Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis.

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

Curcumol inhibits encephalomyocarditis virus by promoting IFN-ß secretion.

BACKGROUND: Encephalomyocarditis virus (EMCV) infection can cause reproductive failure in sows and acute myocarditis and sudden death in piglets. It has caused huge economic losses to the global pig industry and that is why it is necessary to develop effective new treatment compounds. Zedoary turmeric oil has been used for treating myocarditis. Curcumol extracted from the roots of curcuma is one of the main active ingredient of zedoary turmeric oil. The anti-EMCV activity of curcumol along with the molecular mechanisms involved with a focus on IFN-ß signaling pathway was investigated in this study. METHOD: 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the maximum non-toxic concentration (MNTC), 50% cytotoxic concentration (CC50), maximum inhibition rate (MIR) and 50% effective concentration (EC50) against EMCV. Through EMCV load, the anti-viral effect of curcumol was quantitatively determined using real-time quantitative PCR (qPCR). The effect of curcumol on the expression of IFN-ß was investigated using real-time quantitative PCR and ELISA. Western blot was used to determine the amounts of MDA5, MAVS, TANK, IRF3 and P-IRF3 proteins in human embryonic kidney 293 T (HEK-293 T) cells infected with EMCV. RESULTS: The results of MTT showed that compared with the ribavirin positive control group, the maximum inhibition ratio (MIR) of curcumol was greater but the selection index (SI) value was much smaller than that of ribavirin. The results of qPCR showed that curcumol and ribavirin significantly reduced the replication of EMCV in HEK-293 T cells. The curcumol (0.025 mg/mL) treatment has significantly increased IFN-ß mRNA expression in the EMCV-infected HEK-293 T cells while ribavirin treatment did not. The results of ELISA showed that curcumol (0.025 mg/mL and 0.0125 mg/mL) has significantly increased the expression of IFN-ß protein in EMCV-infected HEK-293 T cells. The results of Western blot showed that curcumol can inhibit the degradation of TANK protein mediated by EMCV and promote the expression of MDA5 and P-IRF3, while the protein expression level of MAVS and IRF3 remain unchanged. CONCLUSION: Curcumol has biological activity against EMCV which we suggest that IFN-ß signaling pathway is one of its mechanisms.

A polymorphic residue that attenuates the antiviral potential of interferon lambda 4 in hominid lineages.

As antimicrobial signalling molecules, type III or lambda interferons (IFNλs) are critical for defence against infection by diverse pathogens, including bacteria, fungi and viruses. Counter-intuitively, expression of one member of the family, IFNλ4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural frameshift mutation that abrogates IFNλ4 production improves HCV clearance. To further understand how genetic variation between and within species affects IFNλ4 function, we screened a panel of all known extant coding variants of human IFNλ4 for their antiviral potential and identify three that substantially affect activity: P70S, L79F and K154E. The most notable variant was K154E, which was found in African Congo rainforest 'Pygmy' hunter-gatherers. K154E greatly enhanced in vitro activity in a range of antiviral (HCV, Zika virus, influenza virus and encephalomyocarditis virus) and gene expression assays. Remarkably, E154 is the ancestral residue in mammalian IFNλ4s and is extremely well conserved, yet K154 has been fixed throughout evolution of the hominid genus Homo, including Neanderthals. Compared to chimpanzee IFNλ4, the human orthologue had reduced activity due to amino acid K154. Comparison of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFNλ4 correlates with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data show that the human-specific K154 negatively affects IFNλ4 activity through a novel means by reducing its secretion and potency. We thus demonstrate that attenuated activity of IFNλ4 is conserved among humans and postulate that differences in IFNλ4 activity between species contribute to distinct host-specific responses to-and outcomes of-infection, such as HCV infection. The driver of reduced IFNλ4 antiviral activity in humans remains unknown but likely arose between 6 million and 360,000 years ago in Africa.

Large-scale purification of high purity α1-antitrypsin from Cohn Fraction IV with virus inactivation by solvent/detergent and dry-heat treatment.

α1-Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large-scale purification of AAT from it. liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry-heat. Virus inactivation by dry-heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5 IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry-heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1 H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry-heat. The advantages of this process, including suitability for large-scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.

Antiviral Resistance of Splenocytes in Aged Mice.

We compared the susceptibility to viral infection of splenocytes, isolated from young versus old CBA mice, and evaluated the antiviral actions of lactoferrin in splenocytes infected with Encephalomyocarditis virus (EMCV). Recombinant mouse lactoferrin (rmLF) and bovine lactoferrin (bLF) were used. There were no differences in the susceptibility to EMCV infection in the studied age categories. Both types of lactoferrins were protective in young and old mice. The study confirmed the undisturbed viral resistance in old mice and the protective actions of lactoferrin in viral infection. The antiviral action of the homologous mouse lactoferrin was demonstrated for the first time.

