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PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 °C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.

We addressed the question of whether furin is the endoprotease primarily responsible for processing the human immunodeficiency virus type I (HIV-I) envelope protein gp160 in mammalian cells. The furin-deficient Chinese hamster ovary (CHO)-K1 strain RPE.40 processed gp160 as efficiently as wild-type CHO-K1 cells in vivo. Although furin can process gp160 in vitro, this processing is probably not physiologically relevent, because it occurs with very low efficiency. PACE4, a furin homologue, allowed processing of gp160 when both were expressed in RPE.40 cells. Further, PACE4 participated in the activation of a calcium-independent protease activity in RPE.40 cells, which efficiently processed the gp160 precursor in vitro. This calcium-independent protease activity was not found in another furin-deficient cell strain, 7.P15, selected from the monkey kidney cell line COS-7.

The low-density-lipoprotein receptor-related protein (LRP) is a multifunctional receptor involved in the clearance of a large number of diverse ligands, including proteases, protease-inhibitor complexes and lipoproteins. The mature receptor is composed of a 515 kDa and a 85 kDa subunit generated by proteolytic cleavage from a 600 kDa precursor polypeptide in a trans-Golgi compartment. Proteolytic processing occurs C-terminal to the tetrabasic amino acid sequence RHRR, a consensus recognition site for precursor processing endoproteases or convertases. In this study we have identified furin, a subtilisin-type protease, to be necessary for efficient processing of LRP in cells. Furin-deficient RPE.40 cells exhibited an impaired processing of endogenous LRP and of a recombinant soluble form of the receptor containing the processing site. The processing defect in RPE.40 cells could be complemented by expression of furin from a transfected cDNA in cultured cells and by purified furin in vitro. The impaired maturation of LRP in RPE.40 cells did not affect its intracellular transport, and correlated with a slight but consistent reduction in the endocytosis of LRP-specific ligands. These data suggest that proteolytic processing of LRP by furin is not necessary for intracellular trafficking but might be required for normal receptor activity.

Both diphtheria toxin and Pseudomonas exotoxin A inhibit eukaryotic protein synthesis by ADP-ribosylating diphthamide, a posttranslationally modified histidine residue present in the elongation factor 2 (EF-2) protein. Elongation factor 2 cannot be ADP-ribosylated by the toxins unless this histidine is modified. In this report we identify three new point mutations in toxin-resistant alleles of the Chinese hamster ovary cell elongation factor 2 gene. The mutations resulted in amino acid substitutions at positions 584 (serine to glycine), 714 (isoleucine to asparagine), and 719 (glycine to aspartic acid). All three amino acid substitutions prevented the biosynthesis of diphthamide. The amount by which the toxins reduced protein synthesis in each of these mutant cell strains suggested that all three mutations also either impaired the function of EF-2 or reduced its steady state level in the cytoplasm. Western blot analysis showed that equal amounts of EF-2 were present in each of the cell strains, indicating that the mutations impaired the catalytic function of EF-2.

We have demonstrated that the native proenzymatic form of Pseudomonas exotoxin A can be cleaved at its specific activation site by furin in intact Chinese hamster ovary cells or in vitro by furin in isolated membrane fractions from these cells. We have compared the activity of furin in cell membrane fractions with that of purified, recombinant human furin. We have verified that RPE.40, a Pseudomonas toxin-resistant mutant cell strain, is mutant in the fur gene, and we have demonstrated that these cells are deficient in cleavage of the toxin. We have also determined that this cleavage of Pseudomonas toxin by furin takes place at the authentic activation site to release the 37-kDa active fragment.

Characterization of an endoprotease-deficient mutant Chinese hamster ovary (CHO) cell, designated RPE.40, revealed that it bound less than 10% as much insulin as did its parent, CHO-K1. We examined processing of the endogenous insulin receptor in CHO-K1 and RPE.40 cells, and processing of the human insulin receptor expressed in these cells. RPE.40 cells did not process the endogenous insulin proreceptor to its subunit forms, and processed the human insulin proreceptor inefficiently. Accumulation of the proreceptor form of the insulin receptor was seen in both cases. Furin is a mammalian endoprotease that cleaves proproteins at a consensus sequence of basic amino acids found in the insulin proreceptor. Expression of mouse furin in RPE.40 cells restored normal processing of the endogenous and the human insulin receptor in these cells. In addition, expression of mouse furin corrected the reduced binding of insulin in RPE.40 cells, indicating that receptor function as well as processing was restored.

