Effects of temperature on viral glycoprotein mobility and a possible role of internal "viroskeleton" proteins in Sendai virus fusion.

The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20-22 degrees C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10-40 degrees C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.

Thyroid hormone analogues potentiate the antiviral action of interferon-gamma by two mechanisms.

L-thyroxine (L-T4) potentiates the antiviral activity of human interferon-gamma (IFN-gamma) in HeLa cells. We have added thyroid hormone and analogues to cells either 1) for 24 h pretreatment prior to 24 h of IFN-gamma (1.0 IU/ml), 2) for 24 h cotreatment with IFN-gamma, 3) for 4, after 20 h cell incubation with IFN-gamma, alone, or 4) for 24 h pretreatment and 24 h cotreatment with IFN-gamma. The antiviral effect of IFN-gamma was then assayed. L-T4 potentiated the antiviral action of IFN-gamma by a reduction in virus yield of more than two logs, the equivalent of a more than 100-fold potentiation of the IFN's antiviral effect. 3,3 of the IFN's antiviral effect. 3,3',5-L-triiodothyronine (L-T3) was as effective as L-T4 when coincubated for 24 h with IFN-gamma but was less effective than L-T4 when coincubated for only 4 h. D-T4, D-T3, 3,3',5-triiodothyroacetic acid (triac), tetraiodothyroacetic acid (tetrac), and 3,5-diiodothyronine (T2) were inactive. When preincubated with L-T4 for 24 h prior to IFN-gamma treatment, tetrac blocked L-T4 potentiation, but, when coincubated with L-T4 for 4 h after 20 h IFN-gamma, tetrac did not inhibit the L-T4 effect. 3,3',5-L-triiodothyronine (rT3) also potentiated the antiviral action of IFN-gamma, but only in the preincubation model. Furthermore, the effects of rT3 preincubation and L-T3 coincubation were additive, resulting in 100-fold potentiation of the IFN-gamma effect. When L-T4, L-T3, or rT3, plus cycloheximide (5 micrograms/ml), was added to cells for 24 h and then removed prior to 24 h IFN-gamma exposure, the potentiating effect of the three iodothyronines was completely inhibited. In contrast, IFN-gamma potentiation by 4 h of L-T4 or L-T3 coincubation was not inhibited by cycloheximide (25 micrograms/ml). These studies demonstrate two mechanisms by which thyroid hormones can potentiate IFN-gamma's effect: 1) a protein synthesis-dependent mechanism evidenced by enhancement of IFN-gamma's antiviral action by L-T4, L-T3, or rT3 preincubation, and inhibition of enhancement by tetrac and cycloheximide, and 2) a protein synthesis-independent (posttranslational) mechanism, not inhibited by tetrac or cycloheximide, demonstrated by 4 h coincubation of L-T4 or L-T3, but not rT3, with IFN-gamma. The protein synthesis-dependent pathway is responsive to rT3, a thyroid hormone analogue generally thought to have little effect on protein synthesis. A posttranslational mechanism by which the antiviral action of IFN-gamma can be regulated has not previously been described.

Potentiation by thyroxine of interferon-gamma-induced HLA-DR expression is protein kinase A- and C-dependent.

L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) potentiate the antiviral state induced by interferon-gamma(IFN-gamma) in homologous cells by a mechanism that is dependent upon calcium/phospholipid-dependent protein kinase (PKC). L-T4 and T3 also potentiate induction by IFN-gamma of MHC class II HLA-DR antigen expression in HeLa cells. In the present studies of HLA-DR expression, the PKC inhibitor staurosporine (0.1-1 nM) enhanced the expression of HLA-DR when the inhibitor was added simultaneously with IFN-gamma, 100 IU/ml. In the presence of IFN-gamma and 10(-7) M T4, the same concentrations of staurosporine inhibited potentiation of HLA-DR expression by thyroid hormone. A more specific PKC inhibitor, CGP41251 (0.5-5 nM), similarly enhanced HLA-DR expression in the presence of IFN-gamma but inhibited thyroid hormone potentiation of antigen expression. Both actions of CGP41251 were suppressed when cells were also treated with phorbol 12-myristate 13-acetate (PMA). A phospholipase C inhibitor, U73122 (1-1000 nM), did not alter the potentiating ability of T4, although it inhibited in a concentration-dependent manner the expression of HLA-DR induced by IFN-gamma. The potentiating effect of T4 was much more sensitive to a cyclic AMP-dependent protein kinase (PKA) inhibitor,KT5720 (1-1000nM), than was the induction of HLA-DR by IFN-gamma. The inhibitory effects of KT5720 were reversed by concurrent 8-bromo-cAMP treatment. The calmodulin antagonist W-7 (5-50 microM) did not alter IFN-gamma induction of HLA-DR in either the presence or absence of T4. HLA-DR expression in HeLa cells appears to be under PKC-associated inhibition; IFN-gamma reverses this inhibition to promote the appearance of the DR antigen. In contrast, potentiation by T4 of induction of HLA-DR by IFN-gamma requires activation of PKC. PKA is involved both in DR induction by IFN-gamma and in potentiation of the latter by T4. Thus, PKA and PKC have discrete roles in IFN-gamma-induced MHC class II antigen expression and its modulation by thyroid hormone.

