We have studied the relative rate of transcription across the Epstein-Barr virus genome in the Burkitt’s lymphoma cell line Raji by nuclear run-on analysis during latency and after induction of an abortive lytic cycle with 12-O-tetradecanoylphorbol 13-acetate (TPA) and 5-iodo-2′-deoxyuridine (IUdR). During latency the entire, or almost the entire, viral genome was found to be transcriptionally active to a low or intermediate extent, with some variation in activity along the genome. The fragment with the highest transcriptional activity was EcoRI J, which contains the genes encoding the small nuclear RNAs EBER1 and -2, transcribed predominantly by RNA polymerase III. An intermediate level of transcription was observed between positions 10 and 138 (kb), with areas of slightly higher activity on the large internal repeats and the left duplicated region (DL). The remaining part of the viral genome, between position 138 and the termini, and the termini and position 10 (kb) (with the exception of the EcoRI J fragment), showed very little transcriptional activity, except for the intermediately active regions carrying the righthand oriLyt (DR) and the terminal repeats. Upon induction of the viral genome with TPA and IUdR, the viral genome was transcriptionally active at a rate at least tenfold that seen during latency. Polymerases were not equally distributed along the genome after induction; the highest density was found in regions 48 to 58 kb, 82 to 84 kb, 102 to 104 kb, 118 to 122 kb and 142 to 145 kb of the viral genome. High transcriptional activity correlated with distinct transcription units in some cases, i.e. BamHI HILF1 (DL), BamHI MLF1, BamHI ZLF1/BamHI RLF1 and BamHI X (thymidine kinase), but not in others (BamHI H2). Besides initiation of transcription, other regulatory processes such as stabilization and processing of primary transcripts may also contribute to regulation of virus gene expression. Addition of cycloheximide completely abolished the transcriptional activation of the genome mediated by TPA and IUdR.
BauerG.,
HoflerP.,
zur HausenH.1982; Epstein-Barr virus induction by a serum factor. I. Induction and cooperation with additional inducers. Virology 121:184–194
BentleyD. L.,
GroudineM.1986; Novel promoter upstream of the human c-myc gene and regulation of c-myc expression in B-cell lymphomas. Molecular Cell Biology 6:3481–3489
Chevallier-GrecoA.,
ManetE.,
ChavrierP.,
MosnierC.,
DaillieJ.,
SergeantA.1986; Both Epstein-Barr virus (EBV)-encoded trans-acting factors, EB1 and EB2, are required to activate transcription from an EBV early promoter. EMBO Journal 5:3243–3249
Chevallier-GrecoA.,
GruffatH.,
ManetE.,
CalenderA.,
SergeantA.1989; The Epstein-Barr virus (EBV) DR enhancer contains two functionally different domains: domain A is constitutive and cell specific, domain B is transactivated by the EBV early protein R. Journal of Viology 63:615–623
CountrymanJ.,
MillerG.1985; Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA. Proceedings of the National Academy of Sciences, U.S.A 82:4085–4089
DambaughT.,
WangF.,
HennessyK.,
WoodlandE.,
RickinsonA.,
KieffE.1986; Expression of the Epstein-Barr virus nuclear protein 2 in rodent cells. Journal of Virology 59:453–462
EickD.,
BornkammG. W.1986; Transcriptional arrest within the first exon is a fast control mechanism in c-myc gene expression. Nucleic Acids Research 14:8331–8346
EickD.,
PolackA.,
KoflerE.,
LenoirG. M.,
RickinsonA. B.,
BornkammG.W.1990; Expression of P0- and P3-RNA from the normal and translocated c-myc allele in Burkitt’s lymphoma cells. Oncogene 5:1397–1402
FahraeusR.,
JanssonA.,
RickstenA.,
SjoblomA.,
RymoL.1990; Epstein-Barr virus-encoded nuclear antigen 2 activates the viral latent membrane protein promoter by modulating the activity of a negative regulatory element. Proceedings of the National Academy of Sciences, U.S.A 87:7390–7394
FarrellP. J.,
RoweD. T.,
RooneyC. M.,
KouzaridesT.1989; Epstein-Barr virus BZLF1 trans-activator specifically binds to a consensus AP-1 site and is related to c-fos. EMBO Journal 8:127–132
FlemingtonE.,
SpeckS. H.1990b; Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BHLF1. Journal of Virology 64:1217–1226
GhoshD.,
KieffE.1990; Cis-acting regulatory elements near the Epstein-Barr virus latent-infection membrane protein transcriptional start site. Journal of Virology 64:1855–1858
HardwickJ. M.,
LiebermanP. M.,
HaywardS. D.1988; A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen. Journal of Virology 62:2274–2284
