Characterization of mutants within the gene for the adenovirus E3 14.7-kilodalton protein which prevents cytolysis by tumor necrosis factor.

The 14,700-Da protein (14.7K protein) encoded by the E3 region of adenovirus has previously been shown to protect mouse cells from cytolysis by tumor necrosis factor (TNF). Delineating the sequences in the 14.7K protein that are required for this activity may provide insight into the mechanism of protection from TNF by 14.7K as well as the mechanism of TNF cytolysis. In the present study, we examined the ability of 14.7K mutants to protect cells from lysis by TNF. In-frame deletions as well as Cys-to-Ser mutations in the 14.7K gene were generated by site-directed mutagenesis and then built into the genome of a modified adenovirus type 5 (dl7001) that lacks all E3 genes. dl7001, which replicates to the same titers as does adenovirus type 5 in cultured cells, has the largest E3 deletion analyzed to date. 51Cr release was used to assay TNF cytolysis. Our results indicate that most mutations in the 14.7K gene result in a loss of function, suggesting that nearly the entire protein rather than a specific domain functions to prevent TNF cytolysis.     

The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis

We have previously described a 14,700 m.w. protein (14.7K) encoded by the E3 region of adenovirus that prevents TNF-mediated cytolysis of adenovirus-infected C3HA mouse fibroblasts. In the studies described here we have extended our analysis of TNF cytolysis of C3HA cells and the circumstances under which 14.7K protects these cells from cytolysis. C3HA cells were killed by TNF in the presence of inhibitors of protein synthesis, in the presence of cytochalasin E (which disrupts the microfilaments), and when adenovirus E1A was expressed. As described for other cell types, pretreatment of C3HA cells with TNF prevented cytolysis by TNF plus cycloheximide or TNF plus cytochalasin E, indicating that TNF induces a response that protects against these treatments. Remarkably, when 14.7K was expressed in virus-infected cells, it also prevented TNF-induced lysis whether sensitivity to TNF was induced by inhibition of protein synthesis, disruption of the cytoskeleton by cytochalasin E, or expression of adenovirus E1A. The 14.7K protein also prevented TNF lysis of cells that are spontaneously sensitive to TNF lysis. Thus, 14.7K appears to be a general inhibitor of TNF cytolysis, and as such should be an important tool in unraveling the mechanism of TNF cytolysis. There was one exception; NCTC-929 cells were spontaneously sensitive to TNF lysis and that lysis was not affected by 14.7K even though the protein was made in large quantities and was metabolically stable in these cells. This suggests that there is heterogeneity among TNF-sensitive cell lines. The 14.7K protein was found in both the nuclear and cytosol fractions of TNF resistant as well as all spontaneously sensitive cells suggesting that 14.7K may have more than one site of action within the cell.     

The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model.

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumor necrosis factor alpha (TNF-alpha)-mediated lysis of cells in tissue culture experiments, but the relevance of this effect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to block TNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vaccinia virus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene for murine TNF-alpha was inserted into the same VV containing the 14.7K gene to ensure that each cell infected with the VV recombinant would express both the agonist (TNF) and its antagonist (14.7K). VV was utilized as the vector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expression of the protein products. In addition, infection of mice with VV induces disease with quantifiable morbidity, mortality, and virus replication. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that the Ad 14.7K protein increases the virulence of VV carrying the TNF-alpha gene by reversing the attenuating effect of TNF-alpha on VV pathogenicity. This was demonstrated by increased mortality, pulmonary pathology, and viral titers in lung tissue following infection with VV coexpressing the 14.7K protein and TNF-alpha, compared with the control virus expressing TNF-alpha alone. These results suggest that the 14.7K protein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein which functions in vivo to help counteract the antiviral effects of TNF-alpha.     

The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha.