RPE.40 is a strain of mutated CHO-K1 cells with elevated resistance to Pseudomonas exotoxin A, Sindbis virus, and Newcastle disease virus. Virus resistance is due to an inability to cleave precursor viral membrane glycoproteins and produce infectious virions. Transfection of RPE.40 cells with cDNA for mouse furin causes them to lose all resistance and become as sensitive as wild-type cells to the toxin and viruses. Transfection of RPE.40 cells with cDNA for the related yeast protease Kex2 reduces their resistance to the toxin and viruses, but does not completely eliminate it.

The histidine residue at position 715 of elongation factor 2 (EF-2) is posttranslationally modified in a series of enzymatic reactions to 2-[3-carboxyamido-3-(trimethylammonio)-propyl]histidine, which has been given the trivial name diphthamide. The diphthamide residue of EF-2 is the target site for ADP ribosylation by diphtheria toxin and Pseudomonas exotoxin A. ADP-ribosylated EF-2 does not function in protein synthesis. EF-2 that has not been posttranslationally modified at histidine 715 is resistant to ADP ribosylation by these toxins. In this report we show that a G-to-A transition in the first position of codon 717 of the EF-2 gene results in substitution of arginine for glycine and prevents addition of the side chain of diphthamide to histidine 715 of EF-2. EF-2 produced by the mutant gene is fully functional in protein synthesis.

Anti-[ADP-ribosylated elongation factor 2 (EF-2)] antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Natl Acad. Sci. USA 86, 554–557]. This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, chinese hamster ovary CHO-K1 cells was further characterized. The time course of labelling of ADP-ribosylated EF-2 with [32P]orthophosphate was similar in pyBHK cells and in CHO-K1 cells. The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2. The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2. When purified EF-2 from pyBHK cells was incubated with [carbonyl-14C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, [14C]NAD was generated. The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP-ribose acceptor site in EF-2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.

RPE.40, a mutant strain of CHO-K1 cells isolated for resistance to Pseudomonas exotoxin A and cross-resistant to alphaviruses, is also highly resistant to virulent strains of Newcastle disease virus. The resistance of RPE.40 cells to Newcastle disease virus results from the failure to cleave the viral envelope precursor glycoprotein Fo to fusion glycoprotein F1 at the consensus sequence (Lys/Arg)-Arg-Gln-(Lys/Arg)-Arg.

RPE.40, a mutant CHO-K1 strain selected for resistance to Pseudomonas exotoxin A, is defective in the production of infectious alphaviruses, although viruses are taken in and processed normally (J. M. Moehring and T. J. Moehring, Infect. Immun. 41:998-1009, 1983). To determine the cause of this defect, the synthesis of Sindbis virus proteins was examined. RPE.40 cells produced and glycosylated structural glycoprotein precursors PE2 and immature E1 normally. Mature E1 was formed, but PE2 was not cleaved to E2 and E3. PE2 instead was modified to a higher-molecular-weight form (PE2') in which the high-mannose oligosaccharides were processed to the complex form without proteolytic cleavage. The data suggest that the cleavage which produces E2 occurs within the trans-Golgi or in post-Golgi elements and is closely associated with the addition of sialic acid residues to the asparagine-linked oligosaccharides. RPE.40 cells make and release noninfectious Sindbis virions that contain PE2' and no detectable E2. These virions can be converted to an infectious form by treatment with trypsin. A defect in an intracellular endopeptidase activity in RPE.40 cells is postulated. Comparison of two Sindbis virus strains showed that the requirement for E2 in the virion to ensure infectivity is strain specific.

The amino acid diphthamide is a complex post-translational derivative of histidine that exists in eukaryotic and Archaebacterial elongation factor 2 (EF-2). Diphtheria toxin and Pseudomonas exotoxin A catalyze the transfer of an ADP-ribose residue from NAD to diphthamide, causing the inactivation of EF-2. We have used cytosolic extracts of mutant CHO-K1 cells to study the biosynthesis of diphthamide in vitro. We have identified chromatographically a precursor form of diphthamide that exists in one complementation group of mutant cells and have documented the addition of 3 methyl residues from S-adenosylmethionine to this precursor. We have identified the presence of methyltransferase capable of carrying out this reaction in vitro in cells of 15 diverse eukaryotic species.

