Adenovirus early region 1A modulation of interferon antiviral activity.

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.     

Effect of nucleosides on interferon production and development of antiviral state induced by poly I.poly C.

Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic-polyribocytidylic acid (poly I.poly C) was studied. Addition of inosine or a fifty-fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I.poly C) was enhanced 1.5- to 4.0-fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I.poly C. The nucleoside effect may be applicable for production of high titered interferon.     

Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection. The growth of a viral mutant that is unable to produce the RNA is inhibited by interferon, while wild-type virus is not affected. VAI RNA prevents activation of the interferon-induced P1/eIF-2 alpha kinase. This inhibition can be reproduced in extracts of interferon-treated cells where purified VAI RNA prevents activation of latent kinase by double-stranded RNA.     

Morphological changes of human neuroblastoma cells induced by human gamma interferon

The treatment of GOTO cells, originated from human neuroblastoma, with recombinant human interferon-gamma (rHuIFN-gamma) induced the morphological changes: the extension and bifurcation of neurites and the multinucleated giant cell formation. The treatment of KP-N-RT cells, originated from human neuroblastoma, with rHuIFN-gamma also induced the similar morphological changes. The treatment of these cells with natural HuIFN-gamma also induced the same morphological changes, but those with recombinant human leukocyte interferon (rHuIFN-alpha A), recombinant human fibroblast interferon (rHuIFN-beta) and recombinant murine interferon-gamma (rMuIFN-gamma) did not induce it. The rHuIFN-gamma and the rHuIFN-beta inhibited more strongly the growth of GOTO and KP-N-RT cells than the rHuIFN-alpha A. This suggests that the morphological changes of these neuroblastoma cells are not simply due to the cell growth inhibition, but due to the property which only the rHuIFN-gamma possesses.     

Use of Getah virus for antiviral assay of human interferon

Antiviral assay is used routinely for measuring the biological activity of interferon (IFN). However, the challenge viruses used in these assays are considered dangerous to the animal industry and pose a risk of human infection. For example, the vesicular stomatitis virus (VSV) is an important exotic disease agent in domestic animals, and the sindbis virus provokes rash, arthralgia, and fever in humans. Therefore, biosafety needs to be considered when antiviral assays are performed. We chose Getah virus as a candidate challenge virus because it is less hazardous to animals and humans. Crystal violet staining 50% CPE inhibition antiviral assay of human IFN using Getah virus was studied. Antiviral assay using Getah virus and FL cells gave a higher titer of human IFN than did assay using VSV. The titer of human IFN alpha was almost the same as that given by standardized control samples. The titer of human IFN by antiviral assay using Getah virus on the sheet method (IFN reacted the sheeted FL cells) was higher than those of the simultaneous reaction method (IFN reacted the suspending FL cells before sheeted). We therefore consider the sheet method useful for detection of small amounts of IFN. Antiviral assay using Getah virus on MDBK cells gave a lower titer of human IFN alpha than did assay using VSV. However, the adjusting the number of MDBK cells and the titer of Getah virus to get the best condition for CPE appearance, gave similar results in the assays using Getah virus and VSV. We consider that Getah virus is a potentially useful challenge virus for antiviral assay of human IFN.     

Microinjection of anti-interferon antibodies into cells does not inhibit the induction of an antiviral state by interferon.

Affinity-purified polyclonal antibodies directed against human lymphoblastoid interferon (IFN), Escherichia coli-derived human IFN-alpha 2, or two synthetic fragments of human IFN-alpha 1 all neutralized the antiviral activity of human alpha IFNs when added to the culture medium of MDBK cells together with IFNs. However, when these antibodies were microinjected into the cytoplasm or the nucleus of cells, subsequent treatment of the cells with IFNs induced full protection against vesicular stomatitis virus. This suggests that IFNs themselves need not act in the cytoplasmic compartment or the nucleus to induce an antiviral state.     

Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells

Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.     

Failure to induce an antiviral state in preimplantation bovine embryos treated with interferon

Bovine blastocysts hatched from their zonae pellucidae were cultured for 24 h in the presence or absence of interferon and then challenged with either vesicular stomatitis virus or bluetongue virus to assess the induction of an antiviral state. In contrast to its application to fetal bovine cells, where significant antiviral effects were induced, interferon treatment of embryos failed to reduce virus yield and had no effect on virus-induced cytopathology. This lack of biologic activity of interferon in bovine embryos is similar to that previously observed with undifferentiated murine embryonal carcinoma cells and is probably a manifestation of a more general mechanism regulating gene expression in the early mammalian embryo.     

