dsRNA-dependent Protein Kinase Research Articles

Hepatitis delta virus replication in vitro is not affected by interferon- or - despite intact cellular responses to interferon and dsRNA

The hepatitis delta virus (HDV) genome consists of circular ssRNA which has extensive intramolecular complementarity and can form a dsRNA rod-like structure. If such RNA species were to exist in an unmasked form in cells, they would be expected to induce interferon (IFN) expression and activate two IFN-inducible dsRNA-dependent enzymes with anti-viral activity, namely the dsRNA-dependent protein kinase (PKR) and 2',5' oligoadenylate (2',5' A) synthetase. Since the virus replicates to high copy number for prolonged periods in infected cells it is apparently able to evade these antiviral mechanisms. The RNA genome may be masked and fail to induce or activate the antiviral response, or the virus may inhibit such a response. Treatment of a hepatoma cell line, Huh7, and a fibrosarcoma cell line, HT1080, stably transfected with a trimeric HDV cDNA construct, with IFN-alpha or IFN-gamma for up to seven days failed to influence the level of expression of genomic or antigenomic HDV RNA, or delta antigen (Ag). This is consistent with either failure of activation or inhibition of the IFN response. However the induction of several IFN-responsive genes, including PKR, 2',5' A synthetase and class I MHC is normal and cotransfection of a construct expressing delta Ag did not affect expression from an IFN-inducible chloramphenicol acetyltransferase construct. In addition, the activation of PKR is not inhibited in HDV-expressing cells and antiviral assays suggest that the ability of these cells to mount an antiviral response to at least two cytopathic viruses is unaffected. IFN-beta is inducible normally by dsRNA in cells transfected with the delta cDNA trimer. We conclude that HDV replication is not inhibited by IFN-alpha or IFN-gamma, even though the responses of cells expressing HDV RNA and antigen to IFN and dsRNA are intact.

Increased thermal aggregation of proteins in ATP-depleted mammalian cells.

In an attempt to understand the influence of the intracellular environment on protein stability, the thermal denaturation of various reporter proteins was examined within cultured mammalian cells. Loss of solubility and of enzymatic activities were taken as indicators of thermal denaturation. Photinus pyralis luciferase, Escherichia coli beta-galactosidase, the 70-kDa constitutive heat-shock proteins and the 68-kDa dsRNA-dependent protein kinase are found mostly in the supernatant fractions of centrifuged lysates from control unshocked mammalian cells. However, when cells are lysed after heat shock, a proportion of the reporter molecules is found to be aggregated to the nuclear pellets. This insolubilization does not affect all cellular proteins; many of them remain unaffected by heat shock. The heat-induced insolubilization of all four reporter proteins is markedly enhanced when the intracellular ATP concentration is drastically decreased after inhibition of both oxidative phosphorylation and glycolysis. Although ATP molecules bind to luciferase and protect it from thermal inactivation in vitro, the consequences of strong ATP depletion on luciferase thermal stability within the cells are found to be much greater than expected from in vitro data. The 70-kDa constitutive heat-shock proteins and the 68-kDa protein kinase are ATP-binding proteins but ATP depletion also considerably increases the aggregation of beta-galactosidase to the nuclear pellets, although this enzyme is not known to be an ATP-binding molecule. Insolubilization of all four reporter proteins occurs in ATP-depleted cells even at normal growing temperatures (37 degrees C). Protein denaturation may be enhanced either by the aggregation and disappearance of the intracellular 'free' chaperones or by the trapping of unfolded protein molecules on chaperones; the chaperone/unfolded protein complexes could not dissociate in the absence of ATP. Enhanced protein denaturation due to ATP depletion is proposed to account for the greater heat sensitivity of ATP-depleted cells and for the ability of mitochondrial uncouplers to trigger a heat-shock response in some cells.

Endogenous inhibitors of the dsRNA-dependent eIF-2α protein kinase PKR in normal and ras-transformed cells

The serine/threonine kinase PKR is activated by autophosphorylation in response to nanomolar concentrations of double-stranded RNA and other polyanions. We have previously shown that expression of an oncogenic ras gene induces an endogenous protein inhibitor of PKR activation in murine fibroblasts, as measured by a greatly reduced ability of PKR to autophosphorylate in response to double-stranded RNA in lysates from these ras-expressing cells. Immunoprecipitation of PKR away from the ras-transformed cell lysate restored the ability of PKR to become autophosphorylated. However, the autophosphorylation was no longer dsRNA-dependent. In the present work, PKR immobilized either by immunoprecipitation or by affinity precipitation on Agpoly(I) poly(C) was found to autophosphorylate in a dsRNA-independent manner when incubated in the presence of a detergent lysis buffer and ATP. When lysis buffer was replaced by cytoplasmic extract from normal or ras-transformed cells, autophosphorylation of the immobilized PKR was inhibited in the presence or absence of dsRNA, even though it could be shown that PKR remained bound and intact in the precipitate, and able to autophosphorylate if rewashed with lysis buffer. These findings suggest that PKR activation is regulated by an endogenous inhibitor in murine fibroblasts as well as by dsRNA or other polyanions.

