Epstein-Barr virus (EBV) infects human B lymphocytes, transforming the infected cells into dividing blasts that can proliferate indefinitely. The viral genome of 172 kilobase pairs (kbp) is a plasmid in most transformed cells. We have identified a region of EBV DNA, termed oriP (nucleotides 7,333-9,109 of strain B95-8), which acts in cis to permit linked DNAs to replicate as plasmids in cells containing EBV DNA. We have postulated the existence of a trans-acting gene allowing oriP function. Here we report that this gene lies in a 2.6-kbp region of the viral genome (nucleotides 107, 567-110, 176) which encodes the EBNA-1 antigen. We show that circular DNAs containing oriP, the EBNA-1 gene and a selectable marker replicate autonomously in cells derived from at least four developmental lineages and from at least three species. We also find that the one-third of the EBNA-1 gene repetitive in sequence is not essential for the trans-acting function that EBNA-1 gives oriP.

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The Epstein-Barr viral (EBV) genome of -170 kilobase pairs (kbp) is maintained as a plasmid in human B lymphoblasts transformed by the virus. We have identified a cis-acting element within 1.8 kbp of the viral genome that allows recombinant plasmids carrying it to be selected at high frequency and maintained as plasmids in cells latently infected by EBV. This functional element(s) requires a segment of DNA at least 800 bp and at most 1800 bp long, which contains a family of 30-bp tandem repeats at one end. Since this region confers efficient stable replication only to plasmids transfected into cells containing EBV genomes, its function probably requires trans-acting products encoded elsewhere in the viral genome.Epstein-Barr virus (EBV) is a human lymphotropic herpesvirus that causes infectious mononucleosis and lymphoproliferative disorders in immunosuppressed individuals. It is also probably a causative agent of Burkitt lymphoma and nasopharyngeal carcinoma in some parts of the world (for a review, see ref. 1). The causal association of EBV to diseases is likely a result of its capacity to induce unlimited proliferation in infected cells (2). Progress in understanding cell transformation by EBV has been limited by its large genome [172 kilobase pairs (kbp)], the likelihood that more than one gene is required for transformation (3), and the difficulty of introducing DNA stably into lymphocytes.Our approach in studying transformation by EBV has been to seek to define the genomic location of functions required for viral DNA replication in transformed cells. Since the viral genome is maintained as an independently replicating plasmid (4) in most cells transformed in vivo or in vitro (4-7), plasmid replication functions are probably required for efficient transformation. In addition, mapping the location of viral replication functions would allow the construction of a small EBV plasmid replicon suitable for the manipulations of molecular cloning, which in turn could aid in locating the remaining genes involved in transformation. Using a method described below, we scanned the EBV genome for cis-acting functions that would permit stable plasmid replication, and we found one such element in the short unique region of the genome. MATERIALS AND METHODSCell Lines. D98/Raji Cl 5 and D98/HR1 Cl 1 are hybrids between the human epithelial cell line D98 and the EBV genome-positive lymphoma lines Raji and P3JHR1, respectively (8,9). Both contain multiple copies of the EBV genome (ref. 10; unpublished results). These lines were grown in RPMI 1640 medium except during transfections, when they were in Dulbecco's modified Eagle's medium.Human fibrosarcoma 143 cells (11) and mouse BALB 3T3 fibroblasts (12) were grown in Dulbecco's modified Eagle's medium. Both media were supplemented with 10% fetal bovine serum.Transfections. About 1 x 106 cells in 6-cm dishes were transfected without carrier DNA, essentially as described by Graham and Van der Eb (13), with plasmid DNAs at 10 ,g/ml in Hepes-buffered saline (14). After 4 to ...

Epstein-Barr virus (EBV), a herpes virus, infects human B lymphocytes in vitro and efficiently immortalizes them. About 10 of the approximately 100 genes of EBV are expressed in recently immortalized B cells and although there is circumstantial evidence that at least three of these may contribute to the process of immortalization, there is no direct evidence that any particular gene is required. We have developed a genetic analysis of EBV that uses a transformation-defective strain of the virus as a helper virus in conjunction with DNA that contains all of the viral cis-acting elements required for replication, cleavage and packaging during the lytic phase of the viral life cycle. This DNA can include viral genes required for immortalization that complement the transformation-defective virus strain. The DNA can be amplified and packaged by the products of the helper virus and the packaged DNA is infectious. We have analysed two viral genes expressed in immortalized cells and find that the gene encoding EBV nuclear antigen-2 is required for immortalization, whereas the gene for the EBV nuclear antigen leader protein is not.