Tyrphostin AG1478 Inhibits Encephalomyocarditis Virus and Hepatitis C Virus by Targeting Phosphatidylinositol 4-Kinase IIIα.

Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known epidermal growth factor receptor (EGFR) inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously regarded only as a cancer chemotherapy agent.

New small-molecule inhibitors effectively blocking picornavirus replication.

UNLABELLED: Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited both the formation and functioning of the replication complexes, while E5(1) and E7(2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7(2) had a G5318A mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target a phosphatidylinositol 4-kinase IIIß (PI4KIIIß)-dependent step in viral replication. Analysis of host protein recruitment showed that E7(2) reduced the amount of GBF1 on the replication complexes; however, the level of PI4KIIIß remained intact. E7(2) as well as another enviroxime-like compound, GW5074, interfered with viral polyprotein processing affecting both 3C- and 2A-dependent cleavages, and the resistant G5318A mutation partially rescued this defect. Moreover, E7(2) induced abnormal recruitment to membranes of the viral proteins; thus, enviroxime-like compounds likely severely compromise the interaction of the viral polyprotein with membranes. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. IMPORTANCE: Diverse picornaviruses can trigger multiple human maladies, yet currently, only hepatitis A virus and poliovirus can be controlled with vaccination. The development of antipicornavirus therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here, we describe three novel compounds that effectively block replication of distantly related picornaviruses with minimal toxicity to cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants, making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis.

Total chemical synthesis of human interferon alpha-2b via native chemical ligation.

Interferon-alpha (IFNα) is a cytokine that orchestrates innate and adaptive immune responses and potently inhibits proliferation of normal and tumor cells. These properties have warranted the use of IFNα in clinical practice for the treatment of several viral infections and malignancies. However, overexpression of IFNα leads to immunopathology observed in the context of chronic viral infections and autoimmune conditions. Thus, it is desirable to develop therapeutic approaches that aim at suppressing excessive IFNα production. To that end, artificial evolution of peptides from phage display libraries represents a strategy that seeks to disrupt the interaction between IFNα and its cell surface receptor and thus inhibit the ensuing biological effects. Mirror-image phage display that screens peptide libraries against the D-enantiomer is particularly attractive because it allows for identification of proteolysis-resistant D-peptide inhibitors. This approach, however, relies on the availability of chemically synthesized D-IFNα composed entirely of D-amino acids. Here, we describe the synthesis and biological properties of IFNα2b of 165 amino acid residues produced by native chemical ligation, which represents an important first step toward the discovery of D-peptide antagonists with potential therapeutic applications.

Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.

Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.

Effect of arginine on pre-nucleus stage of interferon beta-1b aggregation.

Understanding the mechanism of aggregation of a therapeutic protein would not only ease the manufacturing processing but could also lead to a more stable finished product. Aggregation of recombinant interferon (IFNß-1b) was studied by heating, oxidizing, or seeding of unformulated monomeric solution. The formation of aggregates was monitored by dynamic light scattering (DLS) and UV spectroscopy. The autocatalytic monomer loss model was used to fit the data on aggregation rates. The influence of pre-nucleation on aggregation step was demonstrated by inducing the liquid samples containing a monomer form of folded IFNß-1b by heat and also an oxidizing agent. Results tend to suggest that the nucleus includes a single protein molecule which has been probably deformed. Seeding tests showed that aggregation of IFNß-1b was probably initiated when 1.0% (w/w) of monomers converted to nucleus form. Chemiluminescence spectroscopy analysis of the sample indicated the generation of 3.0 µM of hydrogen peroxide (H2O2) during nucleation stage of IFNß-1b aggregation. Arginine with a concentration of 200 mM was sufficient to suppress aggregation of IFNß-1b by decreasing the rate of pre-nucleation step. We proposed the formation of pre-nucleus structures prior to nucleation as the mechanism of aggregation of IFNß-1b. Furthermore, we have showed the positive anti-aggregation effect of arginine on pre-nucleation step.

[Substances from pyrolised tissues of reptile carcases differently modulate immunity of mammals of both sexes].

Substances with gender action on immunity were detected in water soluble hydrolised matter from reptile carcases. The gender action was shown on isolated blood neutrophils, whole blood and in vivo by the antiviral activity on experimental animals, contaminated with three types of viruses: Herpes simplex type 1, the virus of encephalomyocarditis and the virus of hepatitis of mice. The possible mechanism of the inhibitory action on the male immunity was associated with the protein kinase cascade, including protein kinase C, activated by phorbolmyristate in the cells of the immune system.

