Wolfgang Hammerschmidt

Epstein–Barr virus‐mediated B‐cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor

The Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for the immortalization of human B cells and is linked etiologically to several human tumors. LMP1 is an integral membrane protein which acts like a constitutively active receptor. It binds tumor necrosis factor (TNF)‐receptor‐associated factors (TRAFs), activates NF‐κB and triggers the transcription factor AP‐1 via the c‐Jun N‐terminal kinase (JNK) cascade, but its specific contribution to B‐cell immortalization has not been elucidated fully. To address the function of LMP1, we established B cell lines with a novel mini‐EBV plasmid in which the LMP1 gene can be regulated at will without affecting the expression of other latent EBV genes. We demonstrate here that continuous expression of LMP1 is essential for the proliferation of EBV‐immortalized B cells in vitro. Re‐induction of LMP1 expression or activation of the cellular CD40 receptor both induce the JNK signaling cascade, activate the transcription factor NF‐κB and stimulate proliferation of these B cells. Our findings strongly suggest that LMP1 mimics B‐cell activation processes which are physiologically triggered by CD40–CD40 ligand signals. Since LMP1 acts in a ligand‐independent manner, it replaces the T cell‐derived activation signal to sustain indefinite B‐cell proliferation.

Latent membrane protein 1 of Epstein–Barr virus mimics a constitutively active receptor molecule

Latent membrane protein 1 (LMP1) of Epstein–Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B‐cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF‐R) ‐associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF‐κB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF‐R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to β‐adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy‐terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild‐type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF‐2 receptor. In EBV, immortalized B cells cross‐linking in conjunction with membrane targeting of the carboxy‐terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand‐independent.

Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins.

Latent membrane protein 1 (LMP1) acts like a permanently activated receptor of the tumor necrosis factor (TNF)‐receptor superfamily and is absolutely required for B cell immortalization by Epstein–Barr virus. Molecular and biochemical approaches demonstrated that LMP1 usurps cellular signaling pathways resulting in the induction of NF‐κB and AP‐1 via two C‐terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of LMP1s C‐terminus is required for the activation of Janus kinase 3 (JAK3). The interaction of LMP1 and JAK3 leads to the enhanced tyrosine auto/transphosphorylation of JAK3 within minutes after crosslinking of a conditional NGF‐R:LMP1 chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the LMP1 C‐terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.

The Epstein–Barr virus lytic program is controlled by the co-operative functions of two transactivators

The propagation of herpesviruses has long been viewed as a temporally regulated sequential process that results from the consecutive expression of specific viral transactivators. As a key step in this process, lytic viral DNA replication is considered as a checkpoint that controls the expression of the late structural viral genes. In a novel genetic approach, we show that both hypotheses do not hold true for the Epstein–Barr virus (EBV). The study of viral mutants of EBV in which the early genes BZLF1 and BRLF1 are deleted allowed a precise assignment of the function of these proteins. Both transactivators were absolutely essential for viral DNA replication. Both BZLF1 and BRLF1 were required for full expression of the EBV proteins expressed during the lytic program, although the respective influence of these molecules on the expression of various viral target genes varied greatly. In replication‐defective viral mutants, neither early gene expression nor DNA replication was a prerequisite for late gene expression. This work shows that BRLF1 and BZLF1 harbor distinct but complementary functions that influence all stages of viral production.

Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade.

The Epstein–Barr virus latent membrane protein‐1 (LMP‐1) is an integral membrane protein which transforms fibroblasts and is essential for EBV‐mediated B‐cell immortalization. LMP‐1 has been shown to trigger cellular NF‐κB activity which, however, cannot fully explain the oncogenic potential of LMP‐1. Here we show that LMP‐1 induces the activity of the AP‐1 transcription factor, a dimer of Jun/Jun or Jun/Fos proteins. LMP‐1 effects on AP‐1 are mediated through activation of the c‐Jun N‐terminal kinase (JNK) cascade, but not the extracellular signal‐regulated kinase (Erk) pathway. Consequently, LMP‐1 triggers the activity of the c‐Jun N‐terminal transactivation domain which is known to be activated upon JNK‐mediated phosphorylation. Deletion analysis indicates that the 55 C‐terminal amino acids of the LMP‐1 molecule, but not its TRAF interaction domain, are essential for AP‐1 activation. JNK‐mediated transcriptional activation of AP‐1 is the direct output of LMP‐1‐triggered signaling, as shown by an inducible LMP‐1 mutant. Using a tetracycline‐regulated LMP‐1 allele, we demonstrate that JNK is also an effector of non‐cytotoxic LMP‐1 signaling in B cells, the physiological target cells of EBV. In summary, our data reveal a novel effector of LMP‐1, the SEK/JNK/c‐Jun/AP‐1 pathway, which contributes to our understanding of the immortalizing and transforming potential of LMP‐1.

Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus

We have identified a cis-acting element of Epstein-Barr virus (EBV) that mediates viral DNA replication during the lytic phase of this viruss life cycle. This lytic origin of DNA replication, termed oriLyt, is complex in structure in that it contains multiple regions that are required for replication and additional DNA sequences that increase replication. One of the required regions of oriLyt can be functionally substituted by a transcriptional enhancing element. DNA replication mediated by oriLyt depends on EBV DNA polymerase and yields a concatemeric molecule. A vector, which contains both oriP (the EBV plasmid origin of replication) and oriLyt, can be maintained as a plasmid in latently EBV-infected cells and can be amplified 100- to 1000-fold in cells in which the lytic phase of the viral life cycle is induced.

B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2.

Infection of primary B‐lymphocytes by Epstein‐Barr virus (EBV) leads to growth transformation of these B‐cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B‐cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B‐cells by mutant virus resulted in estrogen‐dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non‐proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B‐cells. These data suggest that EBV is using a common pathway for B‐cell activation bypassing the requirement for antigen, T‐cell signals and growth factors.

Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein–Barr virus

Epstein–Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP‐dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co‐immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.

Micro RNAs of Epstein-Barr Virus Promote Cell Cycle Progression and Prevent Apoptosis of Primary Human B Cells

Cellular and viral microRNAs (miRNAs) are involved in many different processes of key importance and more than 10,000 miRNAs have been identified so far. In general, relatively little is known about their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification. The recent discovery that Epstein-Barr virus (EBV) and other herpesviruses produce their own, barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects. We have systematically introduced mutations in EBVs precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs. Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells. Our findings also indicate that EBVs miRNAs are not needed to control the exit from latency. The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBVs lytic phase.

The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the earliest viral proteins expressed after infection and is the only latent protein consistently expressed in viral-associated tumors. EBNA1s crucial role in viral DNA replication, episomal maintenance, and partitioning is well examined whereas its importance for the immortalization process and the tumorgenicity of EBV is unclear. To address these open questions, we generated, based on the maxi-EBV system, an EBNA1-deficient EBV mutant and used this strain to infect primary human B cells. Surprisingly, lymphoblastoid cell lines (LCL) emerged from these experiments, although with very low frequency. These cell lines were indistinguishable from normal LCLs with respect to proliferation and growth conditions. A detailed analysis indicated that the entire viral DNA was integrated into the cellular genome. At least 5 of the 11 latent EBV proteins were expressed, indicating the integrity of the EBV genome. EBNA1-positive and ΔEBNA1-EBV-LCLs were injected into severe combined immunodeficient (SCID) mice to examine their tumorgenicity in comparison. Both groups supported tumor growth, indicating that EBNA1 is not mandatory for EBVs oncogenic potential. The results shown provide genetic evidence that EBNA1 is not essential to establish LCLs but promotes the efficiency of this process significantly.