Sarbecovirus ORF6 proteins hamper induction of interferon signaling

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Epidermal growth factor and bombesin differ strikingly in the induction of early responses in Swiss 3T3 cells

Swiss 3T3 cells express receptors for both the polypeptide epidermal growth factor (EGF) and the tetradecapeptide bombesin and respond mitogenically to these substances. These cells thus provide a system to analyze potential signal transduction pathways involved in mitogenic stimulation. Here we have determined and compared the early ionic responses elicited by EGF and bombesin and their relation to diacylglycerol (DG) and inositolphosphate (InsPn) production. Whereas EGF fails to cause any significant change in intracellular Ca2+, bombesin effectively induces prompt and transient Ca2+ mobilization from intracellular stores. Further support of the idea that these receptors utilize distinct signalling pathways comes from the measurements of cytoplasmic pH (pHi). As in most target cells, EGF induces a delayed (1 min) but sustained intracellular alkalinization that reaches a new steady state after approximately 10 min. Bombesin, in contrast, elicits a biphasic response; within seconds, a rapid but transient rise in pHi is observed, followed by a further slower sustained alkalinization. Inhibition of the Na+/H+ exchanger prevents both EGF as well as bombesin-induced alkalinization. However, under these conditions, bombesin evokes a rapid and sustained acidification related to the Ca2+ response. Apparently, bombesin initiates a Ca2(+)-dependent acidifying process immediately after binding of the hormone to its receptor. Furthermore, we could demonstrate that the bombesin-induced alkalinization depends on protein kinase C activation whereas the EGF response does not. Determination of the total DG and InsPn accumulation revealed that EGF is ineffective in stimulating phospholipase C-mediated production of these second messengers. In contrast, bombesin causes a rapid DG and InsPn production coinciding with the Ca2+ response and the first phase of the rise in pHi followed by a slower DG accumulation coinciding with the second alkalinization phase. Our results show that in Swiss 3T3 cells the bombesin receptor activates the hydrolysis of inositol lipids as a mechanism of signal transduction, which consequently causes changes in Ca2+i and pHi. Clearly, the EGF receptor utilizes different pathways to evoke mitogenesis and stimulates Na+/H+ exchange independently of DG production and protein kinase C activation.     

The two subfamilies of rice glutelin differ in both primary and higher-order structures

Rice glutelin, which accounts for 70-80% of the total proteins of the seeds, consists of two nutritionally different subfamilies (A and B types). Although the similarity in primary sequences between the two subfamilies is as high as 60%, we established conditions to discriminate the two subfamilies when low amounts of antigen are analyzed by immunoblot methods. The glutelin alpha polypeptides can be resolved into six bands labeled alpha1 to alpha6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration analysis showed that glutelin exists as a polymerized and a smaller molecular weight form. Immunoblot analysis of SDS-PAGE resolved polypeptides showed that alpha2, alpha3, and alpha4 are an A type and that these A types as well as alpha1, a B type, are polymerized. The polymerization tendency clearly differed between the two subfamilies except for alpha1, which may be derived from GluB-4 as suggested by analysis using Escherichia coli expression systems of glutelin cDNA regions corresponding to alpha polypeptides. GluB-4 and all the A type subunits have an extra Cys residue in the hypervariable regions, corresponding to the C-terminal region of alpha polypeptide. Accordingly, the extra Cys residue is hypothesized to be responsible for the polymerization of glutelin.     