Retrovirus-induced lymphoproliferative disease in mice undergoing graft-versus-host reaction.

The effect of graft-versus-host reaction on the course of concommitant retrovirus-induced lymphoproliferative disease was investigated. The graft-versus-host reaction was elicited by a single i.v. injection of 1.2 x 10(8) parental spleen cells into adult F1 mice. Lymphoproliferative disease was induced by a single transfusion of 0.2 ml of whole blood from donors with fully developed disease, induced by infection with retrovirus LP-BM5 MuLV. Graft-versus-host reaction and the lymphoproliferative disease each separately produced similar syndrome consisting of splenomegaly, lymphadenopathy, leukopenia, neutrophilia, reduced in vitro proliferation of spleen cells and suppression of in vivo immune responsiveness. The above symptoms were usually less pronounced during graft-versus-host reaction. Ongoing graft-versus-host reaction neither aggravated nor accelerated the course of the virus-induced lymphoproliferative disease in genetically susceptible F1 hybrids. Likewise, an ongoing graft-versus-host reaction in genetically resistant F1 hybrids did not alter their susceptibility to the retrovirus infection. The apparent lack of the effect of graft-versus-host reaction -dependent immunosuppression on the severity and the course of the concommitant retrovirus-induced lymphoproliferative disease suggests pathogenic differences between the murine syndrome and human AIDS for which the murine disease is considered by some to be an animal model.

Retrovirus-induced lymphoproliferative disease in mice: role of humoral immunity in perinatally exposed mice.

The mice born to female mice infected with LP-BM5 MuLV, the etiologic agent for lymphoproliferative disease and nursed for 4-6 weeks by them were less susceptible upon reinfection by i.v. transfusion of blood or plasma from infected donors with fully developed disease. Sera of 7 week or older perinatally exposed mice were capable of a complete in vitro neutralization of virus in plasma or blood from mice with fully developed disease. In contrast, sera from 3-week old perinatally exposed mice were ineffective. The neutralizing ability of the sera was drastically reduced or abrogated after their absorption with anti-mouse IgM. These observations are consistent with the notion that perinatally exposure results ina moderate form of the disease of the offspring. This perinatal infection is followed by a production of neutralizing antibodies of predominantly the IgM class that significantly alters the course of the lymphoproliferative disease and, in some instances, even prevents its development.

Quantative aspects of transfusion-transmitted retrovirus-induced lymphoproliferative disease in mice.

Healthy, adult C57BL/6Kh mice of both sexes were transfused with blood or blood products from syngeneic donors with retrovirus (LP-BM5)-induced lymphoproliferative disease. The disease produced in the recipients 8 weeks after transfusion was characterized by splenomegaly, disseminated lymphadenopathy, leukopenia with neutrophilia, abrogation of the primary immune response to SRBC, decreased in vitro proliferation of spleen cells co-stimulated with phorbol ester and IL-2 or ionomycin and abrogation of synergistic effect of the co stimulators. Quantitative analysis of the blood or blood products used for transfusion show that a single transfusion of 0.2 ml of PBS containing 0.2 mu 1 of whole blood or 2 microliters of plasma or 400 Ficoll-isolated peripheral blood mononuclear cells was sufficient for the inducing the disease. The results suggest that the retroviruses were present in preparations of peripheral blood mononuclear cells and plasma of mice with the disease. However, the latter was 10-fold less efficient in inducing the disease. Transfusion of 1.8 x 10(6) isolated erythrocytes failed to induce the disease suggesting a marginal role, if any, in transmission of the disease via transfusion of these cells. Thus, a simple, reliable and reproducible method for propagation of the murine lymphoproliferative disease in the laboratory has been elaborated. These results also point to some important differences with regard to blood transfusion between human and murine AIDS.