Type III interferon (IFN-lambda) antagonizes the antiviral activity of interferon-alpha in vitro.

Type III interferons (IFN-lambda) are the most recently discovered members of IFN family. Synergism between different IFN types is well established, but for type I and type III IFNs no conclusive evidence has been reported so far. Possible synergism/antagonism between IFN-alpha and IFN-lambda in the inhibition of virus replication (EMCV, WNV lineage 1 and 2, CHIKV and HSV-1), and in the activation of intracellular pathways of IFN response (MxA and 2'-5' OAS) was evaluated in different cell lines (Vero E6, A549 and Wish cells). The antiviral potency of IFN-lambda1 and -l2 was lower than that of IFN-alpha. When IFN-alpha and -lambda were used together, the Combination Index (CI) for virus inhibition was greater than 1 virtually for all virus/host cell systems, indicating antagonistic effect. Antagonism between IFN-alpha and -l was also observed for the induction of mRNA for both MxA and 2'-5'OAS. Elucidating the interplay between IFN-alpha and -lambda may help to better understand innate defence mechanisms against viral infections, including the molecular mechanisms underlying the influence of IL-28B polymorphisms in the response to HCV and other viral infections.

Antiviral effects of a transgenic RNA-dependent RNA polymerase.

Transgenic expression of the RNA-dependent RNA polymerase 3D(pol) inhibited infection of Theiler's murine encephalitis virus (TMEV), a picornavirus from which it was derived. Here, we infected 3D(pol) transgenic mice with another picornavirus, as well as an alphaherpesvirus and a rhabdovirus. 3D(pol) transgenic FVB mice had significantly lower viral loads and survived longer after infection with all three types of viruses than nontransgenic FVB mice. Viral inhibition among three different types of virus by transgenic 3D(pol) suggests that the mechanism of action is not the direct interference with picornaviral 3D(pol) but instead may be the changing of host cells to an antiviral state before or after viral infection occurs, as basal interferon levels were higher in 3D(pol) transgenic mice before infection. Further study of this mechanism may open new possibilities for future antiviral therapy.

Expression of biologically active human interferon alpha 2 in Aloe vera.

Methods necessary for the successful transformation and regeneration of Aloe vera were developed and used to express the human protein, interferon alpha 2 (IFNα2). IFNα2 is a secreted cytokine that plays a vital role in regulating the cellular response to viral infection. Transgenic plants were regenerated from callus cultures initiated from zygotic embryos. Expression of the IFNA2 transgene in transformed plants was confirmed by RT-PCR and IFNα2 protein was detected by immunoblot analysis. Human A549 cells treated with transgenic aloe extracts for 6 h induced expression of the interferon stimulated gene 54, indicating activation of the IFN signaling pathway. The biological activity of the aloe produced IFNα2 was assessed using an antiviral assay with A549 cells treated with extracts from both the rind and pulp fractions of the shoot and subsequently infected with the lytic encephalomyocarditis virus. The highest level of activity attributable to recombinant IFNα2 was determined to be 625 IU/mg of total soluble protein (TSP) in the rind and 2,108 IU/mg TSP in the pulp. Two daughter plants that vegetatively budded during the course of this study were also confirmed to express IFNα2. These results confirm that Aloe vera is capable of expressing a human protein with biological activity, and that a secreted protein targeting the apoplast can be detected in the pulp fraction of the plant.

Effects of free immunoglobulin light chains on viral myocarditis.

RATIONALE: In recent work, we have demonstrated a crucial role of mast cells in the development of viral myocarditis. Viral infection could lead to increased synthesis of free immunoglobulin light chains (FLC) and our earlier work showed that FLC can trigger mast cell activation. OBJECTIVE: We studied the possible involvement of FLC in the pathogenesis of viral myocarditis, and therapeutic effects of FLC using an animal model of viral myocarditis. METHODS AND RESULTS: DBA/2 mice were inoculated intraperitoneally with encephalomyocarditis (EMC) virus. Serum levels and concentrations in the heart of kappa FLC on day 14 in mice inoculated with EMC virus were significantly increased compared with controls. Myocardial viral concentration was significantly inhibited, the area of myocardial lesions was smaller in mice treated with kappa or lambda FLC, and survival of mice given FLC significantly improved. In contrast, an FLC antagonist deteriorated myocarditis. kappa and lambda FLC chains inhibited EMC viral replication in human amnion cells in vitro. lambda FLC significantly increased the gene expression of interleukin-10 in the heart which was previously shown to improve viral myocarditis when given exogenously. FLC also tended to increase the gene expressions of interferon-alpha and -gamma in the heart mice. CONCLUSIONS: FLC have antiviral and antiinflammatory effects and improved viral myocarditis in mice. FLC may be promising agents for the treatment of viral myocarditis.

