1 had increased expression of MMP-9 and showed greater invasion through reconstituted basement membrane compared with vector-transfected C33A cells (P < 0.02). Treatment with aspirin or sodium salicylate inhibited invasiveness of the LMP1-expressing C33A cells (P < 0.03) and suppressed both the LMP1-induced MMP-9 expression in zymographic analyses and LMP1-induced MMP-9 promoter activity in CAT reporter assays (P < 0.01). Endogenous MMP-2 levels were unaffected by either drug. Both drugs repressed the CAT activity of the truncated MMP-9 promoter construct, which only contained a binding site for AP-1, to the basal level (P < 0.05). Moreover, EMSA indicated that the effects of the salicylates were through the inhibition of not only NF-kappaB but also AP-1 binding activity. Inhibitory effect of salicylates could be reversed by p50/p65 subunits of NF-kappaB or c-Jun overexpression. The inhibitory effect of aspirin on NF-kappaB activity was attributable to the inhibition of IkappaB kinase activity. Finally, tumors derived from C33A cells stably expressing LMP1 grown in nude mice showed enhanced MMP-9 levels compared with tumors derived from vector-transfected C33A cells. This enhancement was inhibited by treatment of the mice with aspirin. These results suggest that aspirin may be able to suppress invasion and metastasis of EBV-associated tumors that express LMP1 by suppression of MMP-9.

Animals↗
PubMed2000

Interferon regulatory factor 7 is induced by Epstein-Barr virus latent membrane protein 1.

Infection by Epstein-Barr virus (EBV) generates several types of latency with different profiles of gene expression but with expression of Epstein-Barr nuclear antigen 1 (EBNA-1) in common. The BamHI Q promoter (Qp) is used for the transcription of EBNA-1 mRNA in type I latency, which is an EBV infection state exemplified by Burkitt's lymphoma (BL). However, Qp is inactive in type III latency, and other promoters (C/Wp) are used for transcription of EBNA-1, which raises the question of how usage of these promoters is governed. Interferon (IFN) regulatory factor 7 (IRF-7) was identified first as a negative regulator of Qp. Expression of IRF-7 is associated with EBV type III latency, where Qp is inactive, but not with type I latency, raising the possibility that a viral gene product(s) expressed in type III latency might induce IRF-7 and repress Qp. Here, detailed analysis of the expression of IRF-7 revealed that it is associated with the expression of EBV latent membrane protein 1 (LMP-1) and that LMP-1 stimulates the expression of IRF-7 in type III latency in which Qp is inactive. In contrast, LMP-1 is not expressed in type I latency cells in which Qp is active. LMP-1 represses the constitutive activity of Qp reporter constructs. Mutational analysis of Qp reporter constructs revealed that the Qp IFN-stimulated response element (ISRE) is essential for the repression by LMP-1. Furthermore, LMP-1 reduced EBNA-1 mRNA derived from Qp only in type I cells in which IRF-7 could be induced. Finally, IFN-alpha, but not IFN-gamma, repressed endogenous Qp activity, which is consistent with the ability of IFN-alpha to induce IRF-7. Thus, IRF-7 may mediate repression of Qp by LMP-1. The induction of IRF-7 by LMP-1 may be relevant to the silencing of Qp in EBV type III latency.

Blotting, Western↗
PubMed1999

Matrix metalloproteinase 9 is induced by the Epstein-Barr virus BZLF1 transactivator.