Thyroid hormone potentiates the antiviral action of interferon-gamma in cultured human cells.

Human interferon-gamma (hIFN gamma) exhibits a number of biological effects, including antiviral activity in homologous cells. The antiviral activity of recombinant (r) hIFN gamma, estimated by inhibition of vesicular stomatitis virus yield, was potentiated up to 120-fold in human fibroblast (BG-9) cells exposed to free L-T4 (0.5 x 10(-10) mol/L). Thyroid hormone alone did not induce the antiviral state in BG-9 cells. L-T3 also potentiated the antiviral action of rhIFN gamma, but D-T4 and D-T3 were ineffective. The antiviral effect of hIFN alpha in BG-9 cells was not influenced by thyroid hormone. Exposure of rhIFN gamma-treated BG-9 cells to L-T4 for only 3 h was sufficient to potentiate hIFN gamma-mediated antiviral activity. Similar potentiation by L-T4 of the antiviral effect of rhIFN gamma in HeLa cell cultures was also observed. Although the mechanism of potentiation of rhIFN gamma action by thyroid hormone is incompletely understood, the absence of antiviral activity of thyroid hormone alone indicates that the iodothyronine effect does not depend upon hormonal action on genes able to be stimulated by IFN gamma.

Acquired immunodeficiency in murine lymphoproliferative disease: considerations on pathogenesis.

C57BL/6Kh mice were infected with a single i.p. injection of 1 x 10(5) FFU of LP-BM5 MuLV. The development and progress of the virus-induced lymphoproliferative disease was followed for 12 weeks after infection. As anticipated, progressive splenomegaly and lymphadenopathy, as well as almost total abrogation of immune responsiveness ensued. In contrast to previous reports, there was a dramatic increase in the frequency of CD4+ cells in spleens among which over 20% expressed V beta 5 TCR, as compared with fewer than 3% in spleens of normal mice. Spleen cells from infected mice retained their in vitro ability to proliferate upon stimulation with IL-2 and anti-CD3, but were unable to respond when stimulated with phorbol ester and either a low dose of IL-2 or calcium ionophore (ionomycin). A similar pattern of in vitro proliferative responses was obtained when normal spleen cells were treated with K252a compound, a known inhibitor of protein kinase C activity. Together with the observations that viral infection impaired down-regulation of the phorbol-induced kinase activity and that the kinase inhibitor only marginally enhanced suppression of virus-infected cells proliferation, this finding suggests that disturbances of protein kinase C activity may underly the pathological effects seen after viral infection. However, since no apparent quantitative and qualitative changes in protein kinase C itself and its translocation were observed, it is more likely that the virus may interfere with either the substrate or product of kinase activity.

Murine AIDS: a model for the human disease or a distinct entity?

The LP-BM5 mixture of murine retroviruses elicits a disease in mice referred to as murine immunodeficiency syndrome (MAIDS) that is considered by some to be an animal homologue of human AIDS. In this article, we present and discuss some recent findings on the pathogenesis of the murine disease and their implications for the proposed homology between murine and human syndromes. The murine disease seems to display as many similarities to as it does differences from human AIDS. Among the latter are: definitive and exclusive viral etiology, a strong genetic effect on susceptibility to infection, expansion of the CD4+ cell population in spleen and peripheral blood, consistent transmissibility by a single transfusion of the minute amounts of blood or plasma from infected donors, and striking similarity between virus-induced alteration of the in vitro spleen cell proliferation and those caused by treatment with a protein kinase inhibitor K252a. With this in mind, the use of the noncommittal term retrovirus-induced murine lymphoproliferative disease instead of MAIDS appears to be more appropriate at this time.

Blood transfusion as a means for transmission of retrovirus-induced lymphoproliferative disease in mice.