A genetic element of Epstein-Barr virus, oriP, when present on recombinant plasmids allows those plasmids to replicate and to be maintained in cells that express the Epstein-Barr virus-encoded nuclear antigen EBNA-1.Here we define the DNA sequences required for oriP activity. Two noncontiguous regions of oriP are required in cis for activity. One consists of approximately 20 tandem, imperfect copies of a 30-base-pair (bp) sequence. The other required region, approximately 1,000 bp away, is at most 114 bp in length and contains a 65-bp region of dyad symmetry. When present together on a plasmid, these two components supported plasmid replication even when the distance between them was varied or their relative orientation was altered, or both. When present alone on a plasmid that expresses a selectable marker, the family of 30-bp repeats efficiently conferred a transient drug-resistant phenotype in human 143 cells that is dependent on the presence of EBNA-1. This result leads us to suggest that EBNA-1 interacts with the 30-bp repeated sequence to activate oriP. To test whether the 30-bp repeats might cause the increased transient expression of drug resistance by enhancing transcription, the family of 30-bp repeats was tested for the ability to activate the simian virus 40 early promoter present in plasmid pAjoCAT2 (Laimins, et Epstein-Barr virus (EBV) is a human lymphotropic herpesvirus which is the causative agent of infectious mononucleosis and is associated with two human cancers, Burkitt's lymphoma and nasopharyngeal carcinoma (for a review see reference 43). B-lymphoid cells that have been transformed in vitro by EBV, or lymphoid cells derived from Burkitt's lymphoma biopsies, generally express the nuclear antigen EBNA (27) and contain multiple copies of the EBV genome (usually 1 to 100 copies per cell). Most or all of the EBV DNA is present as supercoiled DNA plasmids of approximately 172,000 base pairs (bp) (20). These multiple copies appear to arise soon after infection, by amplification of the viral DNA relative to cell DNA (36).For the most part, the study of DNA replication and the structure of origins of DNA replication in higher eucaryotic cells has been limited to lytic DNA viruses such as herpes simplex virus type 1 (HSV-1), simian virus 40 (SV40), polyomavirus, and the adenoviruses (3,4,25,29,33). Recently, however, studies of DNA replication have been extended to include stable extrachromosomal genetic elements such as those derived from bovine papillomavirus type 1 (23) and EBV (41). A cis-acting element, designated oriP, that allows replication and maintenance of recombinant plasmids in cells harboring either the EBV genome or the EBNA-1-encoding sequence from EBV has been isolated from the EBV genome (41, 42). In the presence of EBNA-1, oriP permits plasmid replication in a variety of mammalian cells that EBV cannot infect in culture (42).To determine the DNA sequence requirements for oriP, we generated and tested deletion derivatives of oriP. Our analysis revealed two cis-acting regions with...