A disulfide-linked conjugate of concanavalin A (Con A) and fragment A from diphtheria toxin has been synthesized and shown to be toxic for HeLa (human), Chinese hamster ovary (CHO), and SV3T3 (murine) cells. The conjugate was constructed by first coupling cystamine to Con A with a carbodiimide reagent and then reacting the modified Con A with reduced fragment A under conditions promoting disulfide interchange. The desired conjugate, obtained in nearly 50% yield relative to input of fragment A, was purified by affinity chromatography on Sephacryl S-200 and NAD-Sepharose; on analysis, it gave an average of 1.4 molecules of fragment A per tetrameric Con A molecule. The conjugate proved to be about equally active in inhibiting protein synthesis in HeLa, CHO, or SV3T3 cells in culture but was inactive relative to controls in a toxin-resistant strain of CHO cells containing altered elongation factor 2, the target protein of fragment A. With toxin-sensitive strains the conjugate was 100- to 1000-fold more active than controls, including fragment A, cystaminyl-Con A, and mixtures thereof, but was 1/50th to 1/500th as toxic as diphtheria toxin itself. Similar activity relative to controls was observed after intradermal inoculations in rabbits, and intravenous injections of the conjugate were lethal for mice. The activity of the conjugate in tissue culture was inhibited by Con A or α-methylmannoside but not by galactose. This and similar conjugates should be useful in studying mechanisms of entry of biologically active proteins into cells.

Cells that possess the morphology and collagen synthetic capacity of fibroblasts were recovered by bronchofiberscopic subsegmental pulmonary lavage from patients with pulmonary fibrosis, from patients with miscellaneous nonfibrotic lung diseases and from healthy volunteers. Lavage cells were placed in tissue culture, observed for 2 to 6 weeks, and compared with human lavage pulmonary alveolar macrophages (PAM), WI-38 and IMR-90 human fetal lung fibroblasts, and adult lung tissue fibroblasts (CLAC-76). Lavage fibroblasts (LF) were identified as proliferating clones in monolayers of nonproliferating PAM and could be subcultured repeatedly. Fibroblasts were propagated from 28 of the 92 lavage specimens cultured. Time-lapse cinematography showed similar distributions of interdivision times for LF, CLAC-76 and WI-38, but the LF and CLAC-76 lines had slower mean migration rates than the fetal line. Light, scanning, and transmission electron microscopy of LF showed attenuated spindle-shaped cells with interdigitating filopodia, flat surfaces with few microvilli, and containing numerous cytoplasmic polyribosomes and rough endoplasmic reticulum. Extracellular fibrils with the appearance of collagen were seen. Collagen synthesis by LF was measured as 3.9% to 4.9% of the cell-associated protein sensitive to bacterial collagenase. This protein was rich in hydroxyproline, and had an electrophoretic migration pattern identical to known collagen. LF did not contain lysozyme although this enzyme was abundant in fresh and 1-week cultured PAM. Thus LF were similar to human fetal and adult lung tissue fibroblasts in their morphology, tissue culture characteristics, constitutive enzymes and collagen synthetic properties but were distinctly different from PAM.

SummaryThe ability of several strains of RS virus to stimulate interferon production in vitro was studied. All RS strains studied were capable of inducing at least low titers of interferon in WI-38 and HF fibroblasts. The concentration of serum in the medium was of importance, the best yields of interferon being produced in medium with 2% fetal calf serum and little or none in 10% fetal calf serum. RS virus was shown to be sensitive to the antiviral action of interferon in WI-38 cells. It appears that the prolific multiplication of RS virus in young children is not due to lack of interferon-stimulating potential of the virus. The possibility that the interferon system is less efficient in young children than in adults was considered.

Conclusions and SummaryThe presence of constant low levels of interferon in the growth medium does not affect the ability of primary human foreskin cells to establish themselves in tissue culture. In addition, continuous exposure of human foreskin fibroblasts to up to 12 units/ml of human interferon does not alter the limited lifespan of these cells in vitro (7). Cells continuously exposed to these levels of interferon are still able to respond normally and in a linear fashion to dilutions of interferon by becoming resistant to virus challenge.No change in the ability to produce interferon in response to poly I:C was noted in continuous-interferon cell lines. Production of interferon in response to NDV challenge was unaltered by cells exposed to a constant 5 units/ml of interferon, but exposure to 12 units/ml caused moderately reduced yields.