The adenovirus E1A and E1B proteins are required for transformation of primary rodent cells. When expressed in the absence of the 19,000-dalton (19K) E1B protein, however, the E1A proteins are acutely cytotoxic and induce host cell chromosomal DNA fragmentation and cytolysis, analogous to cells undergoing programmed cell death (apoptosis). E1A alone can efficiently initiate the formation of foci which subsequently undergo abortive transformation whereby stimulation of cell growth is counteracted by continual cell death. Cell lines with an immortalized growth potential eventually arise with low frequency. Coexpression of the E1B 19K protein with E1A is sufficient to overcome abortive transformation to produce high-frequency transformation. Like E1A, the tumoricidal cytokine tumor necrosis factor alpha (TNF-alpha) evokes a programmed cell death response in many tumor cell lines by inducing DNA fragmentation and cytolysis. Expression of the E1B 19K protein by viral infection, by transient expression, or in transformed cells completely and specifically blocks this TNF-alpha-induced DNA fragmentation and cell death. Cosegregation of 19K protein transforming activity with protection from TNF-alpha-mediated cytolysis demonstrates that both activities are likely the consequence of the same function of the protein. Therefore, we propose that by suppressing an intrinsic cell death mechanism activated by TNF-alpha or E1A, the E1B 19K protein enhances the transforming activity of E1A and enables adenovirus to evade TNF-alpha-dependent immune surveillance.     

Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis.

A 14,700-kDa protein (14.7K) encoded by the E3 region of adenovirus has been shown to protect adenovirus-infected mouse C3HA cells from lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). These infected cells are sensitized to TNF by expression of the adenovirus E1A proteins (P. Duerksen-Hughes, W. S. M. Wold, and L. R. Gooding, J. Immunol. 143:4193-4200, 1989). In this study we show that 14.7K suppresses TNF cytolysis independently of adenovirus infection. Mouse C3HA and C127 cells were transfected with the 14.7K gene controlled by the mouse metallothionein promoter, and permanent 14.7K-expressing cell lines were tested for sensitivity to TNF cytolysis. Transfected cells which were sensitized to TNF either by inhibitors of protein synthesis, microfilament-destabilizing agents, or adenovirus infection were found to be resistant to TNF cytolysis. Two monoclonal antibodies were isolated and used to quantitate 14.7K in transfected and infected cells. Enzyme-linked immunosorbent assay (ELISA) analysis with these monoclonal antibodies and 14.7K immunoblots showed that 14.7K expression can be induced with cadmium in C3HA and C127 transfectants. The 14.7K induction correlated with a dose-dependent decrease in sensitivity to TNF cytotoxicity. The 14.7K protein does not substantially alter cell surface TNF receptor numbers or affinity on C3HA mouse fibroblasts, as determined by Scatchard analysis of 125I-TNF binding. The 14.7K protein also does not alter TNF signal transduction in general, because TNF induction of cell surface class I major histocompatibility complex molecules on 14.7K transfectants was unmodified. Our findings indicate that the adenovirus 14.7K protein functions as a specific inhibitor of TNF cytolysis in the absence of other adenovirus proteins and thus is a unique tool to study the mechanism of TNF cytotoxicity.     

An adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis complexes with dynein and a small GTPase

Adenoviruses (Ad) code for immunoregulatory and cytokine regulatory proteins, one of which is the early region 3, 14.7-kDa protein (Ad E3-14.7K), which has been shown to inhibit tumor necrosis factor alpha-induced apoptosis. In an effort to understand the mechanism of action of Ad E3-14.7K, we previously searched for cell proteins with which it interacted. Three Ad E3-14.7K-interacting proteins (FIP-1, -2, and -3) were isolated. FIP-1 is a small GTPase which was used in this report as bait in the yeast two-hybrid system to find other interacting cell targets. The search resulted in the isolation of a protein, which we called GIP-1 (GTPase-interacting protein) that subsequently was shown to be identical to one of the light-chain components of human dynein (TCTEL1). FIP-1 was able to bind both TCTEL1 and Ad E3-14.7K simultaneously and was necessary to form a complex in which the viral protein was associated with a microtubule-binding motor protein. The functional significance of these interactions is discussed with respect to the steps of the Ad life cycle which are microtubule associated.     