Inhibition of the dsRNA-Dependent Protein Kinase by a Peptide Derived from the Human Immunodeficiency Virus Type 1 Tat Protein

The human immunodeficiency virus (HIV) is the etiologic agent leading to the development of acquired immunodeficiency syndrome (AIDS). Interferons (IFNs) are known for eliciting antiviral responses from cells, and studies have indicated that infection with HIV induces the production of IFN. Previous studies have shown that the trans-acting response element (TAR) sequence of HIV-1 mRNA can activate the IFN-induced double-stranded (ds) RNA-dependent protein kinase (DAI). DAI, when activated, is a potent inhibitor of protein synthesis and has been implicated in mediating part of IFN's antiviral activity. Here, we report that a synthetic peptide containing the basic region of HIV Tat protein is effective in preventing the activation of DAI. Evidence is presented that indicates that the Tat peptide exerts its effect by binding to the TAR RNA sequence and thus preventing this RNA from binding to and activating DAI. It appears that in addition to its role in trans-activation, the tat protein may also function to overcome the antiviral activity of IFN by regulating DAI activity. Thus, inhibition of DAI by the Tat protein early in the life cycle of HIV may provide a mechanism by which the virus can escape a translational block imposed by the kinase.

Mode of Action of the Anti-AIDS Compound Poly(I)·Poly(C12U) (Ampligen): Activator of 2′,5′-Oligoadenylate Synthetase and Double-Stranded RNA-Dependent Kinase

The mismatched double-stranded RNA (dsRNA), poly(I).poly(C12U), also termed Ampligen, exhibits a strong antiviral and cytoprotective effect on cells (human T-lymphoblastoid CEM cells and human T-cell line H9) infected with the human immunodeficiency virus type 1 (HIV-1). Untreated H9 cells infected with HIV-1 start to release the virus 3 days post-infection, while in the presence of 40 micrograms/ml (80 micrograms/ml) of poly(I).poly(C12U) the onset of virus production and release is retarded and does not occur before day 5 (day 6). We demonstrate that poly(I).poly(C12U) markedly extends the duration of the transient increase of 2',5'-oligoadenylate (2-5A) synthetase mRNA level and activity preceding virus production after infection of cells with HIV-1. Treatment of HeLa cells with poly(I).poly(C12U) was found to cause a significant increase in total (activated plus latent) 2-5A synthetase activity; no evidence was obtained that the level of latent (nonactivated) 2-5A synthetase is changed in cells treated with dsRNA plus interferon (IFN). Poly(I).poly(C12U) is able to bind and to activate 2-5A synthetase(s) from HeLa cell extracts. Addition of poly(I).poly(C12U) to HeLa cell extracts results in production of longer 2-5A oligomers (> or = 3 adenylate residues), which are better activators of RNase L. Both free and immobilized poly(I).poly(C12U) also bind to the dsRNA-dependent protein kinase (p68 kinase), resulting in autophosphorylation of the enzyme. Activation of the kinase by the free RNA occurs within a limited concentration range (10(-7) to 10(-6) grams/ml). Addition of HIV-1 Tat protein does not affect binding and activation of p68 kinase to poly(I).poly(C12U)-cellulose but strongly reduces the binding of the kinase to immobilized TAR RNA of HIV-1. We conclude that poly(I).poly(C12U) may antagonize Tat-mediated down-regulation of dsRNA-dependent enzymes.

The mouse antiphosphotyrosine immunoreactive kinase, TIK, is indistinguishable from the double-stranded RNA-dependent, interferon-induced protein kinase, PKR.

The mouse TIK protein, a serine/threonine kinase, was originally isolated from a murine pre-B cell expression library by its ability to bind anti-phosphotyrosine antibodies (Icely et al., J. Biol. Chem. 266, 16073-16077, 1991). The 67 kDa protein was found to have an associated autophosphorylation activity when incubated with ATP. Our results show that TIK is actually the mouse interferon-induced, dsRNA-dependent protein kinase, PKR. We demonstrate that the TIK message is interferon-inducible in mouse L-cells and in vitro transcription and translation of the TIK cDNA produces a protein that is capable of binding double-stranded RNA. The in vitro synthesized TIK protein migrated as a 65 kDa protein on SDS-PAGE when incubated with ATP, but migrated as a 60 kDa protein when incubated with an inhibitor of PKR, 2-aminopurine. We further show that proteolytic digestion of TIK with Staphylococcus aureus V8 protease results in a cleavage pattern identical to that obtained by V8 digestion of authentic PKR. Antiserum to TIK specifically recognized PKR. Cloned TIK had inhibitory activity for replication of EMCV but not VSV. From these observations we conclude that TIK kinase is the mouse interferon-induced, double-stranded RNA-dependent kinase, PKR.

