Interferon priming. Effects on interferon messenger RNA.

The effects of priming mouse cells with interferon on the production of interferon and its mRNA were investigated. Interferon-treated (primed) mouse L929 cells produce 3 to 10 times more interferon than do nonprimed cells following induction with Newcastle disease virus. Interferon appears 2 to 4 h sooner in the primed cultures than in nonprimed cultures and interferon production by primed cells becomes resistant to inhibition by actinomycin D about 4 h sooner than interferon production in nonprimed cells. Interferon mRNA is detected in primed-induced cells about 2 h earlier than in nonprimed-induced cells. It reaches peak levels about 2 to 4 earlier in primed cells, but it also disappears sooner in primed cells. The total amounts of interferon mRNA isolated from primed-induced cells and nonprimed-induced cells were indistinguishable, by the methods utilized. Therefore, although primed cells can produce significantly more interferon and make interferon mRNA sooner than nonprimed cells, the total amount of interferon mRNA produced is apparently not increased, nor is its half-life prolonged in primed cells. Thus, enhanced interferon production in primed cells may result from enhanced efficiency of translation of interferon mRNA in the primed cells.     

Priming: a Nonantiviral Function of Interferon

No interferon is made by L cells when they are infected with MM virus. However, several thousand units of interferon are produced when interferon-treated L cells are infected with MM virus. We call the conversion of cells, from nonproducers to producers, priming. The time required for cells to become fully primed is dependent on the interferon concentration with which they are incubated. Primed cells produced interferon earlier than normal cells stimulated by other inducers. Cells which were exposed to interferon in the presence of inhibitors of protein synthesis became fully primed yet developed no virus resistance. Also, primed cells produced interferon in response to low concentrations of polyriboinosinic acid . polyribocytidylic acid that did not induce interferon in normal cells. Therefore, priming appears to be a function of interferon separable from its antiviral activity. Several other picornaviruses that failed to induce interferon in L cells, human embryonic lung cells, or monkey kidney cells did induce interferon when these cells had been primed by homologous interferons.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Down-regulation of the interferon receptor

The binding of 125I-labeled alpha A interferon to human lymphoblastoid Daudi cells decreased when these cells were incubated with unlabeled alpha or beta interferon. This decrease could not be accounted for by the occupancy of interferon receptors with unlabeled interferon and it apparently resulted from the loss or down-regulation of receptors. The binding activity gradually increased when Daudi cells were incubated in fresh medium after a treatment with interferon, but inhibition of protein synthesis with cycloheximide prevented this recovery. Treatment of Daudi cells with this inhibitor resulted in the loss of half the interferon binding activity within 5 h. These findings suggested that the interferon receptors turn over at a basal rate in interferon-free medium and at an increased rate in cells incubated with interferon. The dose-response for the down-regulation was investigated by treating Daudi cells with different concentrations of alpha interferon. Down-regulation was observed in cells treated with relatively low doses of interferon, sufficient to elicit a biological response. The synthesis of the enzyme (2',5')oligo(A) polymerase was induced at the lowest interferon concentrations tested which caused receptor down-regulation.     

Delayed cytosolic exposure of Japanese encephalitis virus double-stranded RNA impedes interferon activation and enhances viral dissemination in porcine cells