Type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP9 are implicated in tumor invasion and metastasis. In patients with nasopharyngeal carcinoma (NPC), poor prognosis due to development of local and distant metastasis has been reported to be predicted by antibody titers against the Z protein which is an AP-1 family transcription factor encoded by the EBV BZLF1 immediate-early gene. Here we report that in patients with NPC, expression of Z in tumor cells correlates with advanced cervical lymph node metastasis which may suggest that Z affects tumor invasion and metastasis. We therefore tested if Z would induce expression of type IV collagenases. Transfection of Z expression plasmid into the C33A epithelial cell line increased expression of MMP9, but MMP2 expression was unaltered. Mutational analysis of the Z protein revealed that, in addition to all three functional domains of Z (dimerization domain, DNA binding domain, and activation domain), the carboxyl terminal 17 amino acids which stabilize the Z protein were necessary for induction of MMP9 expression. Analysis of the MMP9 promoter demonstrated that only AP-1 site close to the transcriptional start-site was essential for transactivation by Z. Previously we reported that Epstein-Barr virus latent membrane protein 1 (LMP1) stimulates MMP9 expression (Yoshizaki et al. Proc. Natl. Acad. Sci. 1998; 95: 3621-6). Thus, Z together with LMP1 may contribute to invasion and metastasis of NPC by inducing expression of MMP9.

DNA-Binding Proteins↗
PubMed1999

Expression of EBNA-1 mRNA is regulated by cell cycle during Epstein-Barr virus type I latency.

Expression of EBNA-1 protein is required for the establishment and maintenance of the Epstein-Barr virus (EBV) genome during latent infection. During type I latency, the BamHI Q promoter (Qp) gives rise to EBNA-1 expression. The dominant regulatory mechanism for Qp appears to be mediated through the Q locus, located immediately downstream of the transcription start site. Binding of EBNA-1 to the Q locus represses Qp constitutive activity, and repression has been reported to be overcome by an E2F family member that binds to the Q locus and displaces EBNA-1 (N. S. Sung, J. Wilson, M. Davenport, N. D. Sista, and J. S. Pagano, Mol. Cell. Biol. 14:7144-7152, 1994). These data suggest that the final outcome of Qp activity is reciprocally controlled by EBNA-1 and E2F. Since E2F activity is cell cycle regulated, Qp activity and EBNA-1 expression are predicted to be regulated in a cell cycle-dependent manner. Proliferation of the type I latently infected cell line, Akata, was synchronized with the use of the G2/M blocking agent nocodazole. From 65 to 75% of cells could be made to peak in S phase without evidence of viral reactivation. Following release from G2/M block, EBNA-1 mRNA levels declined as the synchronized cells entered the G1 phase of the cell cycle. As cells proceeded into S phase, EBNA-1 mRNA levels increased parallel to the peak in cell numbers in S phase. However, EBNA-1 protein levels showed no detectable change during the cell cycle, most likely due to the protein's long half-life as estimated by inhibition of protein synthesis by cycloheximide. Finally, in Qp luciferase reporter assays, the activity of Qp was shown to be regulated by cell cycle and to be dependent on the E2F sites within the Q locus. These findings demonstrate that transcriptional activity of Qp is cell cycle regulated and indicated that E2F serves as the stimulus for this regulation.

Animals↗
PubMed1999

Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus latent membrane protein 1 C-terminal activation regions 1 and 2.

Nasopharyngeal carcinoma (NPC), which is closely associated with the Epstein-Barr virus (EBV), is a highly metastatic malignant tumor. An important activity in tumor invasion and metastasis is that of the 92-kDa type IV collagenase or gelatinase, matrix metalloproteinase 9 (MMP-9), which mediates the degradation of the basement membrane and extracellular matrix. The expression of MMP-9 has been shown to be enhanced by the EBV oncoprotein, latent membrane protein 1 (LMP-1). LMP-1, which is expressed in NPC, has two essential signaling domains within the carboxy terminus, termed C-terminal activation regions 1 (CTAR-1) and CTAR-2. This study reveals that either signaling domain can activate the MMP-9 promoter and induce MMP-9 activity; however, LMP-1 deletion mutants lacking either CTAR-1 or CTAR-2 had a decreased ability to induce MMP-9 expression. The deletion of both activation regions completely abolished the induction of MMP-9 activity, while the cotransfection of both the CTAR-1 and CTAR-2 deletion mutants restored MMP-9 activity to levels produced by wild-type LMP-1. The NF-kappaB and activator protein 1 (AP-1) binding sites in the MMP-9 promoter were essential for the activation of MMP-9 gene expression by both CTAR-1 and CTAR-2. The induction of MMP-9 expression by LMP-1 and both CTAR-1 and CTAR-2 mutants was blocked by the overexpression of IkappaB. The tumor necrosis factor receptor-associated factor (TRAF) pathway also contributed to the activation of the MMP-9 promoter as shown by the use of TRAF-2 and TRAF-3 dominant-negative constructs. These data indicate that the activation of both the NF-kappaB and AP-1 pathways by LMP-1, CTAR-1, and CTAR-2 is necessary for the activation of MMP-9 expression. In NPC, LMP-1 may contribute to invasiveness and metastasis through the induction of MMP-9 transcription and enzymatic activity.