Mechanism of action of a cellular inhibitor of the dsRNA-dependent protein kinase from 3T3-F442A cells.

When mouse 3T3-F442A preadipocyte fibroblasts reach confluence in the appropriate culture medium, their growth is arrested, and the cells undergo terminal differentiation to adipocytes. Two proteins that may be involved in this process are interferon and the interferon-induced double-stranded RNA (dsRNA)-dependent protein kinase (DAI). In 3T3-F442A cells, interferon and DAI are transiently expressed with a maximum level of active kinase appearing at confluence. Interestingly, the level of active DAI was found to be low when cells were maintained under conditions nonpermissive for differentiation. This reduction in DAI was at least partly because of the presence of elevated levels of a specific inhibitor of DAI, termed dRF, which appeared to be a reversible inhibitor of the autophosphorylation (activation) of DAI. In the present study, the mechanism of action of dRF was investigated. Photocross-linking experiments indicated that dRF prevented the binding of ATP to DAI. Since the binding of ATP to DAI is dsRNA-dependent, we examined the effect of dRF on the binding of dsRNA to the kinase using RNA mobility shift assays. dRF was found to prevent the formation of DAI-dsRNA complexes without a direct effect on the dsRNA. This suggests that dRF exerts its effect through an interaction with DAI.

Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.

The double-stranded RNA-dependent protein kinase (dsRNA-PK) is thought to be a key mediator of the antiviral and antiproliferative effects of interferons (IFNs). Studies examining the physiological function of the kinase suggest that it participates in cell growth and differentiation by regulating protein synthesis. Autophosphorylation and consequent activation of dsRNA-PK in vitro and in vivo result in phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) and inhibition of protein synthesis. Expression of a functionally defective mutant of human dsRNA-PK in NIH 3T3 cells resulted in malignant transformation, suggesting that dsRNA-PK may function as a suppressor of cell proliferation and tumorigenesis.

The war against the interferon-induced dsRNA-activated protein kinase: can viruses win?

The war against the interferon-induced dsRNA-activated protein kinase: can viruses win?

Viral inhibition of the interferon system

In response to interferon (IFN), cells develop an antiviral state in which the replication of a wide spectrum of RNA and DNA viruses is inhibited. Viruses have evolved a variety of mechanisms to inhibit the production and action of the interferons. Interferon action may be blocked by inhibition of the post-receptor signalling pathway, which prevents the expression of a number of proteins with antiviral properties. Other viruses prevent the action of specific, interferon-induced antiviral systems. In particular, the action of the dsRNA-dependent protein kinase (DAI) is inhibited by a variety of different viruses, indicating the fundamental importance of this enzyme to the antiviral response.

Inhibitor of Interferon‐Induced Double‐Stranded RNA‐ Dependent Protein Kinase and Its Relevance to Alteration of Cellular Protein Kinase Activity Level in Response to External Stimuli

In FL cells, interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) was found to be present in a form complexed with a potent inhibitor of its dsRNA-dependent activation. The inhibitor was readily dissociated from PK-I by DEAE-cellulose chromatography to yield a dsRNA-responsive PK-I. The inhibitor was also dissociated easily from PK-I by gel filtration through Sephacryl S-200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5 hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA-activatable form of PK-I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK-I activity. Longer IFN treatment (24 hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK-I protein.

Temporal regulation of tobacco mosaic virus-induced phosphorylation of a host encoded protein

The in vitro and in vivo phosphorylation of a plant encoded protein (p68) associated with dsRNA-dependent protein kinase activity was stimulated at specific time intervals following infection by tobacco mosaic virus or electroporation with dsRNA. The level of p68 phosphorylation in infected and mock inoculated protoplasts did not differ significantly until 6 hr. post-infection, when the basal level of phosphorylation increased 2–3 fold in infected protoplasts. Maximum phosphorylation of p68 occurred between 8–12 hr post-infection and then declined but, at least until 72 hr. post-infection, it was significantly greater than in mock inoculated protoplasts.