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Using highly sensitive microarray-based procedures, we identified eight microRNAs (miRNAs) showing robust differential expression between 31 laser-capture-microdissected nasopharyngeal carcinomas (NPCs) and 10 normal healthy nasopharyngeal epithelial samples. In particular, miRNA mir-29c was expressed at one-fifth the levels in tumors as in normal epithelium. In NPC tumors, the lower mir-29c levels correlated with higher levels of multiple mRNAs whose 3 UTRs can bind mir-29c at target sequences conserved across many vertebrates. In cultured cells, introduction of mir-29c down-regulated these genes at the level of mRNA and inhibited expression of luciferase encoded by vectors having the 3 UTRs of these genes. Moreover, for each of several genes tested, mutating the mir-29c target sites in the 3 UTR abrogated mir-29c-induced inhibition of luciferase expression. Most of the mir-29c-targeted genes identified encode extracellular matrix proteins, including multiple collagens and laminin ␥1, that are associated with tumor cell invasiveness and metastatic potential, prominent characteristics of NPC. Thus, we identify eight miRNAs differentially expressed in NPC and demonstrate the involvement of one in regulating genes involved in metastasis.microarray ͉ collagen ͉ metastasis ͉ miRNA M icroRNAs (miRNAs) are short (Ϸ22 nucleotides) noncoding RNAs involved in posttranscriptional silencing of target genes. In animals, miRNAs control expression of target genes by inhibiting translation, by degrading target mRNAs, or both, through binding to their 3Ј UTRs with varying degrees of sequence complementarity (1). miRNAs have been found to regulate genes involved in diverse biological functions, including development, differentiation, proliferation, and stress response (2). Recently, a growing number of miRNAs have been implicated in cancers, including mir-15 and mir-16 in B cell chronic lymphocytic leukemias (3, 4); mir-143 and mir-145 in colorectal cancer (5); mir- 17-5p, mir-21, mir-125b, mir-145, and mir-155 in breast cancer (6, 7); mir-19, mir-146, mir-181b, mir-221, mir-222, and mir-346 in thyroid cancer (8-10); and mir-21 in glioblastoma (11). A significant number of miRNAs also have been mapped to cancer-associated genomic regions (12). Expression of miRNA let-7 has been correlated with prognosis in lung cancer (13) and found to regulate Ras in the same tumor (14). Very recently, mir-10b has been shown to contribute to metastasis in breast cancer (15). Although many miRNAs have been implicated in regulating cancers, very few of their target genes, and hence their downstream mode of action, have been identified.We developed a sensitive microarray-based assay to profile miRNA expression and used it to analyze human miRNAs in laser-microdissected tumor and normal cells from biopsies of a highly invasive cancer, nasopharyngeal carcinoma (NPC), and site-matched normal tissues. Eight miRNAs were differentially expressed. One of them, mir-29c, down-regulated in NPC, was shown to target multiple mRNAs encoding extracellular ma...

Two regions of the Epstein-Barr virus (EBV) genome together make up an element, oriP, which acts in cis to support plasmid replication in cells that express the EBV nuclear antigen 1 (EBNA-1). The two components of oriP are a region containing a 65-base-pair (bp) dyad symmetry and a region containing 20 copies of a 30-bp direct repeat. Here we show that the 30-bp family of repeats of oriP can function as a transcriptional enhancer that is activated in trans by the EBNA-1 gene product. In either EBV-genome-positive cells or in cells that express EBNA-1, the 30-bp family of repeats, when positioned in either orientation upstream or downstream, enhances expression of the chloramphenicol acetyltransferase (CAT) gene expressed from either the simian virus 40 early promoter or the herpes simplex virus type 1 thymidine kinase promoter. The extent of transcriptional enhancement varies with the promoter and cell type. This enhanced CAT expression reflects an increased level of CAT mRNA and does not result from amplffication of the plasmids expressing CAT. In addition, plasmids carrying the gene for resistance to hygromycin B and the 30-bp family of repeats yielded 10 to 100 times more hygromycin B-resistant colonies than the vector lacking the 30-bp family of repeats in both EBV-genome-positive cells and cells that express EBNA-1. EBNA-1 is known to bind to sequences within the 30-bp family of repeats (D. R. Rawlins, G. Milman, S. D. Hayward, and G. S. Hayward, Cell 42:859468, 1985), and these trans-and cis-acting elements together have at least two functional roles: (i) they are required for DNA replication dependent upon oriP, and (ii) they can enhance expression of genes linked to the 30-bp family of repeats of oriP.Epstein-Barr virus (EBV) is a human herpesvirus that infects B lymphocytes and transforms them into cells capable of indefinite proliferation in culture (for a recent review, see reference 31). B cells that have been transformed by EBV usually express nuclear antigens known as EBNAs and contain multiple copies of the EBV genome (35,41). In general, the viral genome is maintained as a supercoiled DNA plasmid of approximately 172,000 base pairs (bp) (2, 27). Three regions of the viral genome are known to be transcribed into poly(A)+ mRNA in transformed cells (1,45). One region codes for a 62,000-dalton membrane protein (11), and the two others code for the nuclear antigens 18,43). Little is known about the regulation of expression of these genes, and apart from the role of EBNA-1 in EBV plasmid replication (29, 48), little is known about the role of the other expressed EBV genes in B-cell transformation.A cis-acting element, oriP, isolated from the EBV genome, allows the replication and maintenance of recombinant plasmids in cells that express EBNA-1 (29,47,48 To characterize further the properties of this transcriptional enhancer element, we measured its activity in different cell types and its effect on RNA synthesis from two promoters, the SV40 early promoter and the herpes simplex virus type 1 thymidine...