Masayoshi Kohase

A cytokine network in human diploid fibroblasts: interactions of beta-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1.

Earlier studies demonstrated the induction of beta 2-interferon (IFN-beta 2) in human diploid fibroblasts (FS-4 strain) exposed to tumor necrosis factor (TNF). These studies suggested that IFN-beta 2 mediates an antiviral effect in TNF-treated cells and exerts a feedback inhibition of the mitogenic effect of TNF. Here we demonstrate that the expression of the antiviral action of TNF can be enhanced by prior exposure of FS-4 cells to trace amounts of IFN-beta 1. IFN-beta 1, at a higher concentration, can directly increase the expression of IFN-beta 2. Exposure of cells to TNF enhanced IFN-beta 2 (but not IFN-beta 1) mRNA expression in response to poly(I).poly(C), an IFN inducer which is also known to stimulate FS-4 cell growth. Platelet-derived growth factor and interleukin-1 also led to the increased expression of IFN-beta 2. However, platelet-derived growth factor and interleukin-1 could override the antiviral effect of TNF and also that of exogenously added IFN-beta 1. Our data suggest that a complex network of interactions that involves the endogenous production of IFN-beta 2 is triggered by several growth-modulatory cytokines. Cellular homeostasis is likely to represent a balance between the induction of IFN-beta 2 by these cytokines and their ability to override the inhibitory actions of IFN-beta 2.

Y2, the Smallest of the Sendai Virus C Proteins, Is Fully Capable of both Counteracting the Antiviral Action of Interferons and Inhibiting Viral RNA Synthesis

ABSTRACT An open reading frame (ORF) overlapping the amino-terminal portion of the Sendai virus (SeV) P ORF in the +1 frame produces a nested set of carboxy-coterminal proteins, C′, C, Y1, and Y2, which are referred to collectively as the C proteins. The C proteins are extremely versatile triple-role players; they counteract the antiviral action of interferons (IFNs), inhibit viral RNA synthesis, and are involved in virus assembly. In this study, we established HeLa cell lines stably expressing the C, Y1, and Y2 proteins individually and examined the capacities of these cells to circumvent the antiviral action of alpha/beta IFN (IFN-α/β) and IFN-γ and to inhibit viral transcription. The assay protocols included monitoring of IFN-α/β-mediated signaling by interferon-stimulated response element-driven reporter gene expression and of the antiviral state induced by IFN-α/β and IFN-γ and measurement of reporter gene expression from an SeV minigenome, as well as quantification of SeV primary transcripts. When necessary, the activities measured were carefully normalized to the expression levels of the respective C proteins in cells. The data obtained clearly indicate that the smallest protein, Y2, was as active as the C and Y1 proteins in both counteracting the antiviral action of IFNs and inhibiting viral transcription. The data further show that intracellular transexpression of either C, Y1, or Y2 rendered HeLa cells moderately or only poorly permissive for not only wild-type SeV but also 4C(−) SeV, which expressed none of the four C proteins. On the basis of these findings, the roles of SeV C proteins in the natural life cycle are discussed.

Protective effect of antibodies to two viral envelope glycoproteins on lethal infection with Newcastle disease virus

SummaryThe protective effect of humoral immunity against lethal infection of chickens with Newcastle disease virus was studied.Chickens hatched from eggs laid by hens vaccinated with live attenuated Newcastle disease virus vaccine possessed antibody to various components of the virus, and were resistant to a challenge with a virulent strain of Newcastle disease virus which was 100 per cent fatal for the offspring of nonvaccinated hens.Passive administration of antiserum raised against whole virions provided susceptible chickens protection comparable to that seen in the birds with maternal antibody.When administered passively, both anti-HN serum with virus neutralizing activity, and anti-F serum with only marginal virus neutralizing activity significantly prolonged the survival of infected birds but failed to achieve the level of protection as afforded by the anti-whole NDV serum.The protection provided by the simultaneous presence of anti-HN and anti-F serum was significantly greater than that afforded by either alone and comparable to that of anti-whole NDV serum, indicating the complementary effect of anti-HN and anti-F antibodies not only in cell cultures as reported previously (19), but also in a natural host.

Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells

Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10(-10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein.

The Amino-Terminal Half of Sendai Virus C Protein Is Not Responsible for either Counteracting the Antiviral Action of Interferons or Down-Regulating Viral RNA Synthesis

ABSTRACT The Sendai virus C proteins, C′, C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.

Purification and characterization of chick interferon induced by viruses.

Chick interferon (IFN), produced in primary chick embryo (CE) cells stimulated by u.v.-irradiated Newcastle disease virus, was partially purified by two-step chromatography using both controlled pore glass and Blue Sepharose. The specific activity of the IFN increased about 500-fold by this method and the final recovery from starting material was more than 95%. The partially purified IFN was analysed by SDS-PAGE, and two peaks of IFN activity were observed. The molecular weight represented by the sharp peak was estimated to be 18 000 (18K) and a broad peak was found at 20K to 30K. Glycosidase treatment before SDS-PAGE resulted in disappearance of the broad peak and increased the activity of the 18K peak. Anti-CE IFN rabbit serum and a monoclonal antibody against the CE IFN neutralized the antiviral activity of all IFN samples prepared under various conditions.