Two classes of diphtheria toxin-resistant variants were selected from Chinese hamster ovary (CHO-K1) cells: permeability variants, in which uptake of toxin was impaired, and a new class of cytoplasmic variants, which were cross-resistant to Pseudomonas exotoxin. EF-2 prepared from the cytoplasmic variants was resistant to ADP-ribosylation by either toxin. The evidence presented suggests that these are translational variants possessing a mutationally altered EF-2 gene product. These studies also confirmed that Pseudomonas toxin ADP-ribosylates EF-2 in toxin-sensitive intact cells, as well as in cell-free systems.

Publisher Summary This chapter discusses general procedures for the induction and production of avian interferons. Avian interferons may be induced directly in the allantoic fluid of the embryonated egg or in cell cultures produced from whole-embryo digests. The interferon-containing allantoic or culture fluid is then well suited for use as a crude preparation or in further purification schemes. For interferon production in cell cultures, embryo cell cultures are produced by using a standard tissue dissociation method. Newcastle disease virus (NDV) is inactivated by exposure to UV light. The virus is exposed just long enough completely to inactivate its ability to replicate, as monitored by inoculation into eggs or cell cultures. Cell cultures are inoculated with inactivated NDV at a ratio of 10 viral units per cell, determined by plaque assays done prior to inactivation.

KB-R2A, a variant strain of the KB cell line, is highly resistant to the lethal effects of diphtheria toxin. It has proven to be restrictive in the replication of certain viruses as well. The resistance of KB-R2A to 12 different virus types, representing RNA, DNA, enveloped and nonenveloped viruses, is shown. A wide variation in response, from highly restrictive to totally permissive replication, was observed.

Like many physiological ligands, several viruses and toxins enter mammalian cells through receptor-mediated endocytosis. Once internalized, the nucleic acids of several viruses and the toxic subunit of diphtheria toxin gain access to the cytosol of the host cell through an acidic intracellular compartment. In this report, we present evidence that one class of mutants of Chinese hamster ovary (CHO)-K1 cells, which is "cross-resistant" to Pseudomonas exotoxin A, diphtheria toxin, and several animal viruses, has a defect in acidification of the endosome. Cells were allowed to internalize fluorescein isothiocyanate-conjugated dextran before subcellular fractionation. Fluorescence measurements on subcellular fractions permitted measurement of the internal pH of the isolated endosomes and lysosomes. Our results show that (i) endosomes and lysosomes from CHO-K1 cells maintain an acidic pH, (ii) acidification of both endosomes and lysosomes is mediated by a Mg2+/ATP-dependent process, (iii) GTP can satisfy the ATP requirement for acidification of lysosomes but not of endosomes, and (iv) at least one class of mutants that is cross-resistant to toxins and animal viruses has a defect in the ATP-dependent acidification of their endosomes. These studies provide biochemical and genetic evidence that the mechanisms of acidification of endosomes and lysosomes are distinct and that a defect in acidification of endosomes is one biochemical basis for cross-resistance to toxins and viruses.

It has been reported that cell lines which did not orginate from connective tissue cells either synthesize hydroxyproline-containing polypeptides [l-6] or possess prolyl hydroxylase (EC 1.14.11.2) activity [2,3,7]. Since these cell lines had been maintained in culture for some time, the relationship of these observations to the in vivo function(s) of the cells is uncertain [ 11. Few instances of either the formation of hydroxyproline-containing polypeptides [8] or the presence of prolyl hydroxylase in homogeneous cell populations obtained directly from the intact animal have been reported [9,10]. Pulmonary lavage in humans [ 111 and rabbits [ 12,131 yields a cell population which consists primarily of pulmonary alveolar macrophages (PAM). It has been reported that PAM do not either synthesize collagen [ 141 or contain prolyl hydroxylase [7]. The present study demonstrated that rabbit and human PAM do contain significant amounts of prolyl hydroxylase activity.

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a “universal hybridizer” (10).

