Epstein-Barr virus RNA. VIII. Viral RNA in permissively infected B95-8 cells

B-lymphotropic herpesviruses naturally infecting Old World primates share biologic, epidemiologic, pathogenic, and molecular features with the human pathogen Epstein-Barr virus (EBV). These related gammaherpesviruses have colinear genomes with considerable nucleotide homology. The replicative cycle genes share a high degree of homology across species, whereas the transformation-associated EBV latent genes appear to be much more divergent. For example, the EBV BamHI Nhet fragment, which encodes all or part of the EBV latent infection membrane proteins, cross-hybridizes poorly to DNA from nonhuman primate B-lymphotropic herpesviruses. A viral DNA fragment corresponding to this region of the EBV genome was isolated from the baboon B-lymphotropic herpesvirus, herpesvirus papio, and used to clone a herpesvirus papio cDNA corresponding to EBV LMP2A. At least three tyrosine kinase interaction motifs are conserved despite significant amino acid divergence of the herpesvirus papio LMP2A first exon from the EBV homolog. Functionally, the herpesvirus papio LMP2A is tyrosine phosphorylated and induces tyrosine phosphorylation of cell proteins similar to EBV LMP2A. The 12 hydrophobic LMP2 transmembrane domains are well conserved. Two CBP (J) binding sites important for EBNA-2-induced transactivation of the LMP2A promoter are also present in the herpesvirus papio LMP2A promoter, and the simian LMP2A promoter is also responsive to EBV EBNA-2induced transactivation in human B cells. Thus, transcriptional regulation, splicing, kinase interaction sites, and tyrosine phosphorylation of the LMP2A homologs have been conserved despite significant sequence heterogeneity in the preterminal repeat regions of these human and nonhuman primate EBVs. The conservation of the LMP2 gene, despite its apparent nonessential role for in vitro EBV infection, suggests an important role for LMP2A in vivo. The similarities between these human and simian B-lymphotropic herpesviruses, and the LMP2 genes in particular, suggest that the function of LMP2 in vivo could be addressed by using recombinant LMP2A-mutant simian viruses and experimental infection of Old World primates.

Section: Cloning Of a Herpesvirus Papio Dna Fragment Corresponding Tomentioning

confidence: 79%

5' Coding and regulatory region sequence divergence with conserved function of the Epstein-Barr virus LMP2A homolog in herpesvirus papio

Franken

Annis

Ali

et al. 1995

“…This message has not been detected in the latently infected Raji, IB4, or CB4 cell lines, nor was transcription from BamHI A detected by in situ hybridization in specimens of EBV-positive non-Hodgkin's lymphoma (11,34). A related RNA is transcribed from the same sequences early after induction of replication in P3HR1, B95-8, and Akata cells and in superinfected Raji cells (4,16,32). The transcript identified in the lymphoid cell lines differs in size from the mRNA identified in NPC in that it is 4.2 rather than 4.8 kb.…”

Section: Discussionmentioning

confidence: 94%

Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo

Gilligan

Rajadurai

Lin

et al. 1991

106

A family of mRNAs that are transcribed rightward through the BamHI A fragment have been detected in C15, a nasopharyngeal carcinoma (NPC) which has been passaged in nude mice. Northern (RNA) blot hybridizations indicate that these RNAs are also expressed in three other NPCs which have been established in nude mice and in an NPC obtained at biopsy. Moreover, hybridization in situ detected transcription from BamHI A in 12 NPCs and 1 Epstein-Barr virus (EBV)-containing carcinoma of the parotid gland. In each case, transcription was detected in all of the malignant epithelial cells. Transcription was not detected in two cases of EBV-positive lymphoma biopsies by in situ hybridization nor in latently infected EBV-positive lymphoblastoid cell lines by Northern blot hybridization. The consistent transcription of these sequences in latently infected epithelial malignancy but not in lymphoid cells suggests that this viral function is associated with latent EBV infection of epithelial cells. Sequence analysis of a cDNA synthesized from the C15 tumor, representing the 3' end of BamHI A messenger RNA, revealed an open reading frame (ORF). Translation of this ORF in vitro produced several peptides that were immunoprecipitated with antisera from patients with NPC. The detection of antibodies to the protein encoded by the ORF present in the BamHI A cDNA indicates that BamHI A encodes a protein which is expressed in vivo and is antigenic.

“…RNA analysis. Poly(A) ϩ RNA was isolated from cytoplasmic RNA, extracted as described previously (15), by selection with biotinylated oligo(dT)-streptavidin-coupled magnetic beads (PolyATract mRNA Isolation System IV; Promega) according to the manufacturer's instructions. For Northern (RNA) blot analysis, 2.5 g of poly(A) ϩ RNA per sample was fractionated by electrophoresis in a 1.2% agarose-2.2 M formaldehyde gel and transferred to a GeneScreen Plus membrane (DuPont).…”

Section: Methodsmentioning

confidence: 99%

Interferon-independent and -induced regulation of Epstein-Barr virus EBNA-1 gene transcription in Burkitt lymphoma

Nonkwelo

Ruf

Sample

1997

Replication of the Epstein-Barr virus (EBV) genome within latently infected cells is dependent on the EBVEBNA-1 protein. The objective of this study was to identify transcriptional regulatory proteins that mediate EBNA-1 expression via the viral promoter Qp, which is active in EBV-associated tumors such as Burkitt lymphoma and nasopharyngeal carcinoma. Results of a yeast one-hybrid screen suggested that a subset of the interferon regulatory factor (IRF) family may regulate EBNA-1 transcription by targeting an essential cisregulatory element of Qp, QRE-2. Further investigation indicated that the transcriptional activator IRF-1 and the closely related IRF-2, a repressor of interferon-induced gene expression, are both capable of activating Qp. However, the major QRE-2-specific binding activity detected within extracts of Burkitt lymphoma cells was attributed to IRF-2, suggesting that interferon-independent activation of Qp is largely mediated by IRF-2 in these cells. We observed no effect of gamma interferon on Qp activity in transfection assays, whereas we observed a moderate but significant repression of Qp activity in response to alpha interferon, possibly mediated by either the interferon consensus sequence binding protein or IRF-7, a novel alpha interferoninducible factor identified in this study. Since expression of IRF-1 and IRF-2 is increased in response to interferons, the Qp activity observed in the presence of interferon likely represented an equilibrium between IRF factors that activate and those that repress gene expression in response to interferon. Thus, by usurping both IRF-1 and its transcriptional antagonist IRF-2 to activate Qp, EBV has evolved not only a mechanism to constitutively express EBNA-1 but also one which may sustain EBNA-1 expression in the face of the antiviral effects of interferon.