Regulation of human interferon production stimulated with poly(I) · Poly(C): Correlation between shutoff and hyporesponsiveness to reinduction

Abstract The exposure of FS-4 cells to polyinosinate-polycytidylate [poly(I) · poly(C), 2 μg/ml] for 1 hr resulted in rapid interferon production which peaked in 3–4 hr and was rapidly shut off thereafter, declining to low or undetectable levels by 6 hr. Reexposure of cells to 50 μg/ml of poly(I) · poly(C) at 2, 3, 4, or 5 hr after the first induction did not prevent the s shut off of interferon production due to the first induction. However, the restimulated cultures underwent a second round of interferon production peaking at 8–10 hr. The appearance of the second peak could be inhibited with actinomycin D, if the drug was added 3 hr after reexposure to poly(I) · poly(C), i.e., before the actual onset of the second round of interferon production. The second interferon peak resembled the peak seen a after the first induction in that it also showed a rather steep rise and rapid shutoff. The shutoff of the second peak could be prevented by treatment with actinomycin D at 9 hr (5 hr after the second stimulation). A second interferon peak at 9 hr was also seen following a single 1-hr exposure to a high concentration (50 μg/ml) of poly(I) · poly(C). It is suggested that a rapidly turning over repressor system is induced coordinately with interferon. This repressor system causes irreversible inactivation of interferon mRNA and is likely to be responsible for the shutoff of interferon production as well as the phenomenon of hyporesponsiveness to repeated interferon induction.

Biological functions of monospecific antibodies to envelope glycoproteins of Newcastle disease virus

SummaryMonospecific antisera to HN and F glycoproteins of Newcastle disease virus were prepared, and their effects on the biological activities of the virus were investigated. Anti-HN serum inhibited hemagglutinating and neuraminidase activity, as well as hemolysis. Anti-F serum had no effect on hemagglutination or neuraminidase but inhibited hemolysis and virus-induced cell fusion.Anti-HN serum was highly neutralizing, while neutralization by anti-F serum was very inefficient in conventional plaque reduction tests, although both sera were estimated to contain comparable amounts of antibody reacting with the virus as indicated by complement fixation and immunodiffusion tests. The neutralizing activity of anti-F serum was greatly enhanced by the addition of anti-IgG serum or fresh guinea pig serum, whereas that of anti-HN serum was little enhanced.Anti-HN serum incorporated in the agar overlay suppressed the development of plaques to some degree, while anti-F serum had little effect. The combination of anti-HN and anti-F sera resulted in a marked decrease in the number and size of plaques, demonstrating the synergistic effect of the two species of antibody in the containment of the spread of viral infection.

Inhibition of interferon synthesis in phospholipase C treated primary chick embryo cells

Abstract Treatment of primary chick embryo (PCE) cells with subcytotoxic concentrations of phospholipase C (1–2 units/ml) makes them incapable of producing interferon primed by various inducers. The adsorption-penetration steps of viral inducers do not seem to be involved in the mechanism of the inhibition. The inhibitory effect of the enzyme treatment appears to be confined to the cellular mechanism related to interferon synthesis, because the following functions of PCE cells are found to remain intact after exposure to the enzyme which results in nearly complete suppression of interferon production: (1) synthesis of RNA and proteins, (2) capacity to support multiplications of Western equine encephalitis virus (WEE), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), and Semiki Forest virus (SFV). The potency of phospholipase C to suppress interferon production was found to be copurified with the enzyme activity after gel filtration through Sephadex G-100.

Interferon induction with Newcastle disease virus in FS-4 cells: Effect of 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)

SummaryDRB is an inhibitor of heterogeneous nuclear RNA (hnRNA) and messenger RNA (mRNA) synthesis. The effect of DRB on interferon production stimulated by Newcastle disease virus (NDV) in the human FS-4 cells was studied. Interferon production in cells primed by treatment with interferon was markedly enhanced (superinduced) in the presence of DRB. This superinduction was essentially due to an inhibition of the rapid decline (shutoff) of interferon production observed in primed cells not treated with DRB. Continuous presence of DRB was required for maximal superinduction. In this and other respects the interferon response induced by NDV in primed cells resembled poly(I) · poly(C)-induced interferon production. In contrast interferon production in cells not primed with interferon was virtually abolished by DRB treatment. Since neither virus specific RNA synthesis nor virus replication were significantly affected by DRB, the inhibition of interferon production is likely to result from the inhibitory action of DRB on a cellular, rather than viral, function. Apparently some differences exist in the synthesis or processing of the mRNAs for interferons in primed and unprimed cells and these determine the different sensitivities of these two responses to DRB.