Virus Latent Infection Membrane Protein Research Articles

Hostile takeovers: viral appropriation of the NF-kappaB pathway.

Transcriptional regulators of the NF-kB/IkB family promote the expression of well over 100 target genes, the majority of which participate in the host immune response (1). These proteins include a multitude of cytokines and chemokines, receptors required for immune recognition, proteins involved in antigen presentation, and adhesion receptors involved in transmigration across blood vessels walls. Because of this extensive role in immune action, NF-kB has been termed the central mediator of the immune response. Gene knockout and other studies establish roles for NF-kB in the ontogeny of the immune system but also demonstrate that NF-kB participates at multiple steps during oncogenesis (2) and the regulation of programmed cell death (3). For several reasons, the NF-kB pathway provides an attractive target to viral pathogens. Activation of NF-kB is a rapid, immediate early (IE) event that occurs within minutes after exposure to a relevant inducer, does not require de novo protein synthesis, and results in a strong transcriptional stimulation of several early viral as well as cellular genes. In this review, we will describe strategies that viruses have evolved to modulate the NF-kB pathway, to enhance viral replication, host cell survival, and evasion of immune responses. Activation of NF-kB constitutes an obvious target because many of its target genes — growth factors, cytokines and their receptors, and proto-oncogenes — profoundly influence the host cell cycle. In addition, some viruses exploit the antiapoptotic properties of NF-kB to evade the host defense mechanisms that limit replication by killing infected cells, or conversely to trigger apoptosis as a mechanism to increase virus spread. Perhaps not surprisingly, the persistent activation of the NF-kB pathway maintained by certain viruses contributes to oncogenic transformation (2). In addition to the classic studies with the avian REV-T retrovirus which contains the v-Rel oncoprotein and induces a rapid and fatal B-cell lymphoma in young birds (4), several lines of evidence demonstrate that NF-kB family members contribute to human oncogenesis. Localization of NF-kB–encoding genes at sites of chromosomal translocations and genomic rearrangements in cancer, high levels of NF-kB activity in many breast cancer cells, and constitutive nuclear NF-kB complexes in Hodgkin’s lymphoma cells all support this view (2). Furthermore, as discussed below, viral oncogene products, including human T-cell leukemia virus type 1 (HTLV-1) Tax protein and Epstein-Barr virus latent infection membrane protein 1 (EBV LMP1), each act by unique mechanisms to disrupt NF-kB regulation and initiate viral transformation.

Effects of the NIK aly Mutation on NF-κB Activation by the Epstein-Barr Virus Latent Infection Membrane Protein, Lymphotoxin β Receptor, and CD40

Homozygosity for the aly point mutation in NF-kappaB-inducing kinase (NIK) results in alymphoplasia in mice, a phenotype similar to that of homozygosity for deletion of the lymphotoxin beta receptor (LTbetaR). We now find that NF-kappaB activation by Epstein-Barr virus latent membrane protein 1 (LMP1) or by an LMP1 transmembrane domain chimera with the LTbetaR signaling domain in human embryonic kidney 293 cells is selectively inhibited by a wild type dominant negative NIK comprised of amino acids 624-947 (DN-NIK) and not by aly DN-NIK. In contrast, LMP1/CD40 is inhibited by both wild type (wt) and aly DN-NIK. LMP1, an LMP1 transmembrane domain chimera with the LTbetaR signaling domain, and LMP1/CD40 activate NF-kappaB in wt or aly murine embryo fibroblasts. Although wt and aly NIK do not differ in their in vitro binding to tumor necrosis factor receptor-associated factor 1, 2, 3, or 6 or in their in vivo association with tumor necrosis factor receptor-associated factor 2 and differ marginally in their very poor binding to IkappaB kinase beta (IKKbeta), only wt NIK is able to bind to IKKalpha. These data are compatible with a model in which activation of NF-kappaB by LMP1 and LTbetaR is mediated by an interaction of NIK or a NIK-like kinase with IKKalpha that is abrogated by the aly mutation. On the other hand, CD40 mediates NF-kappaB activation through a kinase that interacts with a different component of the IKK complex.