Inhibition of encephalomyocarditis virus and poliovirus replication by quinacrine: implications for the design and discovery of novel antiviral drugs.

The 9-aminoacridine (9AA) derivative quinacrine (QC) has a long history of safe human use as an antiprotozoal and antirheumatic agent. QC intercalates into DNA and RNA and can inhibit DNA replication, RNA transcription, and protein synthesis. The extent of QC intercalation into RNA depends on the complexity of its secondary and tertiary structure. Internal ribosome entry sites (IRESs) that are required for initiation of translation of some viral and cellular mRNAs typically have complex structures. Recent work has shown that some intercalating drugs, including QC, are capable of inhibiting hepatitis C virus IRES-mediated translation in a cell-free system. Here, we show that QC suppresses translation directed by the encephalomyocarditis virus (EMCV) and poliovirus IRESs in a cell-free system and in virus-infected HeLa cells. In contrast, IRESs present in the mammalian p53 transcript that are predicted to have less-complex structures were not sensitive to QC. Inhibition of IRES-mediated translation by QC correlated with the affinity of binding between QC and the particular IRES. Expression of viral capsid proteins, replication of viral RNAs, and production of virus were all strongly inhibited by QC (and 9AA). These results suggest that QC and similar intercalating drugs could potentially be used for treatment of viral infections.

Recombinant protein hydrazides: application to site-specific protein PEGylation.

Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.

Immunogenicity of an interferon-beta1a product.

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Anti-infective activities of Pelargonium sidoides (EPS® 7630): effects of induced NO production on Leishmania major in infected macrophages and antiviral effects as assessed in a fibroblast-virus protection assay.

EPs® 7630 is an aqueous-ethanolic extract of the roots of Pelargonium sidoides, employed in the treatment of upper respiratory tract infections. Its anti-infective activity is supposed to be associated with the activation of the nonspecific immune system. Using Leishmania major GFP-infected murine BMMΦ, the NO production of EPs® 7630-activated macrophages was correlated with the reduction of the GFP signal measured at single cell levels using flow cytometry. The anti-infectious effect of EPs® 7630 (3-10 µg/mL) on its own (NO production: 4-13 µM; signal reduction: 25-73 %) was less prominent than that in combination with IFN- γ (100 U/mL) (NO production: 20-27 µM; signal reduction: 35-78 %). Furthermore, supernatants of EPs® 7630-stimulated BMMΦ (10 µg/mL) significantly reduced the cytopathic effect of EMCV on L929 fibroblasts (antiviral activity 80 U/mL) when compared with an IFN- γ standard (100 U/mL). Direct addition of EPs® 7630 to L929 did not mediate cytoprotective effects. The antiviral components induced in BMMΦ by EPs® 7630 remain to be identified. Detection of any IFNs by ELISA was unsuccessful, which may be due to their very low concentrations in cell supernatants. The current data provide convincing support for the induction of anti-infectious responses by EPs® 7630.

Possible role of phosphoinositide-3-kinase in Mx1 protein translation and antiviral activity of interferon-omega-stimulated HeLa cells.

Interferon ω (IFN-ω), a cytokine released during innate immune activation, is well known for promoting direct antiviral responses; however, the possible signal pathways that are initiated by IFN-ω binding to the type I IFN receptors have not been fully studied. Here, we provide evidence that activation of phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) signaling plays a pivotal role in the generation of IFN-ω-mediated biological responses. We found that LY294002 (PI3K inhibitor)-attenuated antiviral activities are induced by IFN-ω treatment. Although such effects of LY294002 are unrelated to regulatory activities on IFN-ω-dependent Mx1 (myxovirus resistance 1) or Mx2 gene transcriptional regulation, translation of Mx1 protein, which was known as a key mediator of cell-autonomous antiviral resistance, was significantly reduced by PI3K inhibition. Further studies showed that PI3K inhibition using LY294002 leads to a decrease in PI3K substrate Akt and mitogen-activated protein kinase extracellular signal-regulated kinase and p38 phosphorylation/activation. In addition, although LY294002 was not able to reduce STAT1 activation, we found that the mammalian target of rapamycin (mTOR)/p70 S6 kinase pathway was significantly attenuated by inhibition of the PI3K/Akt signaling pathway. These results indicate that the PI3K/Akt pathway is a common and central integrator for antiviral responses in IFN-ω signaling via its regulatory effects on mTOR that are required for initiation of mRNA translation of Mx genes.