Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross‐react immunologically with two species of cell surface TNF receptors (TNF‐R). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2‐terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF‐R. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine‐rich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF‐R closely resemble those of TBPI. The COOH‐terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF‐R matches the COOH‐terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF‐R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF‐R. CHO cells transfected with type I TNF‐R cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNF‐Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.

A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting capdependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.Infection with poliovirus results in a dramatic shutoff of host protein synthesis that is followed by a selective and efficient translation of the viral mRNA (9). Cellular mRNAs contain a 5Ј-terminal cap structure which plays a pivotal role in the process of initiation of their translation (40). In contrast, poliovirus mRNA is uncapped (15,32), and its translation is initiated by an alternative mechanism that involves direct landing of the ribosomes at an internal site termed internal ribosome entry site (IRES) (19,36). An early event occurring during poliovirus infection is cleavage of the eukaryotic translation initiation factor 4G (eIF4G) by the viral 2A protease (10). Since eIF4G acts as an important mediator that bridges eIF3 (which is complexed to the 40S ribosomal subunit) and the cap binding protein eIF4E (18,23), the translation of cap-dependent mRNAs is selectively inhibited (10, 34). Picornavirus RNAs utilize the C-terminal fragment of eIF4G for translation (35,38). A recent study suggested that the translational inhibitory effect of poliovirus infection may under certain conditions trigger apoptotic cell death (44). Since infection with the entire virus is likely to complicate any interpretation concerning the contribution of individual viral proteins to cellular effects, it was important to express individual viral genes in cells. In this respect, the viral 2A protease is of particular interest. Since expression of the 2A protease is likely to be toxic to cells, all previous attempts to express it were by means of transient transfection (2, 33, 39). However, transient transfections are subjected to variations in the proportion of successfully transfected cells, and therefore the effects are measured in a mixed cell population. We have therefore efficiently expressed the poliovirus 2A protease in an inducible ...

The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hup55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4,(-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct.

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