Control of interferon signaling in human papillomavirus infection

Human papillomaviruses (HPV) infect mucosal and cutaneous epithelium resulting in several types of pathologies, most notably, cervical cancer. Persistent infection with sexually transmitted oncogenic HPV types represents the major risk factor for the development of cervical cancer. The development of HPV-associated cervical cancer has been closely linked to the expression of the viral oncogenes E6 and E7 in the tumor cells. The major viral oncoproteins, E6 and E7, target the cellular tumor suppressor gene products p53 and Rb, respectively. As detailed within, these interactions result in the stimulation of proliferation and the inhibition of apoptosis, thus representing major oncogenic insults to the infected cell. In addition to mediating transformation, the E6 and E7 genes also play significant roles in altering the immune response against infected cells by suppressing interferon (IFN) expression and signaling. At the clinical level, IFNs have been used in the treatment of HPV-associated cervical intraepithelial neoplasia (CIN) or cervical cancers with mixed results. The success of the treatment is largely dependent on the subtype of HPV and the immune response of the patients. Despite this inefficiency, the increasing knowledge about the regulation of IFN signaling pathways at molecular level may hold a promise for the use of new therapeutic strategies against HPV infection. Studies on the regulation of the function of IFN-inducible gene products by the E6 and E7 may lead to the development of new therapeutic approaches based on strategies that modify the function of the HPV oncoproteins and restore IFN-signaling pathways through endogenous control mechanisms.     

Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.     

Herpes simplex viruses and induction of interferon responses

Herpes simplex viruses (HSV) are human pathogens responsible for a variety of diseases,including localized mucocutaneous lesions,encephalitis,and disseminated diseases.HSV infection leads to rapid induction of innate immune responses.A critical part of this host response is the type I IFN system including the induction of type I IFNs,IFN-mediated signaling and amplification of IFN response.This provides the host with immediate countermeasure during acute infection to limit initial viral replication and to facilitate an appropriate adaptive immune response.However,HSV has devised multiple strategies to evade and interfere with innate immunity.This review will focus on the induction of type I IFN response by HSV during acute infection and current knowledge of mechanisms by which HSV interferes with this induction process.     

The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1

Rabies virus P protein inhibits alpha interferon (IFN-alpha)- and IFN-gamma-stimulated Jak-STAT signaling by retaining phosphorylated STAT1 in the cytoplasm. Here, we show that P also blocks an intranuclear step that is the STAT1 binding to the DNA promoter of IFN-responsive genes. As P is a nucleocytoplasmic shuttling protein, we first investigated the effect of the cellular distribution of P on the localization of STAT1 and consequently on IFN signaling. We show that the localization of STAT1 is correlated with the localization of P: in cells expressing a nuclear form of P (the short P3 isoform or the complete P in the presence of the export inhibitor leptomycin B), STAT1 is nuclear, whereas in cells expressing a cytoplasmic form of P, STAT1 is cytoplasmic. However, the expression of nuclear forms of P inhibits the signaling of both IFN-gamma and IFN-alpha, demonstrating that the retention of STAT1 in the cytoplasm is not the only mechanism involved in the inhibition of IFN signaling. Electrophoretic mobility shift analysis indicates that P expression in the cell extracts of infected cells or in stable cell lines prevents IFN-induced DNA binding of STAT1. The loss of the DNA binding of STAT1 and ISGF3 was also observed when purified recombinant P or P3 was added to the extracts of IFN-gamma- or IFN-alpha-treated cells, indicating that P directly affects the DNA binding activity of STAT1. Then products of the rabies virus P gene are able to counteract IFN signaling by creating both cytoplasmic and nuclear blocks for STAT1.     

ERKs activation and calcium signaling are both required for VEGF induction by vanadium in mouse epidermal Cl41 cells