Characterization of TNFRSF19, a novel member of the tumor necrosis factor receptor superfamily

By searching the expressed sequence tag database, a novel murine tumor necrosis factor receptor designated TNFRSF19 was identified. TNFRSF19 cDNA encodes a putative membrane protein of 348 amino acids with one incomplete and two complete cysteine-rich motifs within its extracellular region and a large cytoplasmic domain. TNFRSF19 mRNA can be detected in most murine tissues examined, particularly in brain, reproductive organs, and late developmental stages of murine embryo, but not in tissues of the immune system. The cell surface expression of the ligand of TNFRSF19 is highly restricted. Of 22 human and murine cell lines examined by FACS analysis, only Raji (B cell lymphoma cell line), GM847 (fibroblast cell line), 293 (embryonic kidney cell line), and K562 (chronic myeloid leukemia) were positive. TNFRSF19 did not bind newly cloned TNF ligands, including TWEAK (HGMW-approved symbol TNFSF12), VEGI/TL1 (HGMW-approved symbol TNFSF15), TL6/endokine (HGMW-approved symbol TNFSF18), APRIL (HGMW-approved symbol TNFSF13), OPGL (HGMW-approved symbol TNFSF11), LIGHT (HGMW-approved symbol TNFSF14), or BAFF/THANK (HGMW-approved symbol TNFSF13B) by enzyme-linked immunosorbent assay and FACS analyses. Overexpression of TNFRSF19 transduced neither apoptotic signaling nor signals leading to NF-kappaB induction. Taken together with the data that the TNFRSF19 extracellular domain-immunoglobulin fusion protein did not affect the allogeneic mixed lymphocyte reaction, our data indicate that TNFRSF19 is not involved in the modulation of immune responses.     

The adenovirus E3 14.5-kilodalton protein, which is required for down-regulation of the epidermal growth factor receptor and prevention of tumor necrosis factor cytolysis, is an integral membrane protein oriented with its C terminus in the cytoplasm.

We previously reported that the adenovirus type 5 E3 14.5-kilodalton protein (14.5K) forms a complex with E3 10.4K and that both proteins are required to down-regulate the epidermal growth factor receptor in adenovirus-infected human cells. Both proteins are also required to prevent cytolysis by tumor necrosis factor of most mouse cell lines infected by adenovirus mutants that lack E3 14.7K. The E3 14.5K amino acid sequence suggests that 14.5K is an integral membrane protein with an N-terminal signal sequence for membrane insertion. Here we show that 14.5K was found exclusively in cytoplasmic membrane fractions. Radiochemical sequencing of 14.5K indicated that the N-terminal signal sequence is cleaved predominantly between Cys-18 and Ser-19. With a mutant that does not express 10.4K, cleavage occurs predominantly between Phe-17 and Cys-18, indicating that the presence or absence of 10.4K affects the signal cleavage site. 14.5K was extracted into the detergent phase with Triton X-114, it remained associated with membranes after extraction with Na2CO3 at pH 11.5, and it was partially protected by membranes from proteinase K digestion; these observations indicate that 14.5K is an integral membrane protein. Proteinase K digestion followed by immunoprecipitation with antipeptide antisera directed against the N or C terminus of mature 14.5K indicated that 14.5K is oriented in the membrane with its N terminus in the lumen and its C terminus in the cytoplasm. Thus, 14.5K is a type I bitopic membrane protein. Previous studies indicated that 10.4K is also an integral membrane protein oriented with its C terminus in the cytoplasm. Altogether, these findings suggest that cytoplasmic membranes are the site of action when 10.4K and 14.5K down-regulate the epidermal growth factor receptor and prevent tumor necrosis factor cytolysis.     

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.     

Purification and biologic characterization of a specific tumor necrosis factor alpha inhibitor

The purpose of the present investigation was to purify a urine-derived tumor necrosis factor alpha inhibitor (TNF alpha INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNF alpha INH purified by ion-exchange chromatographies, gel filtration, TNF alpha affinity column, and reverse-phase chromatography. The TNF alpha INH migrates with an apparent Mr of approximately 33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNF alpha INH activity from the gel yields also a approximately 33,000-Da inhibitory fraction. Besides inhibiting TNF alpha-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNF alpha INH impeded in a dose-dependent manner prostaglandin E2 production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNF alpha INH is active on both actinomycin D-treated and untreated cells. In contrast to TNF alpha, TNF beta-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1 alpha nor interleukin-1 beta biologic activities. The mechanism of action of TNF alpha INH involves blocking of 125I-TNF alpha binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNF alpha with TNF alpha INH increased binding inhibition, suggesting an interaction between TNF alpha and the inhibitor.     