Interferon is a principal component of the host antiviral defense system. In this study, abortive focus formation by Japanese encephalitis virus (JEV) in primate cells was accompanied by early interferon induction, while productive focus formation in porcine cells was associated with a late interferon response. Neutralization antibodies against interferon relieved the restricted infection in primate cells, and increasingly larger foci were generated as treatment with exogenous interferon was delayed, thereby establishing a solid correlation between interferon response and viral dissemination. However, delayed interferon induction in JEV-infected porcine cells occurred in the absence of active inhibition by the virus. We further demonstrated that JEV mediates interferon activation through double-stranded RNA and cytosolic pattern recognition receptors. Immunofluorescence and subcellular fractionation studies revealed that double-stranded RNA is concealed in intracellular membranes at an early phase of infection but eventually appears in the cytosol at later periods, which could then allow detection by cytosolic pattern recognition receptors. Interestingly, cytosolic exposure of double-stranded RNA was delayed in porcine cells compared to primate cells, independent of total double-stranded RNA levels and in correlation with the timing of the interferon response. Furthermore, when double-stranded RNA was artificially introduced into the cytosol of porcine cells, more rapid and robust interferon activation was triggered than in viral infection. Thus, cytosolic exposure of JEV double-stranded RNA is imperative for interferon induction, but in cell lines (e.g., porcine cells) with delayed emergence of cytosolic double-stranded RNA, the interferon response is late and viral dissemination is consequently enhanced.     

The induction of interferon by vesicular stomatitis virus in mouse L cells.

Although no detectable interferon was produced when L cells were infected with wild-type VSV (VSV-o), considerable amounts of interferon were produced when cells were infected with UV-irradiated VSV-o at a multiplicity equivalent to 10 PFU/cell. Treatment of VSV-o with UV-light resulted in the marked reduction of the RNA synthesizing capacity and cytotoxity of the virus, and the UV-irradiated virus had neither infectivity nor interfering activity against homologous viruses. The amount of interferon induced by UV-VSV-o was markedly influenced by multiplicity of infection and incubation temperature. Less-virulent temperature-sensitive mutants (VSV-mp and VSV-sp) derived from L cells persistently infected with VSV induced interferon in L cells without treatment of the viruses with UV-light, but these viruses could not induce interferon if the infected cells were incubated at nonpermissive temperature, or if cells were infected at multiplicities of more than 10 PFU/cell. On the other hand, it was shown that treatment of cells with cycloheximide (100 μg/ml) delayed the expression of cell damage caused by non-irradiated VSV-o and resulted in the production of interferon when cycloheximide was removed from the cultures. These results indicate that VSV has intrinsically interferon-inducing capacity in L cells and can induce interferon if the induction is carried out under such condition that cell damage caused by VSV are suppressed or delayed. Furthermore, the effect of pretreatment of cells by interferon and undiluted passage of VSV-o on interferon induction was discussed in relation to persistent infection.     

The role of cell membrane in the antiviral effect of interferon

The mechanism of interferon action in human fibroblasts has been studied by use of both antisera to human fibroblast interferon and the antisera to the surface of human fibroblast cell. The anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Interferon antiserum prevented the intracellular antiviral state from developing when added to the medium of the cells in which interferon synthesis had already been induced by poly (I · C). This suggests that development of the antiviral state involves interferon interaction with the external part of the producing cell. Treatment with the serum directed against the surface of human fibroblast cells failed to inhibit the antiviral activity of human interferon in these cells. In addition, the effect of gangliosides on the antiviral activity of human interferon was studied and it was found that human interferon binds to gangliosides and that this interaction leads to inactivation of the antiviral effect of interferon. Pretreatment of human fibroblasts with gangliosides had no effect on the sensitivity of these cells to exogenous interferon.     

Target cells for interferon production in human leukocytes stimulated by sendai virus.

Whole leukocytes, mononuclear cells, polymorphonuclear cells (PMN), MONOCYTES, PURIFIED LYMPHOCYTES, AND T (rosette-forming cells, RFC) and non-T (nonrosette-forming cells, nonRFC) lymphocytes isolated from the human peripheral blood were stimulated by Sendai virus, respectively, and examined for interferon production in their culture fluids. High levels of interferon were produced by mononuclear cells, but not by PMN. Removal of monocytes from the mononuclear cell population did not affect at all the levels of interferon produced, although it strongly suppressed interferon induction by polyinosinic-polycytidylic acid (poly IC) and mitogenic response to phytohemagglutinin (PHA) of the lymphocytes. Purified monocytes and T lymphocytes were unresponsive to the virus. In contrast, a population of purified non-T lymphocytes produced high levels of interferon. Addition of monocytes to the interferon-producing non-T lymphocytes did not affect the levels of interferon produced. No detectable levels of interferon were produced in the mixture of T lymphocytes and monocytes. It is concluded that non-T lymphocytes may be a major target for interferon induction of human leukocytes by Sendai virus.     