Variations in two general classes of diphtheria toxin-resistant mutants which may be selected from Chinese hamster ovary (CH0-K1) cells and the conditions for their selection are described. The resistance of class I mutants can be overcome with increasing concentrations of toxin. Their entire complement of EF-2 is susceptible to ADP-ribosylation by toxin. Class I includes those strains in which resistance resides at the level of the plasma membrane. The resistance of class II, translational, mutants cannot be overcome by high concentrations of toxin, as all, or a portion, of their EF-2 is insensitive to the action of diphtheria toxin and Pseudomonas exotoxin A. Adjustment of the concentration of toxin used to select resistant mutants can be used to regulate the class of mutant recovered. Metabolic cooperation between cells does not affect recovery of either class I or class II mutants. Resistance is stable in class I strains, but class IIb strains, which possess 50 % resistant and 50 % sensitive EF-2, display a transient high level of resistance which is retained for varying lengths of time following exposure to toxin. Class IIa strains, which possess 100% resistant EF-2, grow normally in saturating concentrations of toxin, but class IIb strains grow at a reduced rate. Evidence is presented which suggests that the gene for EF-2 is functionally diploid in CH0-K1 cells.

We have investigated the cellular fate of epidermal growth factor (EGF) in KB cells and a variant, KB-R2A, that was isolated based on its resistance to diphtheria toxin and subsequently was shown to be resistant to infection by RNA viruses (Moehring and Moehring, 1972, Infect. Immunity. 6:487-492). Both cell lines bind 125I-EGF and internalize the cell-bound hormone at the same rate. However, when the degradation of internalized 125I-EGF was measured by the release of low molecular weight (mw) hydrolysis products into the medium, the toxin-resistant KB-R2A cells degraded the hormone at a drastically reduced rate; 50% and 3% of the cell-bound 125I-EGF was degraded and released by 80 min in the KB and KB-R2A cells, respectively. To investigate the fate of cell-associated EGF prior to release into the medium, the radioactivity in extracts of cells labeled with 125I-EGF was fractionated by native gel electrophoresis. In KB cells three peaks of radioactivity other than native 125I-EGF were resolved. Time course and subcellular fractionation studies showed that the first processed product appeared while the hormone was located in the endocytic vesicles and the appearance of the other two peaks correlated with the arrival of the hormone in the lysosomal compartment. KB-R2A cells also produced the first intermediate but they produced only very low amounts of the other two peaks. These studies show that endocytic vesicles in both cell lines contain enzymes capable of processing EGF prior to the arrival of the hormone in the lysosomes and show that the KB-R2A cells have a lesion that prevents the complete degradation of the hormone. We propose that the KB-R2A cell line has a defective mechanism for the intracellular processing of a number of ligands that are internalized by the process of receptor-mediated endocytosis and that this defect is located beyond the initial endocytic step.

We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.

We have identified two types of mutants of Chinese hamster ovary cells in which the unique ADP-ribose attachment site in elongation factor 2 (EF-2) is altered, thereby rendering them resistant to diphtheria and Pseudomonas toxins (TOXR). The first is mutant in the gene for EF-2 and possesses a permanently altered, TOXR gene product. The second lacks a component of a posttranslational modification system that converts TOXR EF-2 to the toxin-sensitive (TOXS) state. We postulate that this modification system is involved in the conversion of a single histidine residue in EF-2 to the specific target of toxin-catalyzed ADP-ribosylation, the novel amino acid X. We have designated the second type MOD- mutants. The missing of nonfunctional component in the MOD- mutants can be restored by hybridizing them with either normal TOXS cells or with EF-2 structural gene mutants. The TOXR EF-2 from MOD- mutants is also converted to toxin sensitivity in vitro by incubation with extracts of TOXS or EF-2 gene mutant cells in the presence of an energy-generating system. Our results demonstrate that EF-2 can be synthesized and released from ribosomes in a toxin-resistant form and then converted to toxin sensitivity by posttranslational modification.