Binding Sites↗
PubMed1999

Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.

Acyclovir↗
PubMed1999

Interferon regulatory factor 2 represses the Epstein-Barr virus BamHI Q latency promoter in type III latency.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is the essential protein for maintenance of the EBV episome and establishment of latency. The BamHI Q promoter (Qp) is used for the transcription of EBNA-1 mRNA in type I and type II latency, which are EBV infection states exemplified by Burkitt's lymphoma and nasopharyngeal carcinoma. However, Qp is inactive in type III latency, and other promoters (the BamHI C promoter and/or the BamHI W promoter) are used for EBNA-1. The involvement of interferon regulatory factors (IRFs) in the regulation of Qp is suggested by the presence of an essential interferon-stimulated response element (ISRE) in the promoter. In this work, expression of IRF-2 is shown to be inversely associated with Qp status, i.e., IRF-2 levels are high in type III latency (when Qp is inactive) and low in type I latency (when Qp is active). Also, IRF-2 is identified by electrophoretic mobility shift assay as the major protein binding to the Qp ISRE in type III latency. In transient transfection assays, IRF-2 represses the activity of Qp-reporter constructs. Overexpression of IRF-2 in a type I latency cell line did not activate the endogenous Qp but marginally reduced the EBNA-1 mRNA level. Switching from type III latency (Qp inactive) to type II latency (Qp active), as produced by cell fusion, is directly associated with greatly reduced expression of IRF-2. These data strongly suggest that IRF-2 is a negative regulator of Qp and may contribute to the silencing of Qp in type III latency.

DNA-Binding Proteins↗
PubMed1998

The expression of matrix metalloproteinase 9 is enhanced by Epstein-Barr virus latent membrane protein 1.

Matrix metalloproteinases (MMPs) are frequently expressed in malignant tumor cells and are thought to play crucial roles in tumor invasion and metastasis. Here we report that expression of MMP9 is increased in Epstein-Barr virus (EBV)-infected type III latency lymphoma cell lines, but not in type I lines where latent viral gene expression is highly restricted. Type III cell lines express abundant EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as the other latency proteins including the transcriptional factor, EBV nuclear antigen 2, which is also required for cell immortalization. Transfection of an LMP1 expression plasmid in the C33A cell line increased MMP9 expression, whereas overexpression of EBV nuclear antigen 2 did not. Three motifs, homologous to the binding sites of NF-kappaB, SP-1, and AP-1 proteins, contribute to induction of the MMP9 promoter by 12-O-tetradecanoyl-phorbol-13-acetate and tumor necrosis factor alpha. Here we report that binding sites for NF-kappaB, SP-1, and AP-1 also contribute to induction of the MMP9 promoter by the viral protein, LMP1, mainly through the NF-kappaB and, to a lesser extent, the SP-1 and AP-1 sites. Moreover the AP-1 binding site is essential in that mutation of it abolished reporter gene induction by LMP1. The enhancement of MMP9 expression was blocked by cotransfection of an IkappaB expression plasmid. Thus in addition to its transforming properties, the oncoprotein LMP1 may contribute to invasiveness and metastasis of EBV-associated tumors such as nasopharyngeal carcinoma.

