Use of thyrotropin and cholera toxin to probe the mechanism by which interferon initiates its antiviral activity.

Abstract

Thyrotropin (10 gM) inhibited the antiviral activity of interferon. When added after interferon, thyrotropin (TSH) had no effect on antiviral activity. There was also no inhibition of interferon action in cells washed with medium between incubations with TSH and interferon.125I-Labeled TSH and 125I-labeled cholera toxin could bind to preparations of mouse L-cell plasma membranes. The binding was specific in that it was prevented by unlabeled thyrotropin or cholera toxin, but not by insulin, glucagon, prolactin, growth hormone, human chorionic gonadotropin, or luteinizing hormone.Mouse interferon inhibited 125I-labeled TSH binding to L-cell plasma membranes. The effect of mouse interferon on 125I-labeled cholera toxin binding was more complex, inhibition occurring only after an initial enhancement at low interferon concentrations. A 10-fold higher concentration of interferon was required to inhibit 25I-labeled cholera toxin binding as compared to 25I-labeled TSH binding. Mouse interferon was also able to displace bound 125I-labeled TSH, but not bound 125I-labeled cholera toxin. The interferon interaction with cell membranes was temperature-sensitive.Human interferon could induce changes in binding of 125I-labeled TSH and 125I-labeled cholera toxin to mouse L-cell plasma membranes similar to those induced by mouse interferon. Mouse interferon induced similar changes in plasma membranes of human KB-3 cells, which are insensitive to both human and mouse interferons. In view of these results, the species specificity of interferons does not appear to reside solely at the point of the initial interaction with their binding sites. The mechanism by which thyrotropin (TSH) and cholera toxin transmit their message to the cell is believed to require a multistep sequence of events whose main contributors are, in turn, a specific ganglioside or ganglioside-like receptor structure, a significant alteration in the state of the membrane, an effector-responsive adenylate cyclase, and cell metabolism susceptible to regulation by the increased production of adenosine 3':5'-cyclic monophosphate (1)(2)(3)(4).Interferon initiates an antiviral state by interacting with receptors which, like those for cholera toxin or TSH, may be gangliosides or ganglioside-like structures containing an oligosaccharide moiety as a critical feature of their structure (5-7). The evidence for a ganglioside or a ganglioside-like structure of the interferon binding site is: Phaseolus vulgaris phytohemagglutinin blocks interferon action (5); Sepharose-bound interferon loses its antiviral activity after preincubation with gangliosides (6); soluble interferon binds to Sepharose-bound gangliosides (6); interferon binding to gangliosides is inhibited by phytohemagglutinin (6); and sialyl-lactose reverses the ability of gangliosides to inhibit the action of interferon (7).The B protein of cholera toxin and the # subunit of TSH appear to carry the primary determinants for their interaction with the ganglioside or ganglioside-like structures on the cell memb...

Antiviral Action of Murine Interferons on Heterologous Cells

“…Degré [10] observed, how ever, that human and mouse interferons ad sorbed well to both human and mouse cells, although these cells are relatively insensitive to the biological activity of the heterologous interferon. This finding supports the theory of Kohn et al [11] that the initial site of binding of interferon to a cell does not determine species specificity. Carter [12] proposes that glycosylation of this interferon protein deter mines its activity on heterologous cells.…”

Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Identification of the anti-viral activity as interferon and characterization of the human effector lymphocyte subpopulation.

“…Mouse interferon was similarly measured on monolayers of murine L-F2 cells, by using EMC (50,000 PFU/well) as the challenge virus (35). Assays were standardized against an NIH mouse interferon reference standard.…”

Adsorption of interferon to homologous and heterologous cells

“…Somewhat conflicting results have been reported concerning the adsorption of interferons to heterologous cells. According to some authors heterologous interferons cannot be recovered from cells after extensive treatment (1,16), while others reported interferon adsorption to several types of heterologous ceils (4,13,14,15). Correspondingly, there is no established concensus, whether the species specificity is partly or wholly a function of the adsorption process.…”

“…If this were the ease, then adsorption of homologous interferon should be blocked by pretreatment of cells with high concentrations of heterologous interferon, similarly to that achieved by TSH or cholera toxin which block the ganglioside receptors (6,10,13).…”