Wengen,a member of the Drosophila tumor necrosis factor receptor superfamily,is required for Eiger signaling

We identified Wengen, the first member of the Drosophila tumor necrosis factor receptor (TNFR) superfamily. Wengen is a type III membrane protein with conserved cysteine-rich residues (TNFR homology domain) in the extracellular domain, a hallmark of the TNFR superfamily. wengen mRNA is expressed at all stages of Drosophila development. The small-eye phenotype caused by an eye-specific overexpression of a Drosophila TNF superfamily ligand, Eiger, was dramatically suppressed by down-regulation of Wengen using RNA interference. In addition, Wengen and Eiger physically interacted with each other through their TNFR homology domain and TNF homology domain, respectively. These results suggest that Wengen can act as a component of a functional receptor for Eiger. Our identification of Wengen and further genetic analysis should provide increased understanding of the evolutionarily conserved roles of TNF/TNFR superfamily proteins in normal development, as well as in some pathophysiological conditions.     

A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.

TR6 (decoy receptor 3 (DcR3)) is a new member of the tumor necrosis factor receptor (TNFR) family. TR6 mRNA is expressed in lung tissues and colon adenocarcinoma, SW480. In addition, the expression of TR6 mRNA was shown in the endothelial cell line and induced by phorbol 12-myristate 13-acetate/ionomycin in Jurkat T leukemia cells. The open reading frame of TR6 encodes 300 amino acids with a 29-residue signal sequence but no transmembrane region. Using histidine-tagged recombinant TR6, we screened soluble forms of TNF-ligand proteins with immunoprecipitation. Here, we demonstrate that TR6 specifically binds two cellular ligands, LIGHT (herpes virus entry mediator (HVEM)-L) and Fas ligand (FasL/CD95L). These bindings were confirmed with HEK 293 EBNA cells transfected with LIGHT cDNA by flow cytometry. TR6 inhibited LIGHT-induced cytotoxicity in HT29 cells. It has been shown that LIGHT triggers apoptosis of various tumor cells including HT29 cells that express both lymphotoxin beta receptor (LTbetaR) and HVEM/TR2 receptors. Our data suggest that TR6 inhibits the interactions of LIGHT with HVEM/TR2 and LTbetaR, thereby suppressing LIGHT- mediated HT29 cell death. Thus, TR6 may play a regulatory role for suppressing in FasL- and LIGHT-mediated cell death.     

Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand

Among members of the tumor necrosis factor receptor (TNFR) superfamily, 4-1BB, CD27, and glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) share a striking homology in the cytoplasmic domain. Here we report the identification of a new member, activation-inducible TNFR family member (AITR), which belongs to this subfamily, and its ligand. The receptor is expressed in lymph node and peripheral blood leukocytes, and its expression is up-regulated in human peripheral mononuclear cells mainly after stimulation with anti-CD3/CD28 monoclonal antibodies or phorbol 12-myristate 13-acetate/ionomycin. AITR associates with TRAF1 (TNF receptor-associated factor 1), TRAF2, and TRAF3, and induces nuclear factor (NF)-kappaB activation via TRAF2. The ligand for AITR (AITRL) was found to be an undescribed member of the TNF family, which is expressed in endothelial cells. Thus, AITR and AITRL seem to be important for interactions between activated T lymphocytes and endothelial cells.     

Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells.     

Renewed interest in cancer immunotherapy with the tumor necrosis factor superfamily molecules

Molecules belonging to the Tumor Necrosis Factor (TNF) and TNF receptor superfamilies have explosively expanded through the era of genomics and bioinformatics. Biological investigations of these molecules have explored their potency as attractive targets for cancer therapy. Anti-tumor mechanisms mediated by TNF superfamily molecules (TNFSF) could be classified into direct actions onto tumor cells and indirect effects through immune or non-immune components of tumor-bearing host. In this review, we focus on TRAIL, CD40, 4-1BB (CD137), and LIGHT as promising molecules to mediate powerful and selective anti-tumor responses, and summarize their unique effector mechanisms. In addition, optimal approaches to manipulate these molecules for cancer therapy are also discussed. We try to provide an insight into a role of TNFSF in cancer therapeutics and highlight each of their potency to be an important player in anti-cancer strategies.     

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-X(L). These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.