Differentiation of the immunosuppressive and antiviral effects of interferon.

Virus-induced (virus-type) interferon suppression of the in vitro antibody response of mouse (C57B1/6) spleen cells to sheep red blood cells was blocked by 5 × 10^?5M 2-mercaptoethanol (2-ME). The blockade was not due to a direct effect on interferon since 2-ME was capable of blocking the suppression when added to cultures up to 48 hr after interferon. 2-ME blockade of virus-type interferon immunosuppression was not due to the immunoenhancing property of 2-ME. Similar protective effects of 2-ME were observed during immunosuppression by virus-type interferon inducers, but not T-cell mitogen inducers of interferon (immune interferon). The data suggest that the immunosuppressive properties of virus-type and immune interferon preparations involve different mechanisms. Virus-type interferon inhibited DNA synthesis in unstimulated spleen cell cultures and in 2-ME stimulated cultures, and the degree of inhibition of DNA synthesis appeared to be related to the immunosuppressive property of interferon in the absence or presence of 2-ME. 2-ME did not affect the antiviral properties of either virus-type or immune interferon in nonlymphoid cells. Further, the induction of virustype interferon in spleen cells was neither inhibited nor enhanced by 2-ME, while the induction of immune interferon was enhanced. This enhancement is consistent with 2-ME enhancement of the immunosuppressive effects of immune interferon inducers.There are two possibilities for 2-ME blockade of the immunosuppressive effect of virus-type interferon, while not affecting the antiviral property. Firstly, the immunosuppressive and antiviral properties of virus-type interferon may involve different mechanisms at the subcellular level. Secondly, the selectivity of the blockade by 2-ME could be due to the fact that spleen cells are the target cells in immunosuppression, while L cells are the target cells in inhibition of virus replication. Thus, virus-type interferon may suppress the immune response at the level of the macrophage and 2-ME may reverse this effect by replacing a blocked macrophage function.     

Increase in the sensitivity of leukemia cells, incubated in interferon, to the effect of immune serum]

L-1210 AND P-388 leukemic cells were incubated in three types of interferon, i.e. L-cell interferon and two types of lymphocyte interferon (induced in the lymph node lymphocytes of intact mice and the lymphocytes obtained on the 10th day after intraperitoneal injections of 5-10(7) L-1210 cells). "False" interferon obtained by the method analogous to that of obtaining L-cell interferon, excluding the viral induction, was used for control. Cells incubated in interferon proved to be more sensitive to the action of the cytotoxic antibodies than those treated with "false" interferon. Treatment of the cells with lymphocytic interferon induced on the lymphocytes of immune mice increased their sensitivity even more in comparison with the cells treated with interferon obtained from intact mice.     

Recombinant human leukocyte interferon produced in bacteria has antiproliferative activity

Recombinant human leukocyte interferon synthesized by Escherichia coli possesses antiproliferative activity in addition to antiviral activity. When the ability to inhibit multiplication of lymphoblastoid Daudi cells was examined, the growth-inhibitory capacity of recombinant leukocyte interferon was equivalent to that exhibited by crude human leukocyte interferon or by the homogeneous gamma 2 species of leukocyte interferon synthesized by human cells.     

Bone marrow interferon]

The nucleated cells of the bone marrow of mouse, rat, guinea pig, chick, cattle and humans proved to be capable of producing interferon in vitro following induction with the Newcastle disease virus. The production of interferon by these cells was characterized by high stability. The bone marrow interferon was not inferior in its activity to the corresponding interferon prepared with the blood leukocytes or splenic cells.     