Binding Sites↗
PubMed1998

The antiviral agent cidofovir [(S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine] has pronounced activity against nasopharyngeal carcinoma grown in nude mice.

The effect of the antiviral agent (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (cidofovir) on the EBV-associated tumor nasopharyngeal carcinoma (NPC) was evaluated in NPC xenografts in athymic mice. Intratumoral injection arrested tumor growth within 1 week, and by 4 weeks, tumors regressed to 8-75% (39 +/- 33%) of the original size, whereas control tumors injected with PBS grew to 282 +/- 25% of the original size. Ganciclovir slowed but did not arrest or cause regression of tumor growth. A striking antitumor effect was also produced by systemic administration; at 4 weeks, tumors were 79 +/- 49% of the original size, compared with 635 +/- 91% for the controls. Widespread apoptosis was detected after treatment for 2-6 days in C15 as well as two other NPC xenografts, C17 and C18; the latter NPCs have mutations in the p53 gene. These data indicate that cidofovir induces rapid cell death through apoptosis in EBV-transformed epithelial cells.

Adolescent↗
PubMed1998

The Epstein-Barr virus immediate-early gene product, BRLF1, interacts with the retinoblastoma protein during the viral lytic cycle.

Retinoblastoma protein (Rb) is a key regulator of cellular proliferation, controlling entry into G1/S in the cell cycle, largely through its action in binding the cellular transcription factor E2F, which activates genes important in DNA synthesis. Small DNA tumor viruses encode gene products which can functionally inactivate Rb, promoting cellular proliferation and viral DNA synthesis. In this study, the Epstein-Barr virus (EBV) immediate-early lytic gene product, BRLF1 (R), is shown to bind Rb in vivo, shortly after induction of the viral lytic cycle in EBV-infected Akata cells. Furthermore, the temporal kinetics of R-Rb interaction correlate with displacement of E2F1 from Rb. Mapping of the domains required for the interaction of R and Rb proteins reveals that R binds specifically to the N terminus of Rb, outside the Rb pocket, and that the first 200 amino acids of R are required for this interaction. The interaction of R and Rb may initiate cell cycle progression and facilitate viral DNA synthesis during lytic replication.

Base Sequence↗
PubMed1998

The Epstein-Barr virus (EBV) SM protein enhances pre-mRNA processing of the EBV DNA polymerase transcript.

The Epstein-Barr virus (EBV) DNA polymerase (pol) mRNA, which contains a noncanonical polyadenylation signal, UAUAAA, is cleaved and polyadenylated inefficiently (S. C. S. Key and J. S. Pagano, Virology 234:147-159, 1997). We postulated that the EBV early proteins SM and M, which appear to act posttranscriptionally and are homologs of herpes simplex virus (HSV) ICP27, might compensate for the inefficient processing of pol pre-mRNA. Here we show that the SM and M proteins interact with each other in vitro. In addition, glutathione S-transferase-SM/M fusion proteins precipitate the heterogeneous ribonucleoprotein (hnRNP) C1 splicing protein. Further, the SM protein is coimmunoprecipitated from SM-expressing cell extracts with an antibody to the hnRNP A1/A2 proteins, which are splicing and nuclear shuttling proteins. Finally, the amount of processed EBV DNA polymerase mRNA was increased three- to fourfold in a HeLa cell line expressing SM; this increase was not due to enhanced transcription. Thus, inefficient processing of EBV pol RNA by cellular cleavage and polyadenylation factors appears to be compensated for and may be regulated by the early EBV protein, SM, perhaps via RNA 3'-end formation.

DNA-Directed DNA Polymerase↗
PubMed1997

A noncanonical poly(A) signal, UAUAAA, and flanking elements in Epstein-Barr virus DNA polymerase mRNA function in cleavage and polyadenylation assays.

