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.

The Epstein-Barr virus (EBV) DR promoter is located upstream of the PstI repeats, and besides the TATA box, it contains two cis-acting regulatory elements. One of them has enhancer properties. To define more precisely the functional region(s) in the DR enhancer, we generated 5' and 3' deletion mutants. These deletion mutants, which were transfected into various recipient cells of different origins, allowed us to identify two functionally distinct domains, A and B. Domain A was constitutively active in all cell lines tested, except in lymphoid B cells. Domain B was active in lymphoid B cells, and its activity required both EB1 (the BZLF1-encoded EBV trans-acting factor) and the presence of the EBV genome. This suggested that an EBV-encoded, EB1-inducible factor was activating the enhancer B domain. In effect, the B domain was trans-activated by R, an EBV early product encoded by the open reading frame BRLF1, and the activation by R occurred in epithelial, fibroblastic, and lymphoid cells. The R-responsive element has been reduced to 28 base pairs containing the double palindromic sequence TTGTCCCGTGGACAATGTCC. Both domains A and B act by increasing the initiation of specific RNAs.     

The Epstein-Barr virus BMLF1 promoter contains an enhancer element that is responsive to the BZLF1 and BRLF1 transactivators.

We have previously shown that the Epstein-Barr virus (EBV) immediate-early gene product, BZLF1, can activate expression of the EBV BMLF1 immediate-early promoter in EBV-positive, but not EBV-negative, B cells, suggesting that the BZLF1 effect may be mediated through another EBV gene product (S. Kenney, J. Kamine, E. Holley-Guthrie, J.-C. Lin, E.-C. Mar, and J. S. Pagano, J. Virol. 63:1729-1736, 1989). Here, we show that the EBV BRLF1 immediate-early gene product transactivates the BMLF1 promoter in either EBV-positive or EBV-negative B cells. Deletional analysis revealed that both the BZLF1-responsive region and the BRLF1-responsive region of the BMLF1 promoter are contained within the same 140-base-pair FokI-PvuII fragment located 300 base pairs upstream of the mRNA start site. This FokI-PvuII fragment functions as an enhancer element in the presence of the BRLF1 transactivator and contains the sequence CCGTGGAGA ATGTC, which is strikingly similar to the BRLF1-responsive region of the EBV DR/DL enhancer (A. Chevallier-Greco, H. Gruffat, E. Manet, A. Calender, and A. Sergeant, J. Virol. 63:615-623, 1989). The effect of BZLF1 on the BMLF1 promoter is likely to be indirect and mediated through the BRLF1 transactivator.     

Differential regulation of the human immunodeficiency virus type 2 enhancer in monocytes at various stages of differentiation.

We have demonstrated that stimulation of the human immunodeficiency virus type 2 (HIV-2) enhancer in T cells is dependent upon at least four cis-acting elements, including two purine-rich binding sites, PuB1 and PuB2, which are capable of binding members of the ets family of proto-oncogenes, the pets (peri-ets) site, which lies just upstream of the PuB2 site, and a single kappa B site (D. M. Markovitz, M. Smith, J. M. Hilfinger, M. C. Hannibal, B. Petryniak, and G. J. Nabel, J. Virol. 66:5479-5484, 1992). In this study, we examined the regulation of the HIV-2 enhancer in cells of monocytic lineage. We found that in immature monocytic cell lines, the HIV-2 enhancer is markedly induced by phorbol esters and that all four cis-acting elements are required for activation. In mature monocytic cells, constitutive activity is high, with only modest stimulation following phorbol ester treatment. Mutation of any of the four cis-acting elements resulted in greatly reduced basal expression in mature monocytes. This is in contrast to HIV-1, in which developmentally controlled expression of the enhancer in monocytes is mediated largely through the kappa B sites alone [G. E. Griffin, K. Leung, T. M. Folks, S. Kunkel, and G. J. Nabel, Nature (London) 339:70-73, 1989]. Further, we demonstrated that although both Elf-1, an ets family member with significant similarity to the drosophila developmental regulatory protein E74, and Pu.1, a monocyte- and B-cell-specific member of the ets family, bind the purine-rich enhancer region, Elf-1 is the protein which binds predominantly in vivo. A nuclear factor(s) which binds the pets site, an element which has been described only in HIV-2, was detected in extracts of all of the monocytic cells tested. These findings indicate that the mechanism by which cellular factors regulate HIV-2 enhancer function in monocytic cells differs significantly from that of HIV-1 and may offer a partial explanation for the differences in the biological and clinical characteristics of the two viruses.     