Interferon-Inducing Characteristics of MM Virus

Interferon induction by MM virus in mice and in L cells was studied. In mice the virus readily induced interferon. The time of appearance was dose-dependent. A large virus dose induced interferon by 4 hr, whereas a small dose resulted in interferon production which paralleled virus replication 24 hr after infection. In L cells the interferon-inducing capacity of the virus was rapidly destroyed by ultraviolet light irradiation. Heating (56 C) of the virus, on the other hand, greatly increased its ability to induce interferon. Interferon production could also be increased by prior treatment of the cells with homologous interferon (priming). The increase in interferon production after priming was dependent on the concentration of interferon used for priming, the length of interferon treatment, and the multiplicity of infection. It is suggested that MM virus might be useful for the further study of the mechanisms involved in the production and action of interferon.     

Inhibition of Mengo virus by interferon

Gauntt, Charles J. (The University of Texas, Austin), and Royce Z. Lockart, Jr. Inhibition of Mengo virus by interferon. J. Bacteriol. 91:176-182. 1966.-The inhibition of Mengo virus replication in L cells resulting from interferon was studied quantitatively. Interferon was titrated on L cells with Western equine encephalomyelitis (WEE) virus as the challenge virus. One protective unit (PU) of interferon is the least amount of interferon which prevents cytopathic effects when a large multiplicity of WEE virus is added subsequent to overnight incubation with interferon. Ten PU of interferon reduced the yields of Mengo virus by about 90%. Larger doses of interferon, up to 220 PU, caused no further reduction in the amount of virus produced. Plaque formation by Mengo virus was also reduced in number by about 85 to 90%, but could not be further reduced. The plaques which formed on interferon-treated cells were reduced in size. We were unable to obtain a virus population with increased resistance to interferon action by use of five successive growth cycles in interferon-treated cultures. Analysis of the cell population for the proportion of cells able to act as infectious centers revealed that incubation of cells with 10 PU of interferon decreased the proportion of virus-yielding cells by 80%. The yield of virus per virus-producing cell was decreased by 40 to 60%. Despite the reduction in yields, plaques, and infectious centers resulting from interferon, all doses of interferon failed to prevent the complete destruction of the cells. Experiments with puromycin indicated that the cytopathic effects observed in L cells infected with Mengo virus required that a virus-directed protein be synthesized between 4 and 5 hr postinfection. The evidence suggested, therefore, that the Mengo virus genome was able to code for new protein synthesis in the absence of the production of infectious virus.     

Interferon treatment reduced adherence, invasiveness and intracellular multiplication of Shigella flexneri in coxsackie B1 virus-infected cells.

The effect of interferon treatment on interaction of Shigella flexneri with in vitro cultured cells was investigated. Pretreatment of HEp-2 cells with human interferons had no effect on the susceptibility of cells to S. flexneri, measured by invasiveness and adhesiveness. Human leukocyte interferon and human recombinant interferon-alpha-A reduced adhesiveness, intracellular multiplication and invasiveness of S. flexneri in HEp-2 cells preinfected with coxsackie B1 virus. Also non-receptor mediated-phagocytosis was reduced by interferon treatment in virus infected cells. The interferon effects were dependent on continuous protein synthesis, because they were not expressed when cycloheximide or abrin was added to the virus infected cell cultures. No effect of interferon was detected on intracellular content of Na+ or K+, Na(+)-K+ activated ATPase activity or cytoplasma membrane polarity, in virus infected or control cell cultures. The interferon effect on bacterial invasiveness seems to be dependent on an interferon receptor interaction on cytoplasma membrane level because directly microinjected interferon showed no effect.     

Induction of interferon and erythropoietic differentiation in cells transformed by Friend virus.