Two forms of the Epstein-Barr virus DNA polymerase (pol) mRNA (3.7 and 5.1 kb) have been detected, neither of which contains a canonical poly(A) signal. The 5.1-kb pol mRNA, which contains a rare poly(A) signal, UAUAAA, studied only in transcripts of Hepadnaviridae and a plant pararetrovirus, was analyzed in cleavage and polyadenylation assays. Incubation of the pol transcript in cell extracts produced relatively low efficiency of cleavage (12 to 14%), which was improved by conversion of the poly(A) signal to AAUAAA. Deletion of the UAUAAA signal abolished cleavage and polyadenylation. An auxiliary element, UUUGUA, 3-8 nt upstream of the poly(A) signal and two downstream core elements, a GU-rich sequence 36-46 nt, and an AUUUGUGU sequence 47-53 nt downstream of the signal (8-19 nt and 20-28 nt downstream of cleavage site) facilitated processing of pol mRNA. Replacement of sequences near the cleavage/poly(A) site affected cleavage accuracy. Binding of the 64-kDa cleavage stimulatory factor to the U-rich as well as the GU-rich elements correlated with cleavage efficiency. Thus the UAUAAA hexanucleotide plus the other cis-acting elements are clearly functional in the native pol mRNA, but are relatively inefficient. Implications of the use of an anomalous poly(A) signal and its elements are discussed.

Binding Sites↗
PubMed1997

IRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency.

The Epstein-Barr virus (EBV) BamHI Q promoter (Qp) is the only promoter used for the transcription of Epstein-Barr virus nuclear antigen 1 (EBNA-1) mRNA in cells in the most restricted (type I) latent infection state. However, Qp is inactive in type III latency. With the use of the yeast one-hybrid system, a new cellular gene has been identified that encodes proteins which bind to sequence in Qp. The deduced amino acid sequence of the gene has significant homology to the interferon regulatory factors (IRFs). This new gene and products including two splicing variants are designated IRF-7A, IRF-7B, and IRF-7C. The expression of IRF-7 is predominantly in spleen, thymus, and peripheral blood leukocytes (PBL). IRF-7 proteins were identified in primary PBL with specific antiserum against IRF-7B protein. IRF-7s can bind to interferon-stimulated response element (ISRE) sequence and repress transcriptional activation by both interferon and IRF-1. Additionally, a functional viral ISRE sequence, 5'-GCGAAAACGAAAGT-3', has been identified in Qp. Finally, the expression of IRF-7 is consistently high in type III latency cells and almost undetectable in type I latency, corresponding to the activity of endogenous Qp in these latency states and the ability of the IRF-7 proteins to repress Qp-reporter constructs. The identification of a functional viral ISRE and association of IRF-7 with type III latency may be relevant to the mechanism of regulation of Qp.

Amino Acid Sequence↗
PubMed1996

Activation of the Epstein-Barr virus DNA polymerase promoter by the BRLF1 immediate-early protein is mediated through USF and E2F.

The Epstein-Barr virus (EBV) DNA polymerase (pol) is essential for the replication of viral genomes during productive EBV infection. We have previously reported that the EBV DNA pol promoter, which is TATA-less and constitutively inactive, is activated by a genomic clone expressing both immediate-early viral transactivators, BZLF1Z and BRLF1 (R), in EBV-infected lymphoid cells. Here we demonstrate that R alone is sufficient to activate the pol promoter in EBV-negative B cells. Unlike other early promoters to which the R protein binds directly, its effect on the pol promoter does not appear to involve a direct DNA-binding mechanism. Instead, we found that two cellular transcription factors, an upstream stimulatory factor USF, and a member of the E2F family of proteins, bind directly to the pol promoter at positions -795 to -786 and -186 to -170, respectively, regions previously identified as important for activation of the pol promoter. These two sites contribute to or are essential for transactivation of the pol promoter by R in EBV-noninfected B cells. These data suggest that the R immediate-early protein may activate a key early EBV promoter (pol) through both USF and E2F.

Base Sequence↗