Induction of immunoglobulin G Fc receptors by recombinant vaccinia viruses expressing glycoproteins E and I of herpes simplex virus type 1.

Glycoprotein E (gE) of herpes simplex virus type 1 (HSV-1) will bind immunoglobulin G (IgG) (Fc) affinity columns (R. B. Bauke and P. G. Spear, J. Virol. 32:779-789, 1979), but recent evidence suggests that the HSV-1 Fc receptor is composed of a complex of gE and glycoprotein I (gI) and that both gI and gE are required for Fc receptor activity (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987; D. C. Johnson, M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow, J. Virol. 62:1347-1354, 1988). We have expressed gE and gI, either alone or in combination, on the surface of HeLa cells by using recombinant vaccinia viruses and have measured Fc receptor activity by Fc-rosetting or IgG-binding assays. Expression of gE alone resulted in the induction of Fc receptor activity, while expression of gI alone gave no detectable Fc binding. Coexpression of gE and gI resulted in higher levels of IgG binding than did expression of gE alone, despite the fact that under conditions of coexpression, the levels of surface gE were reduced. We propose that gE and gI together form a receptor of higher affinity than gE alone and that HSV-1 therefore has the potential to induce two Fc receptors of different affinities.     

Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 beta gene.

A site located between -2782 and -2729 of the human prointerleukin-1 beta (IL1B) gene functions as a strong lipopolysaccharide (LPS)-responsive enhancer independent of the previously identified enhancer located between -2896 and -2846 (F. Shirakawa, K. Saito, C.A. Bonagura, D.L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). Although these two enhancers appear to function cooperatively in the native sequence context, they function independently as LPS-responsive elements upon removal of an interposed silencer sequence. The new enhancer is not induced by dibutyryl cyclic AMP (dbcAMP) alone but is superinduced by costimulation with LPS-dbcAMP. This pattern of induction depends upon the nature of the sequence, a composite NF-IL6-cAMP response element (CRE) binding site. This pseudosymmetrical sequence is shown to contrast with a classical symmetric CRE which responds to dbcAMP but not LPS. DNA binding studies using in vivo nuclear extract, recombinant proteins, and specific antibodies show that LPS induces the formation of two different complexes at the enhancer: (i) an NF-IL6-CREB heterodimer and (ii) a heterodimer consisting of NF-IL6 and a non-CREB, CRE-binding protein. Cotransfection studies using NF-IL6 and CREB expression vectors show that NF-IL6 transactivates the enhancer in the presence of LPS, whereas CREB acts either positively or negatively, depending upon its cAMP-regulated phosphorylation state. Our data demonstrate that the newly identified enhancer is a specialized LPS-responsive sequence which can be modulated by cAMP as a result of the involvement of NF-IL6-CRE-binding protein heterodimers.     

The herpes simplex virus 1 gene for ICP34.5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+.

In a previous study, it was reported that herpes simplex virus 1 (HSV-1) strain F contains a transcribed open reading frame situated in the inverted repeats of the L component between the terminal a sequence and the open reading frame that encodes the alpha 0 gene (J. Chou and B. Roizman, J. Virol. 57: 629-637, 1986). By means of an antibody to repeats of the trimer Ala-Thr-Pro predicted to be specified by the open reading frame, it was shown that the open reading frame specifies a protein (M. Ackermann, J. Chou, M. Sarmiento, R. A. Lerner, and B. Roizman, J. Virol. 58: 843-850, 1986). This open reading frame is absent from the reported sequence of HSV-1(17)syn+ (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69: 1531-1574, 1988; L. J. Perry and D. J. McGeoch, J. Gen. Virol. 69: 2831-2846, 1988). To define the extent of variability in this open reading frame, we compared the sequences of the ICP34.5-encoding open reading frames of the genomes of three strains characterized by limited passage in cell culture with that of the HSV-1(17)syn+ strain. Furthermore, to establish unambiguously that the antibody to the Ala-Thr-Pro repeats reacts with the product of this open reading frame, we inserted a short sequence that encodes a known epitope in frame at the 5' terminus of the coding domain. Our results indicate that with minor variations, the open reading frame is conserved in the three HSV-1 genomes analyzed but not in HSV-1(17)syn+. Thus, two strains contain an inserted amino acid and one strain, isolated from a case of human encephalitis, lacks a seven-amino-acid sequence. The recombinant virus carrying the foreign epitope expressed a slightly slower-migrating protein which reacted with both the rabbit polyclonal antibody to the Ala-Thr-Pro trimer repeats and the monoclonal antibody to the inserted epitope. The implications of the results are discussed.     