Cis-acting regulatory elements near the Epstein-Barr virus latent-infection membrane protein transcriptional start site

Epstein-Barr virus (EBV) latent-infection membrane protein (LMP) gene cis-acting regulatory sequences were assayed in human B lymphocytes by using chloramphenicol acetyltransferase (cat) gene expression as a reporter. The activities of progressively longer upstream elements from bases -55 to -2350 were compared. At least two positive cis-activating regulatory components (-155 to -147 and -234 to -205) upstream of the LMP promoter were defined. LMP promoter cat gene constructs were more active in a Burkitt's lymphoma cell line latently infected with the B95 EBV strain than in the same cells latently infected with the P3HR1 EBV strain. Since the P3HR1- and B95-infected cells differ in EBNA-2 and EBNA-LP expression, EBNA-2 or EBNA-LP is a likely transactivator of the LMP promoter. Probable cognate sequences for known transcription factors in the LMP promoter are discussed.

Epstein-Barr virus latent infection membrane protein increases vimentin expression in human B-cell lines

Latent Epstein-Barr virus (EBV) infection activates B-lymphocyte proliferation through mechanisms which are partially known. One approach to further delineate these mechanisms is to identify cellular genes whose expression is augmented in cells latently infected with EBV. Since EBV-negative Burkitt's lymphoma cells can be grown in continuous culture and EBV can establish growth-altering latent infection in these cells, some effects of EBV on B-lymphocyte gene expression can be studied by using this in vitro system. Pursuing this latter approach, we have used cDNA cloning and subtractive hybridization to identify a gene whose expression is increased after EBV infection. This gene encodes the cytoskeletal protein vimentin. Latent infection of established EBV-negative Burkitt's lymphoma cell lines with the transforming EBV strain, B95-8, resulted in dramatic increases in vimentin mRNA and protein levels, while infection with the nontransforming P3HR1 strain failed to do so. Vimentin induction was reproduced by the expression of the single EBV gene which encodes the latent infection membrane protein (LMP). An amino-terminal LMP deletion mutant did not induce vimentin. These results are of particular interest in light of the transforming potential of LMP, as demonstrated in rodent fibroblasts, and the interaction between vimentin and LMP observed in immunofluorescent colocalization and cell fractionation studies.

Epstein-Barr virus latent infection membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity.

A latent infection membrane protein (LMP) encoded by the Epstein-Barr virus (EBV) genome in latently infected, growth-transformed lymphocytes alters the phenotype of a human EBV-negative B-lymphoma cell line (Louckes) when introduced by gene transfer. These LMP-expressing cells exhibit increased homotypic adhesion due to increased expression of the adhesion molecules LFA-1 and ICAM-1. Increased homotypic adhesion could foster B-cell growth by facilitating autocrine growth factor effects. LFA-3 expression is also induced. The induction of LFA-3 and ICAM-1 results in increased heterotypic adhesion to T lymphocytes. This could result in more effective T-cell immune surveillance. Since LMP is expressed in EBV-transformed lymphocytes and has been demonstrated to transform rodent fibroblasts in vitro, a wide range of possible effects on B-lymphoma cell growth were assayed. In the Louckes B-lymphoma cell line, EBV LMP causes increased cell size, acid production, plasma membrane ruffling, and villous projections. Although cell proliferation rate was not greatly affected, the steady-state intracellular free calcium level, transforming growth factor beta responsiveness, and expression of the lymphocyte activation markers (CD23 and transferrin receptor) were increased. Thus, LMP appears to be a mediator of EBV effects on B-cell transformation. In transfected lymphoma cells, LMP localizes to patches at the cell periphery and associates with the cytoskeleton as it does in EBV-transformed B lymphocytes or in rodent fibroblasts. A partially deleted form of LMP (D1LMP) does not aggregate in patches or associate with the cytoskeleton and had little effect on B-cell growth. Thus, cytoskeletal association may be integral to LMP activity.

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.