The truncated form of the Epstein-Barr virus latent-infection membrane protein expressed in virus replication does not transform rodent fibroblasts.

The gene encoding the Epstein-Barr virus membrane protein LMP, expressed in latent infection, is known to induce morphologic changes and some loss of contact inhibition in NIH 3T3 cells as well as profound loss of contact inhibition and of anchorage dependence in Rat-1 cells. Another form of LMP (D1LMP), deleted for the amino terminus and first four putative transmembrane domains of LMP, was recently shown to be expressed late in Epstein-Barr virus replication. We now demonstrate that D1LMP has no transformation-associated phenotypic effect in Rat-1 cells and does not significantly affect LMP-induced Rat-1 cell transformation. LMP activity and D1LMP inactivity in inducing anchorage-independent growth are not restricted to Rat-1 cells, but are also evident in BALB/c 3T3 cells. In both cell types, loss of contact inhibition and anchorage independence are acutely evident after LMP expression. Although newly transfected polyclonal Rat-1 or BALB/c cells have a lower agar cloning efficiency than established LMP-expressing clones, this is attributable, at least in part, to their lower average LMP expression, since among clones of transfected cells, higher cloning efficiencies correlated with higher levels of LMP. LMP is bound to the vimentin cytoskeletal network in rodent fibroblasts as it is in transformed lymphocytes, whereas D1LMP showed no detectable cytoskeletal binding, suggesting that cytoskeletal association may be integral to LMP-mediated cell transformation. LMP association with the cytoskeleton in latently infected, growth-transformed lymphocytes and LMP-transformed rodent fibroblasts, correlated with the lack of both rodent cell-transforming activity and cytoskeletal association of D1LMP supports working hypothesis that cytoskeletal association is important in LMP transforming activity.     

Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit

We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.     

Interferon regulatory factor 7 mediates activation of Tap-2 by Epstein-Barr virus latent membrane protein 1

Transporter associated with antigen processing 2 (Tap-2) is responsible for ATP-dependent transport of peptides from the cytosol to the endoplasmic reticulum, where peptides bind to newly synthesized human leukocyte antigen (HLA) class I molecules, which are essential for cellular immune responses. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) has been shown to induce the expression of Tap-2. In this study, the induction of endogenous Tap-2 by LMP-1 is shown to be associated with and requires the expression of interferon regulatory factor 7 (IRF-7). In DG75 Burkitt's lymphoma (BL) cells, in which LMP-1 induces the expression of IRF-7, LMP-1 induced endogenous Tap-2, and ectopic expression of IRF-7 could enhance the induction. In Akata BL cells, in which LMP-1 could not induce IRF-7, LMP-1 could not induce Tap-2. Addition of IRF-7, which complements the defect in Akata cells, could stimulate the expression of Tap-2. Furthermore, LMP-1 and IRF-7A but not other IRF-7 splicing variants could activate endogenous Tap-2. A Tap-2 promoter reporter construct could be activated by the overexpression of IRF-7A. The activation could be specifically enhanced by LMP-1 and was dependent on an intact interferon-stimulated response element (ISRE) present in the Tap-2 promoter. Also, IRF-7 can bind to the Tap-2 promoter under physiological conditions in vivo, as shown by formaldehyde cross-linking, as well as to the Tap-2 ISRE in vitro, as shown by gel mobility shift assays. Furthermore, LMP-1 facilitates the phosphorylation and nuclear translocation of IRF-7. These data point to the role of IRF-7 as a secondary mediator of LMP-1-activated signal transduction for Tap-2 as follows: LMP-1 stimulates the expression of IRF-7 and facilitates its phosphorylation and nuclear translocation, and then the activated IRF-7 mediates the activation of the cellular Tap-2 gene. The induction of Tap-2 by IRF-7 and LMP-1 may have an important implication for the immune response to EBV and its persistence in vivo.