Characterization of a protein that binds multiple sequences in mammalian type C retrovirus enhancers.

Mammalian type C retrovirus enhancer factor 1 (MCREF-1) is a nuclear protein that binds several directly repeated sequences (CNGGN6CNGG) in the Moloney and Friend murine leukemia virus (MLV) enhancers (N. R. Manley, M. O'Connell, W. Sun, N. A. Speck, and N. Hopkins, J. Virol. 67:1967-1975, 1993). In this paper, we describe the partial purification of MCREF-1 from calf thymus nuclei and further characterize the binding properties of MCREF-1. MCREF-1 binds four sites in the Moloney MLV enhancer and three sites in the Friend MLV enhancer. Ethylation interference analysis suggests that the MCREF-1 binding site spans two adjacent minor grooves of DNA.     

Epstein-Barr virus nuclear antigen 2 trans-activates the cellular antiapoptotic bfl-1 gene by a CBF1/RBPJ kappa-dependent pathway

The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000), and the EBV latent membrane protein 1 (LMP1) and its cellular functional homologue CD40 can both drive bfl-1 via an NF-kappaB-dependent enhancer element in the bfl-1 promoter (B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gélinas, and D. Walls, J. Virol. 78:1800-1816, 2004). Here we show that the EBV nuclear antigen 2 (EBNA2) also upregulates bfl-1. EBNA2 trans-activation of bfl-1 requires CBF1 (or RBP-J kappa), a nuclear component of the Notch signaling pathway, and there is an essential role for a core consensus CBF1-binding site on the bfl-1 promoter. trans-activation is dependent on the EBNA2-CBF1 interaction, is modulated by other EBV gene products known to interact with the CBF1 corepressor complex, and does not involve activation of NF-kappaB. bfl-1 expression is induced and maintained at high levels by the EBV growth program in a lymphoblastoid cell line, and withdrawal of either EBNA2 or LMP1 does not lead to a reduction in bfl-1 mRNA levels in this context, whereas the simultaneous loss of both EBV proteins results in a major decrease in bfl-1 expression. These findings are relevant to our understanding of EBV persistence, its role in malignant disease, and the B-cell developmental process.     

Phenotypes of Epstein-Barr virus LMP1 deletion mutants indicate transmembrane and amino-terminal cytoplasmic domains necessary for effects in B-lymphoma cells.

The Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) has previously been shown to cause EBV-negative B-lymphoma cells to grow in large clumps and to alter expression of surface activation and adhesion molecules (D. Wang, D. Liebowitz, F. Wang, C. Gregory, A. Rickinson, R. Larson, T. Springer, and E. Kieff, J. Virol. 62:1473-4184, 1988; F. Wang, C. Gregory, C. Sample, M. Rowe, D. Liebowitz, R. Murray, A. Rickinson, and E. Kieff, J. Virol. 64:2309-2318, 1990). In order to identify functional elements in the amino-terminal cytoplasmic domain and the first four transmembrane domains which were previously shown to be essential for LMP1 activity, three smaller deletion mutants were constructed and tested for their activity in B-lymphoma cells. The results of the present study indicate that the amino-terminal cytoplasmic domain, the first transmembrane domain, and the third and fourth transmembrane domains each contribute to LMP1's effects on B lymphocytes.