We studied the binding of 125I-labeled diphtheria toxin (DTX) to receptors on monolayer cultures of Chinese hamster ovary cells (CHO-K1) and Vero cells. The number of DTX receptors detected on the cell surface was shown to be dependent on the cell density (number of cells per unit area). Cells at low density (less than 23,000 cells per cm2 for CHO-K1 cells; less than 80,000 cells per cm2 for Vero cells) had more receptors for DTX than cells at higher densities. The difference in receptor number between low- and high-density cells was 33-fold for CHO-K1 cells and 19-fold for Vero cells. We estimated the maximum number of DTX receptors on low-density CHO-K1 and Vero cells to be 50,000 and 370,000 per cell, respectively. The cell density at which the binding of DTX was reduced to 50% of maximum was considerably lower for CHO-K1 cells than for Vero cells (33,000 vs. 220,000 cells per cm2, respectively). Vero cells grown on a surface that had been conditioned by high-density cells bound less DTX, suggesting that interaction of these cells with the underlying extracellular matrix might regulate the number of cell surface receptors for DTX. Low-density cells were more sensitive to DTX than high-density cells, suggesting that low-density cells possessed an increased number of functional receptors that actively transported DTX to the cytosol. CHO-K1 and Vero cells were equally protected by SITS (4-Acetamido-4'-Isothiocyano-Stilbene-2,2'-disulfonic Acid), a compound that has been shown to inhibit the binding and entry of DTX in Vero cells, suggesting that intoxication of CHO-K1 and Vero cells is mediated by a similar mechanism. The data illustrate the importance of taking into account the cell density when measuring the number of DTX receptors on adherent cells.

It has been reported that treatment with homologous interferon of cells in culture interferes with the process of diphtheria intoxication in these cells. This report both confirms and extends this observation. The most sensitive indicator of diphtheria toxin action in cells, i.e., the inhibition of incorporation of labeled amino acids into protein, was employed, and human, mouse, and chicken interferon-cell systems were studied. Interferon treatment afforded a distinct, reproducible protection from the action of toxin, but in no case could total protection be shown. The “antitoxic” component is stable at p H 2, is species-specific, is not sedimented at 100,000 × g , and is trypsin-sensitive. Human cell preparations are heat-labile. Experiments with cell-free amino acid incorporation systems suggest that the “anti-toxic” effect of interferon is concentrated at the level of the cell membrane and subcellular components are not directly involved.

KB cell strains which are resistant to the lethal effects of diphtheria toxin were isolated from the toxin-sensitive KB cell line. For one such strain, KB-R2, the concentration of diphtheria toxin required to cause a 50% reduction of incorporation of amino acids into protein was more than 2,000 times that required in the sensitive KB-S cell. The KB-R2 strain was cloned by a single-cell isolation method to yield uniform strains with an even greater resistance to toxin. Resistance is a stable, inheritable property, providing a good genetic marker, and appears to be related to some alteration in the cell membrane. Preliminary studies showed that these cell strains are resistant to poliovirus and vesicular stomatitis virus as well as to toxin.

It was determined that KB-R cell strains, isolated from the KB cell line and resistant to diphtheria toxin, also resist infection by poliovirus, Mengo virus, vesicular stomatitis virus, and Newcastle disease virus. This resistance manifests itself by reduction in yields of progeny virus (a reduction of more than 2 logs in some cases), reduced production of viral-specific ribonucleic acid (RNA), and delayed cytopathic effect. In three KB-R strains tested, resistance was related to a step which falls between adsorption of virus and uncoating or release of viral messenger RNA. In two of these three strains, a second resistance mechanism was also active, causing a reduced production of viral-specific RNA. A relationship between the resistance to diphtheria toxin and the resistance to viral infection of the KB-R strains is considered. It has been postulated that the native diphtheria toxin molecule must be “activated” at the surface of a susceptible cell by a proteolytic process before it can enter the cell and inhibit protein synthesis. It is also known that the eclipse of some viruses occurs at or near the cell membrane and involves proteolytic activity. Resistance to viruses and toxin in the KB-R strains may result from the loss or modification of related labilizing or activating principles associated with the surface receptors for these agents.

INTERFERON preparations usually show some degree of species specificity in their antiviral effects, exerting their greatest effect on growth of viruses in cells of the species which produced the interferon, and being less active or totally inactive in cells of other species1–3. Recent studies have shown that species specificity is far from absolute: certain human and primate interferons have proved active in rabbit cells4; the activity of monkey interferon in human cells was first noted by Isaacs et al.5, and cross-reactivity of human and monkey interferon systems at the level of the taxonomic family has been demonstrated6. Mouse serum interferon expresses one-twentieth of its mouse cell activity on other rodent cells7.