Integrating Enhancer Mechanisms to Establish a Hierarchical Blood Development Program.

DISCLOSURES: No relevant conflicts of interest to declare.

EBV lytic infection enhances transformation of B-lymphocytes infected with EBV in the presence of T-lymphocytes.

Epstein-Barr virus (EBV) establishes lifelong latency in B-lymphocytes following infection. Although in immune-competent individuals EBV remains in a quiescent state, in immunodeficient individuals, such as those with AIDS or transplant recipients, B-lymphocytes infected with EBV proliferate to give rise to lymphoproliferative diseases. Similarly, in vitro, EBV transforms human B-lymphocytes into indefinitely growing lymphoblastoid cell lines (LCLs) in the absence of cytotoxic T-lymphocytes. Although LCLs harbor the entire EBV genome as an episome, in most cells the virus remains in a latent state expressing a fraction of EBV genes, and lytic infection occurs spontaneously but only in a small percentage of cells. Here, we report that lytic infection contributes to EBV-induced lymphoproliferation by a paracrine mechanism. An EBV immediate-early protein, BZLF1, induces IL-13, thus facilitating the proliferation of EBV-transformed B-lymphocytes in the presence of T-lymphocytes. These data suggest that lytic gene products could contribute to virus-induced oncogenesis by a paracrine mechanism.

Epstein-Barr virus-encoded Bcl-2 homologue functions as a survival factor in Wp-restricted Burkitt lymphoma cell line P3HR-1.

Burkitt lymphoma (BL) is etiologically associated with Epstein-Barr virus (EBV). EBV-positive BL tumors display two latent forms of infection. One is referred to as latency I infection, in which EBV expresses the virus genome maintenance protein EBNA1 as the only viral protein. The other is referred to as Wp-restricted latency and was recently identified in a subset of BL tumors. In these tumors, EBV expresses EBNA1, EBNA3A, EBNA3B, EBNA3C, a truncated form of EBNA-LP, and the viral Bcl-2 homologue BHRF1, all of which are driven by the BamHI W promoter (Wp). To investigate the role of EBV in Wp-restricted BL, we conditionally expressed a dominant-negative EBNA1 (dnEBNA1) mutant which interrupts the virus genome maintenance function of EBNA1 in the P3HR-1 BL cell line. Induction of dnEBNA1 expression caused loss of the EBV genome and resulted in apoptosis of P3HR-1 cells in the absence of exogenous apoptosis inducers, indicating that P3HR-1 cells cannot survive without EBV. Stable transfection of the BHRF1 gene into P3HR-1 cells rescued the cells from the apoptosis induced by dnEBNA1 expression, whereas stable transfection of truncated EBNA-LP, EBNA3A, or EBNA3C did not. Moreover, knockdown of BHRF1 expression in P3HR-1 cells resulted in increased cell death. These results indicate that EBV is essential for the survival of P3HR-1 cells and that BHRF1 functions as a survival factor. Our finding implies a critical contribution of BHRF1 to the pathogenesis of Wp-restricted BLs.

Quantitative evaluation of the role of Epstein-Barr virus immediate-early protein BZLF1 in B-cell transformation.

The Epstein-Barr virus (EBV) immediate-early transactivator BZLF1 plays a key role in switching EBV infection from the latent to the lytic form by stimulating the expression cascade of lytic genes; it also regulates the expression of several cellular genes. Recently, we reported that BZLF1 is expressed in primary human B cells early after EBV infection. To investigate whether this BZLF1 expression early after infection plays a role in the EBV-induced growth transformation of primary B cells, we generated BZLF1-knockout EBV and quantitatively evaluated its transforming ability compared with that of wild-type EBV. We found that the 50% transforming dose of BZLF1-knockout EBV was quite similar to that of wild-type EBV. Established lymphoblastoid cell lines (LCLs) harbouring BZLF1-knockout EBV were indistinguishable from LCLs harbouring wild-type EBV in their pattern of latent gene expression and in their growth in vitro. Furthermore, the copy numbers of EBV episomes were very similar in the LCLs harbouring BZLF1-knockout EBV and in those harbouring wild-type EBV. These data indicate that disrupting BZLF1 expression in the context of the EBV genome, and the resultant inability to enter lytic replication, have little impact on the growth of LCLs and the steady-state copy number of EBV episomes in established LCLs.

Proteasome-dependent decrease in Akt by growth factors in vascular smooth muscle cells.

Akt is activated by growth factors to regulate various aspects of vascular smooth muscle cell function. Platelet-derived growth factor (PDGF) and insulin-like growth factor-1 activated Akt in vascular smooth muscle cells with a rapid reduction of total Akt protein that lasted for several hours. The downregulation of Akt required phosphatidylinositol 3-kinase activity, but not intrinsic Akt activity. The downregulation of Akt was abrogated by MG-132, a proteasome inhibitor, but not by inhibitors of calpain or cathepsins. Akt was found in ubiquitin immune complex after PDGF treatment. Proteasome-dependent degradation of Akt may provide a counter-regulatory mechanism against overactivation of Akt.