Two lines of Friend virus (FV)-transformed mouse spleen cells have been analyzed in respect to their interferon production capacity: neither F4 cells, which liberate infectious FV when kept under tissue culture conditions, nor the thymidine kinase-deficient B8 cells, which do not produce significant amounts of FV, release detectable amounts of autogenous interferon into cell supernatants. However, interferon is produced in these cells in amounts comparable to that in L-929 cells when interferon induction is initiated with UV-inactivated Newcastle disease virus. Conversely poly(I)-poly(C), a potent interferon inducer in L-929 cells, proved ineffective in this capacity in F4 or B8 cells. When erythropoietic differentiation is induced in these cells by dimethyl sulfoxide, no autogenous interferon production occurs, but with NDV-induction a four- to fivefode increase of interferon production is observed. A similar elevation of interferon production is achieved during 5-bromodeoxyuridine stimulation of differentiation in the thymidine kinase-deficient B8 cells. The refractiveness against poly(I)-poly(C) displayed in unstimulated cells is not overcome at any stage of differentiation, indicating major differences of Newcastle disease virus and poly(I)-poly(C) induction mechanisms.     

Radioactive human lymphoblastoid interferon. One-step purification, regulation of heterogeneous species production and its use for radioimmunoassay

Human lymphoblastoid interferon (alpha type), labeled with [3H]leucine added to virus-induced Namalwa cells, was purified quantitatively and in one step from the culture fluid by immune precipitation. The material showed, upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only four radioactive bands with molecular weights ranging from 17000 to 21000, which coincided well with interferon activity. They coincided also with the four interferon protein bands in the electropherogram of unlabeled interferon purified by a different method. The purity of the labeled interferon was ascertained also by polyacrylamide gel electrophoresis in the absence of dodecyl sulfate. Pulse-labeling of interferon with [3H]leucine for 1 h at various times after induction indicated that the cells always synthesized and secreted the four interferon species in parallel during the interferon production period. Competitive radioimmunoassay for human interferon alpha was achieved by the use of purified radioactive interferon, anti-(interferon alpha) serum, and bacterial adsorbent. The immune precipitation of the labeled interferon was inhibited by unlabeled interferon alpha, and 100 international reference units of interferon alpha could be measured in this way.     

Mechanism of the suppressive effect of interferon on antibody synthesis in vivo.

Mouse interferon preparations significantly suppress the in vivo antibody response to sheep red blood cells (SRBC), a thymus-dependent antigen, and to Salmonella typhimurium lipopolysaccharide (LPS), a thymus-independent antigen. It is also possible to effect the late responses of antigen sensitive "memory" cells observed during secondary immunization by administration of interferon prior to primary immunization. The immunosuppressive activity of interferon was time- and dose-dependent. Maximum suppression was produced when animals were given 1.5 times 10-5 units of interferon between 4 and 48 hr before antigenic stimulation. These findings suggested that interferon affects some early event(s) in the process of antibody synthesis which might be related to the general inhibitory effect of interferon on rapidly dividing cells and viral m-RNA translation. In addition, the use of nonadherent spleen cell cultures from interferon-treated mice, immunized in vitro with a thymus-independent antigen, indicated that in this situation the inhibitory effect of interferon was due to an action on B lymphocytes. A variety of soluble "suppressive" factors are secreted by T cells as a consequence of activation by mitogens or specific antigens in vitro. Since T cells are recognized as one of the sources of interferon, it is suggested that interferon should be investigated as a suppressor T cell-produced lymphokine which can regulate B cell expression.     

Influenza virus-induced interferon production in mouse spleen cell culture: T cells as the main producer.

Interferon production by spleen cells from unimmunized C3H mice challenged in vitro with influenza virus AO/PR8 was investigated. Glass-nonadherent cells (lymphocytes) produced significant levels of interferon, although cocultivation of glass-adherent macrophages was needed for optimal production. Treatment of the cells with antithymocyte serum and complement markedly reduced the interferon production. When glass-nonadherent cells were fractionated on a nylon wool column, the T-cell-enriched fraction consistently produced more interferon than the B-cell-enriched fraction. It is concluded that T cells are an important producer of interferon in spleen cell cultures from normal mice upon challenge with influenza virus, although non-T cells (macrophages and B cells) also may produce interferon under suitable conditions.