Studies were conducted to determine the immunomodulatory effects of high dietary ascorbic acid (vitamin C) on growth, serum concentration, non-specific immune response and disease resistance of a commercially important Asian catfish, Clarias batrachus. Four practical diets were formulated to contain 0, 500, 1000 and 2000 mg ascorbic acid (AA) equivalent/kg diet, supplied as L-ascorbyl-2-polyphosphate (LAPP) and were fed for 2, 4, 6 and 8 weeks. Each diet was fed to triplicate groups of catfish with initial body weight of 15.47± 0.59 g. After 2, 4, 6 and 8 weeks, growth, serum concentration of AA, oxidative respiratory burst, lysozyme and natural hemolytic complement activities, myeloperoxidase (MPO) content and natural haemagglutination titre were measured. Ten numbers of fish in duplicate were challenged with Aeromonas hydrophila to measure the level of protection against aeromoniasis at each one of the assayed times. The results showed that AA concentration in serum correlated positively with those in the diets and reached its saturation level after the time period directly proportional to the increase in dose level. Fish fed AA-supplemented diets showed significantly (p < 0.05) higher specific growth rate after 2 weeks of feeding. The superoxide production was enhanced after 8 weeks of feeding fish at a supplemented dose level of 2000 mg/kg. Similarly, MPO content, haemagglutination titre and alternative complement activity in serum enhanced with the increase of dietary AA levels at different duration of feeding. The lysozyme activity was not affected by the dietary AA treatment. On the other hand, feeding of AA at all concentrations significantly increased percent survival against A. hydrophila challenge after 4 weeks compared to control. The non-specific immune parameters as well as percent survival were enhanced as a result of high AA supply particularly at 500 mg/kg diet, although the increase was not maintained but returned to the initial levels after 4 weeks. These results support the possible use of AA as an immunostimulant at a dose of 500 mg/kg diet for a period of 4 weeks in catfish farming. (Mol Cell Biochem xxx: 25–33, 2005)     

Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro

RNA interference occurs when cytoplasmic small interfering RNAs (siRNAs) enter the RNA-induced silencing complex and one strand guides cleavage of the target RNA by the Argonaute 2 protein. A significant concern when applying siRNAs or expressing small hairpin RNAs (shRNAs) in human cells is activation of the interferon (IFN) response. Synthetic siRNAs harboring certain motifs can induce an immune response when delivered to mouse and human immune cells such as peripheral blood mononuclear cells, monocytes, plasmacytoid dendritic cells (pDCs) and nonplasmacytoid dendritic cells (mDCs). In the present study we have tested the immunostimulatory effects of lipid-delivered siRNAs versus Pol III promoter-expressed shRNAs in primary CD34+ progenitor-derived hematopoietic cells. We show that in this system, lipid-delivered siRNAs are potent inducers of IFNalpha and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory.     

Saturated fatty acids activate ERK signaling to downregulate hepatic sortilin 1 in obese and diabetic mice

Hepatic VLDL overproduction is a characteristic feature of diabetes and an important contributor to diabetic dyslipidemia. Hepatic sortilin 1 (Sort1), a cellular trafficking receptor, is a novel regulator of plasma lipid metabolism and reduces plasma cholesterol and triglycerides by inhibiting hepatic apolipoprotein B production. Elevated circulating free fatty acids play key roles in hepatic VLDL overproduction and the development of dyslipidemia. This study investigated the regulation of hepatic Sort1 in obesity and diabetes and the potential implications in diabetic dyslipidemia. Results showed that hepatic Sort1 protein was markedly decreased in mouse models of type I and type II diabetes and in human individuals with obesity and liver steatosis, whereas increasing hepatic Sort1 expression reduced plasma cholesterol and triglycerides in mice. Mechanistic studies showed that the saturated fatty acid palmitate activated extracellular signal-regulated kinase (ERK) and inhibited Sort1 protein by mechanisms involving Sort1 protein ubiquitination and degradation. Consistently, hepatic ERK signaling was activated in diabetic mice, whereas blocking ERK signaling by an ERK inhibitor increased hepatic Sort1 protein in mice. These results suggest that increased saturated fatty acids downregulate liver Sort1 protein, which may contribute to the development of dyslipidemia in obesity and diabetes.     

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.Key words: autophagy, calcium, Fyn, IP₃ receptor, dexamethasone     

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.     

Effects of butyrate on induction and action of interferon

We have identified two different and independent effects of sodium butyrate on induction and action of interferon. In the monkey cell line, GL-V3, simultaneous treatment with interferon and butyrate strongly reduced the antiviral activity of the interferon preparation, Whereas addition of butyrate before interferon or after establishment of the antiviral state had no effect. Interferon production induced by Sendai virus was also reduced by simultaneous treatment with butyrate, but pretreatment resulted in marked enhancement of interferon yields. Whereas the inhibitory effects of simultaneous butyrate treatment were also observed in human (WISH) and bovine (MDBK) cells, pretreatment with butyrate in these cells had no effect on interferon yields.