Lymphoproliferative disease was elicited in C57BL/6KH and (BALB/c x C57BL/6)F1 hybrids by a single intraperitoneal injection of 10(5) FFU of LP-BM5 virus preparation. The disease could reproducibly be transferred by a single intravenous transfusion of 0.2 ml of whole blood as well as 0.1 ml of blood cells, plasma or serum from the infected animals. F1 hybrids displayed a delayed development of the disease when an acellular virus preparation was administered, but they were fully susceptible to the disease when syngeneic blood from infected F1 donors was transfused. Blood from donors in the prodromal stage was as effective in transmission of the disease as blood from donors with fully developed disease. This indicated that in murine lymphoproliferative disease viremia develops very early in the course of the disease. It seems that using the blood transfusion one could develop a reliable semiquantitative assay for the infectiveness of the animals suffering from LP-MB5-induced lymphoproliferative disease.

Production of human interferon-beta by Sendai virus and poly(rI).poly(rC): inhibition by neomycin.

Neomycin inhibits the production of interferon-beta (IFN-beta) in human fibroblast cells in response to Sendai virus or to poly(rI).poly(rC) in a concentration-dependent manner, and to the greatest extent effective when added prior to or up to 2 h after induction. This inhibitory effect is negated when the protein kinase C activator, SC-9, is present during IFN-beta production in response to poly(rI).poly(rC), but not in response to Sendai virus. These results suggest that in human cells both virus and poly(rI).poly(rC) utilize an early neomycin-sensitive signal transduction step for the production of IFN-beta; because neomycin binds specific phosphatidylinositol phosphates, both of these inducers very likely require hydrolysis of these phosphates.

Effect of alcohol on spleen cells and their functions in C57BL/6 mice.

Spleen cells from C57BL/6 mice maintained on alcohol containing liquid diet for two weeks were evaluated for different immune functions. On an average, 22% fewer spleen cells were recovered from alcohol-fed mice when compared to cells from control animals. In alcohol-fed mice, the relative frequency of B cells increased, whereas total T cells including CD4+ cells decreased significantly. Alcoholic mice, when challenged with poly(rI) poly(rC), produced significantly less interferon than control mice. In vitro production of interferon alpha and gamma by the spleen cells of alcoholic mice was reduced by 67-90%. No significant differences were seen in the level of natural killer cell activity in spleen cells of control and alcoholic mice. These results suggest that chronic alcohol intake can result in not only changes in the number of immune cells, but more importantly affect their biological functions such as their ability to produce interferons.

Negative-regulatory role of calmodulin in the expression of interferon-beta gene.

Addition of the calmodulin-antagonist, trifluoperazine (TFP), to human cell cultures producing biologically active IFN-beta in response to Sendai virus, results in a significant increase in IFN-beta production. This increase in IFN-beta production is observed 1 h after addition of TFP. The increase in IFN-beta production is correlated with increase in IFN-beta mRNA synthesis. Results suggest that calmodulin or calmodulin-dependent cellular process is involved in negative regulation of IFN-beta gene.

Effect of protein kinase C inhibitors on interferon-beta production by viral and non-viral inducers.

Induction of interferon-beta (IFN-beta) in human (BG-9), simian (CV-1) and mouse (L-929) cell lines by Sendai virus and by poly(rI). poly(rC) has been studied for its possible dependence on protein kinase C (PKC) through the use of pharmacological inhibitors (K252a and H-7) of PKC. Exposure of BG-9, CV-1 or L-929 cells to K252a (greater than or equal to 0.025 microM), a staurosporine derivative, 24 h before or after induction of IFN with poly(rI).poly(rC), inhibited by greater than 95% the production of IFN-beta. In contrast, virus-induced IFN production was enhanced threefold or more by K252a in BG-9 and L-929 but not in CV-1 cells. A naphthalene sulphonamide inhibitor of PKC, H-7, at greater than or equal to 5 microM, decreased poly(rI).poly(rC)-induced IFN production in BG-9 and CV-1 cells by 75 to 94%, but had no effect on IFN production in L-929 cells. Viral induction of IFN was not affected significantly by H-7 in BG-9, CV-1 and L-929 cells. In contrast to these results, the calmodulin inhibitor, trifluoperazine (5 to 15 microM) did not affect IFN-beta production by poly(rI).poly(rC) but significantly enhanced IFN production by Sendai virus in both human and murine cell lines. Thus, in human and simian fibroblasts the induction of IFN-beta by poly(rI).poly(rC) appears to be PKC-dependent, whereas viral induction of IFN-beta is not. Results with K252a implicate PKC in non-viral induction of IFN in mouse fibroblasts, as well. Direct measurements of PKC activity in BG-9 cells exposed to several concentrations of K252a showed that the membrane PKC activity is significantly more sensitive to inhibition by K252a than is cytosolic PKC activity. In L-929 cells, K252a inhibited membrane PKC activity similarly, but was less effective as an inhibitor of cytosolic enzyme activity than in BG-9. These studies support an integral role for PKC activity, particularly membrane-associated activity, in non-viral [poly(rI).poly(rC)] induction of IFN-beta in human, simian and mouse fibroblasts.