HestonL.,
RabsonM.,
BrownN.,
MillerG.1982; New Epstein-Barr virus variants from cellular subclones of P3J-HR-1 Burkitt lymphoma. Nature, London 295:160–163
HoweJ. G.,
ShuM.-D.1989; Epstein-Barr virus small RNA (EBER) genes: unique transcription units that combine RNA polymerase II and III promoter elements. Cell 57:825–834
HummelM.,
KieffE.1982a; Mapping of polypeptides encoded by the Epstein-Barr virus genome in productive infection. Proceedings of the National Academy of Sciences, U.S.A 79:5698–5702
LauxG.,
FreeseU. K.,
FischerR.,
PolackA.,
KoflerE.,
BornkammG. W.1988a; TPA-inducible Epstein-Barr virus genes in Raji cells and their regulation. Virology 162:503–507
LauxG.,
PerricaudetM.,
FarrellP. J.1988b; A spliced Epstein-Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome. EMBO Journal 7:769–774
LauxG.,
EconomouA.,
FarrellP. J.1989; The terminal protein gene 2 of Epstein-Barr virus is transcribed from a bidirectional latent promoter region. Journal of General Virology 70:3079–3084
LiebermanP. M.,
O’HareP.,
HaywardG. S.,
HaywardS. D.1986; Promiscuous trans activation of gene expression by an Epstein-Barr virus-encoded early nuclear protein. Journal of Virology 60:140–148
LindahlT.,
AdamsA.,
BjursellG.,
BornkammG. W.,
Kaschka-DierichC.,
JehnU.1976; Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. Journal of Molecular Biology 102:511–530
MetzenbergS.1989; Relative rates of RNA synthesis across the genome of Epstein-Barr virus are highest near oriP and oriLyt. Journal of Viology 63:4938–4944
MillerG.,
HestonL.,
CountrymanJ.1985; p3hr-1 ebv heterogeneous DNA is an independent replicon maintained by cell to cell spread. Journal of Virology 54:45–52
MoosM.,
GallwitzD.1983; Structure of two human beta-actinrelated processed genes one of which is located next to a simple repetitive sequence. EMBO Journal 2:757–761
NepveuA.,
MarcuK. B.,
SkoultchiA.,
LachmanH. M.1987; Contributions of transcriptional and post-transcriptional mechanisms.to the regulations of c-myc expression in mouse erythroleukemia cells. Genes and Development 1:938–945
OguroM. O.,
ShimizuN.,
OnoY.,
TakadaK.1987; Both the rightward and the leftward open reading frames within the Bam H1 M DNA fragment of Epstein-Barr virus act as trans-activators of gene expression. Journal of Virology 61:3310–3313
PolackA.,
DeliusH.,
ZimberU.,
BornkammG. W.1984a; Two deletions in the Epstein-Barr virus genome of the Burkitt lymphoma nonproducer line Raji. Virology 133:146–157
PolackA.,
HartlG.,
ZimberU.,
FreeseU. K.,
LauxG.,
TakakiK.,
HohnB.,
GissmannL.,
BornkammG. W.1984b; A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates. Gene 27:279–288
RabsonM.,
HestonL.,
MillerG.1983; Identification of a rare Epstein-Barr virus variant that enhances early antigen expression in Raji cells. Proceedings of the National Academy of Sciences, U.S.A 80:2762–2766
RawlinsD. R.,
MilmanG.,
HaywardS. D.,
HaywardG. S.1985; Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell 42:859–868
SampleJ.,
KieffE.1990; Transcription of the Epstein-Barr virus genome during latency in growth-transformed lymphocytes. Journal of Virology 64:1667–1674
SugawaraK.,
MizunoF.,
OsatoT.1972; Epstein-Barr virus-associated antigens in non-producing clones of human lymphoblastoid cell lines. Nature, London 239:242–243
UrierG.,
BuissonM.,
ChambardP.,
SergeantA.1989; The Epstein-Barr virus early protein EB1 activates transcription from dilferent responsive elements including AP-1 binding sites. EMBO Journal 8:1447–1453
WangF.,
GregoryC. D.,
RoweM.,
RickinsonA. B.,
WangD.,
BirkenbachM.,
KikutaniH.,
KishimotoT.,
KieffE.1987; Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23. Proceedings of the National Academy of Sciences, U.S.A 84:3452–3456
YajimaY.,
NonoyamaM.1976; Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells. Journal of Virology 19:187–194
YatesJ.,
WarrenN.,
ReismanD.,
SugdenB.1984; A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proceedings of the National Academy of Sciences, U.S.A 81:3806–3810
Zimber-StroblU.,
SuentzenichK. O.,
LauxG.,
EickD.,
CordierM.,
CalenderA.,
BillaudM.,
LenoirG. M.,
BornkammG. W.1991; The Epstein-Barr virus nuclear antigen 2 activates transcription of the terminal protein gene. Journal of Virology 65:415–423
Zur HausenH.,
BornkammG. W.,
SchmidtR.,
HeckerE.1979; Tumor initiators and promoters in the induction of Epstein-Barr virus. Proceedings of the National Academy of Sciences, U.S.A 76:782–785