Proteins induced by various types of double-stranded RNA and interferon type I in human fibroblasts. Correlation with antiviral activity of the substances]

Protein induction by new antiviral preparations of dsRNAs (larifan, ridostin, rifastin and poly(A).poly(U)) and recombinant beta-interferon in human fibroblasts (M19) was studied. The common gene products: 88, 80, 68, 58, 56, 52, 50 and 26 kD were detected in the spectra of the induced cytoplasmic polypeptides. At the same time the sets of the induced proteins had individual distinctions in various preparations. Induction of the 56-kD protein was more essential in the action of dsRNAs than that of interferon. The antiviral activity of dsRNAs and interferon preparations correlated with a relative increase in the synthesis of proteins with molecular weights of 88, 80 and 58 kD. The study results are in agreement with the fact that the dsRNAs have interferon-independent pathways of antiviral action with participation of 56- and 58-kD protein genes.     

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.     

Dissociation of protein kinase activity and the induction of the antiviral state in a cell line responsive to the antiviral effects of interferon

Interferons or oxidized glutathione were found to induce double-stranded RNA-dependent protein kinase activity in mouse L cells that phosphorylates the α subunit of eukaryotic peptide initiation factor 2. A mixture of leukocyte/fibroblast interferons as well as immune interferon induced the protein kinase and also suppressed virus replication in the L cells. Oxidized glutathione was equally effective in inducing protein kinase activity, but it did not induce an antiviral state in these cells. The data suggest that a simple cause and effect relationship does not exist between protein kinase induction and the establishment of the antiviral state in a cell that is responsive to the antiviral effects of interferon.     

Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy

Influenza virus infections are major public health threats due to their high rates of morbidity and mortality. Upon influenza virus entry, host cells experience modifications of endomembranes, including those used for virus trafficking and replication. Here we report that influenza virus infection modifies mitochondrial morphodynamics by promoting mitochondria elongation and altering endoplasmic reticulum-mitochondria tethering in host cells. Expression of the viral RNA recapitulates these modifications inside cells. Virus induced mitochondria hyper-elongation was promoted by fission associated protein DRP1 relocalization to the cytosol, enhancing a pro-fusion status. We show that altering mitochondrial hyper-fusion with Mito-C, a novel pro-fission compound, not only restores mitochondrial morphodynamics and endoplasmic reticulum-mitochondria contact sites but also dramatically reduces influenza replication. Finally, we demonstrate that the observed Mito-C antiviral property is directly connected with the innate immunity signaling RIG-I complex at mitochondria. Our data highlight the importance of a functional interchange between mitochondrial morphodynamics and innate immunity machineries in the context of influenza viral infection.     

Effect of oxygen on the antiviral activity of human interferon in cell culture

Antiviral activity of human lymphocytic interferon under conditions of increased oxygen levels in the cell culture was studied. It was found that oxygen had a capacity for increasing the antiviral effect of human interferon in homologous cells. When 20-80% air was replaced by oxygen the interferon titers on an average amounted to 1:113.4-1:124.8 against 1:29.1 in the control. This means that the average titer of interferon in the experiments with oxygen was 4 times higher than that in the control. On the basis of these data it is recommended using interferon in the form of aerosols in conjunction with oxygen for the treatment of viral respiratory infections.     

Apparent dispensability of the carbohydrate moiety of human interferon for antiviral activity.

Human leukocyte and tritium-labeled fibroblast interferons, prepared by induction with Sendai virus and with double-stranded polyinosinic acid.polycytidylic acid respectively, have been studied in relation to the carbohydrate moieties attached to them. These interferons were partially purified by immunoabsorbance and by gel filtration. On treatment with glycosidases, about 80% of the 3H-labeled sugar moieties in this glycoprotein-containing fraction was removed without detectable alteration of the antiviral activity or antibody-binding properties characteristic of interferon. The molecular weight of leukocyte interferon was reduced by about 4000. As others have reported, the heterogeneous character of interferon revealed by isoelectric focusing was greatly reduced by the enzyme treatment.