Role of calmodulin/protein kinase C in interferon production by poly(rI).poly(rC) in primed human cell cultures.

Human cells treated with trifluoperazine (TFP) or K252a prior to and/or during priming with human interferon-alpha (HuIFN-alpha) produce significantly more IFN on induction with poly(rI).poly(rC) than primed cells in the absence of the drug. Results suggest that calmodulin/protein kinase activity may be involved in priming activity of HuIFN-alpha.

Effect of calmodulin antagonists on the interferon system: induction and action of interferons.

Synthesis of interferon (IFN) in response to Sendai virus and the development of the resulting antiviral state were studied in human (BG-9) and murine (L-929) fibroblast cell cultures in the presence of the calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Compared to control cultures, 16-fold and 8-fold more IFN was formed in human and murine cells, respectively, when 10 microM TFP was present in the medium for 24 h prior to IFN induction with Sendai virus. W-7 did not affect IFN production in human cells, but enhanced it in L-929 cells by 4- to 8-fold. TFP inhibited the antiviral state induced by homologous IFN in the two cell systems, and at 20 microM, there was a 3,000-fold increase in vesicular stomatitis virus (VSV) yield. It also reduced the maintenance of the antiviral state in human cells. In contrast, W-7 had no effect on the development of the antiviral state in either of the two cell systems. Thus, calcium-calmodulin dependent cellular processes are involved in both induction of IFN and its action. The several patterns response to TFP and W-7 may reflect different ligand-binding sites on calmodulin.

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity.

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.

Early abnormal development of calmodulin gene expression and calmodulin-resistant Ca2+-ATPase activity in avian dystrophic muscle.

We have reported previously that the pectoralis muscle from three month-old dystrophic chickens with signs of myopathy exhibits increased calmodulin content, elevated calmodulin-specific mRNA (Biochem. Biophys. Res. Commun. 137:507-512, 1986), and reduced sarcoplasmic reticulum (SR) Ca2+-ATPase activity in response to calmodulin exposure in vitro (Clin. Res. 34: 725A, 1986). To determine the early time sequence for development of these abnormalities, we have studied muscle from embryos and post-hatched chickens at various ages. Quantitated by dot blot analysis, there was an approximate two-fold increase in calmodulin-specific mRNA in dystrophic muscle as early as 13 days ex ovo which was maintained throughout development up to three months ex ovo. Similarly, Ca2+-ATPase activity measured in SR membranes from chickens as early as 13 days post-hatch was also found to be resistant to stimulation in vitro by exogenous calmodulin, whereas the enzyme from normal muscle was calmodulin-stimulable. These findings suggest that the genetic lesion expressed in the avian dystrophic animal model involves the loss of normal control of intracellular calcium metabolism early in the maturation of the affected musculature and prior to appearance of disease signs.

Abnormal expression of the calmodulin gene in muscle from the dystrophic chicken.

Compared to that of genetically-related normal chickens, pectoralis muscle from the dystrophic chicken contained increased calmodulin measured by radioimmunoassay. Determined by the dot blot procedure, expression of the calmodulin gene was enhanced in muscle from affected animals. The bioactivity of the gene product was normal. Together with previous studies reporting increased cell Ca2+ content in dystrophic muscle, the current findings of increased sarcoplasmic calmodulin suggest the latter is a cellular response to defective Ca2+ transport at the level of cell efflux or intracellular organelle (sarcoplasmic reticulum) uptake.