We have identified two Epstein‐‐Barr virus (EBV) transacting factors which are involved in the transcriptional activation of EBV early promoters in latently infected Raji cells. In Raji cells, expression of the factor EB1 encoded by the open reading frame (ORF) BZLF1 is necessary and sufficient to disrupt latency. However, factor EB2 encoded by the ORF BMLF1‐ BSLF2 does not disrupt latency when expressed alone in Raji cells. Expression of an EBV activatable early promoter depends on the presence of both EB1 and EB2.

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV BMLF1-KO ). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV BMLF1-KO 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV BMLF1-KO mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.

The Epstein‐Barr virus (EBV) genome codes for several transcriptional trans‐activators. One of them, the BZLF1 open reading frame (ORF)‐encoded product EB1, is able to induce the productive cycle in infected B cells. From the cloning and characterization of full‐length cDNAs, we found that EB1 could be made from three overlapping messenger RNAs expressed under the control of two different promoters that we call P1 and P2. The first mRNA, 1 kb long, is made from the P1 promoter and codes for EB1 alone. The two other mRNAs, respectively 3 and 4 kb long and made by facultative splicing, are bicistronic mRNAs. They code not only for the trans‐activator EB1 but also for a second EBV transcriptional trans‐activator R, encoded by the BRLF1 ORF. In effect, authentic EB1 and R proteins are expressed from the 3 and 4 kb long cDNAs as demonstrated by identification of the proteins with specific antisera. In addition, EB1 and R expressed from the 3 and 4 kb cDNAs activate transcription from their specific targets in the EBV early promoter DR.

During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta-and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta-and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses. IMPORTANCEControl of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC). Herpesviruses are enveloped viruses containing relatively large, double-stranded DNA genomes. They are divided into three subfamilies (alpha-, beta-, and gammaherpesviruses) according to sequence homology, cellular tropism, and productive cycle behavior under laboratory culture conditions. Nine herpesviruses have been identified in humans. Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV) are alphaherpesviruses that due to neurotropism cause recurrent skin lesions, meningitis, and rare but very serious encephalitis in the case of HSV-1; human cytomegalovirus (HCMV), human herpesviruses 6A and 6B (HHV-6A and HHV-6B), and human he...

In cells latently infected with EBV, the switch from latency to productive infection is linked to the expression of two EBV transcription factors called EB1 (or Z) and R. EB1 is an upstream element factor which has partial homology to the AP1/ATF family, whereas R is an enhancer factor. In the R-responsive enhancer of the replication origin only active during the EBV lytic cycle (ORIIyt), R-responsive elements are located in a region of about 70 bp (RRE-DR). Here we show that R, produced either by in vitro translation, or present in nuclear extracts from HeLa cells constitutively producing R, binds directly to and protects against DNAase I digestion, two regions in RRE-DR. Using mobility shift assay and DMS interference, we have characterized the contact-points between R and the DNA. Two binding sites, RRE-DR1 and RRE-DR2, were characterized and are contiguous in RRE-DR. R binds to these two sites probably by simultaneously contacting two sequences within the sites, which are separated by 7 bp in RRE-DR1, cctGTGCCttgtcccGTGGACaatgtccc, and by 6bp in RRE-DR2, caatGTCCCtccagcGTGGTGgctg. Direct interaction of R with its cognate sequences is conferred by its N-terminal 355 amino-acids. Directed mutagenesis in RRE-DR, of either R-binding site, impaired binding of R in vitro and, as assayed by transient expression in HeLa cells, impaired R-activation by a factor of two. This suggests that RRE-DR1 and RRE-DR2 do not respond cooperatively to R.

The Epstein‐Barr virus (EBV) protein EBNA2, which is essential for the immortalization of human primary B cells by EBV, acts as a transcriptional activator of cellular and viral genes. Specific responsive elements have been characterized in several of the promoters activated by EBNA2. They all share the core sequence GTGGGAA. EBNA2 does not, however, bind to these sequences directly, but appears to be targeted to them by a cellular protein. A similar core sequence has recently been identified as a high‐affinity binding site for the human recombination signal sequence binding protein RBP‐J kappa. Here we provide evidence that RBP‐J kappa binds to specific sequences in EBNA2‐responsive elements. Our results also demonstrate that RBP‐J kappa makes direct physical contact with EBNA2 in solution and recruits EBNA2 to its cognate DNA sequences, suggesting that RBP‐J kappa may mediate EBNA2 transactivation of both cellular and viral genes.

A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.In cells infected by human herpesviruses, viral mRNAs and proteins are trafficked through the nuclear pore complex. Several cellular factors that mediate the nucleocytoplasmic transport of mRNAs have now been identified (1-6). Interestingly, some human herpesviruses carry, in their genome, genes whose products are also mRNA export factors, such as HSV-1 1 ICP27 (7) and the EBV BMLF1 early gene product originally called EB2 (8), but later called Mta (9) or SM (10). Such genes are conserved among all human herpesviruses, suggesting a conserved function for their products. At least for HSV-1 and EBV, the inactivation of ICP27 (11) or EB2 (12), respectively, abolishes the production of infectious viral particles, demonstrating that ICP27 and EB2 are essential factors for viral mRNA export and that their function cannot be trans-complemented by cellular factors. Moreover, EB2 appears to have an effect on cellular mRNAs because it has transforming properties when expressed both in established cell lines such as Rat1 and NIH3T3 and in primary rat fibroblasts (13).Most of the HSV-1 and EBV early and late mRNAs are transcribed from intronless genes. However, it is now clearly established that the nuclear export of mRNAs is dramatically increased when a splicing event occurs (14). In effect, splicing leads to the deposition on the mRNA of a multiprotein export complex (called EJC for exon-exon junction complex), including REF/Aly (Yra1 in yeast), Y14, RNPS1, SRm160, and Magoh, 20 -24 nucleotides upstream of the exon-exon junction (2-5). Such a complex is thought to export mRNAs by recruiting TAP/Mex67p (15) to cellular messenger RNPs (16 -18). For cellular mRNAs generated from intronless genes, they are likely to be exported to the cytoplasm by cellular factors through nonspecific interactions with mRNA-bound adapters like REF (19) or through sequence-specific interactions with SRp20 or 9G8 (20) or U2AF (21). It is therefore tempting to speculate that EB2, like its HSV-1 functional homolog ICP27 (7), is an...

In B lymphocytes induced to proliferate in vitro by the EpsteinBarr virus (EBV), extra-chromosomal viral episomes packaged in chromatin persist in the nucleus, and there is no productive cycle. A switch from this latency to the productive cycle is observed after induced expression of the EBV BZLF1 gene product, the transcription factor EB1. We present evidence that, during latency, proteins of the myocyte enhancer binding factor 2 (MEF2) family are bound to the BZLF1 promoter and recruit class II histone deacetylases. Furthermore, we propose that latency is determined primarily by a specific and local recruitment of class II histone deacetylase (HDAC) by MEF2D to the BZLF1 gene promoter. The switch from latency to the productive cycle could be due in part to post-translational modification of MEF2 proteins and changes in the local acetylation state of the chromatin.