A distinct kinase modulates the expression of IFN-inducible genes in human breast cancer cells

The biological activity of interferons (IFNs) is presumed to be mediated through the induction of a number of IFN-inducible genes. IFN-mediated gene induction was examined in two human breast cancer cell lines, MCF-7 and BT-20. Both these cell lines were remarkably responsive to IFNs as a number of IFN inducible genes were rapidly induced. We examined the sensitivity of these genes towards 2-aminopurine (2-AP), a known inhibitor of double-stranded (ds) RNA dependent protein kinase. 2-AP has also been reported to inhibit the induction of IFN- β 1 in response to dsRNA and the genes c-myc and c-fos in fibroblasts. In both MCF-7 and BT-20 cell lines, 2-AP selectively inhibited the IFN-induced gene responses. 2-AP did not affect levels of the oncogene, HER-2/neu. Tamoxifen (TAM), an antiestrogenic drug, which is known to inhibit the activity of protein kinase C at high concentrations, did not affect IFN-mediated gene induction. Our data is consistent with the concept that the 2-AP sensitive kinase is primarily associated with the IFN-induced gene systems and that positive and negative growth regulating stimulii in breast cancer may require the participation of distinct kinases.

Enhancement of the Interferon‐Induced Double‐Stranded RNA‐Dependent Protein Kinase Activity by Sindbis Virus Infection and Heat‐Shock Stress

In extracts of FL cells that were infected with Sindbis virus or treated with heat-shock stress, dsRNA-dependent phosphorylation of 77K protein was markedly increased. The 77K phosphoprotein was indistinguishable from the autophosphorylated and activated form of interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) by two-dimensional gel electrophoresis, and was immunologically related to P68 (Galabru, J. and Hovanessian, A., J. Biol. Chem. 262, 15538 (1987], the HeLa cell counterpart of PK-I. Immunoblotting experiments using monoclonal antibody against PK-I revealed that control cell extracts contained a substantial amount of PK-I protein, although they showed no measurable PK-I activity even when dsRNA was added. The amount of PK-I protein did not increase during a transient dsRNA-dependent enhancement of PK-I activity caused by Sindbis virus infection and heat-shock stress. This implies that the conversion of PK-I protein from a dsRNA-unresponsive form to a responsive form may be important in the regulation of PK-I activity. A similar mode of PK-I regulatory mechanism was operative in the early stages of IFN treatment, although after a prolonged treatment a net synthesis of the PK-I protein did take place.

Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNA1from a T7 vector

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.

Studies on the Mechanism of the Interferon-mediated Antiviral State to Vesicular Stomatitis Virus in Resting Mouse Peritoneal Macrophages

We have analysed the expression of vesicular stomatitis virus (VSV) proteins in virus-infected freshly explanted mouse peritoneal macrophages (resistant to virus replication), macrophages aged in vitro (permissive for virus replication) and freshly explanted macrophages from mice treated with antibody to interferon (IFN) alpha/beta (permissive for VSV replication). Our data showed that some VSV proteins (i.e. N/NS and G) were synthesized in virus-infected (1 p.f.u/cell) freshly harvested macrophages at early times after infection (3 to 6 h); the expression of such viral proteins was subsequently inhibited at 18 h post-infection. In contrast, a progressive increase in the expression of VSV proteins was observed in the macrophages aged in vitro and infected with VSV at 1 p.f.u./cell. Infection with a higher m.o.i. (16 p.f.u./cell) resulted in similar viral protein electrophoresis patterns for both aged macrophages and freshly explanted macrophages. Even at low m.o.i. a marked and progressive expression of all VSV proteins was observed in freshly harvested macrophages from mice treated with antibody to mouse IFN-alpha/beta. Higher levels of oligo-2',5'-adenylate synthetase (2-5AS) were found in freshly harvested macrophages than in either aged macrophages or those from mice treated with antibody to IFN. No dsRNA-dependent 67K protein kinase was detected in freshly harvested macrophages or peritoneal cells from untreated mice or mice treated with poly(rI).poly(rC) or Newcastle disease virus. The following conclusions can be drawn from these results. Low levels of spontaneous IFN-alpha/beta are responsible for the time-dependent inhibition of VSV protein synthesis in virus-infected freshly harvested macrophages; high levels of 2-5AS (in the absence of detectable levels of 67K protein kinase) appear to correlate with the progressive inhibition of VSV proteins; this natural antiviral state is highly effective only at low m.o.i.

Activation of interferon-regulated, dsRNA-dependent enzymes by human immunodeficiency virus-1 leader RNA.

Human immunodeficiency virus-1 (HIV-1) leader RNA, which contains double-stranded regions due to inverted repeats, was shown to activate the dsRNA-dependent enzymes associated with the interferon system. HIV-1 leader RNA produced in vitro using SP6 RNA polymerase was characterized using probes for antisense and sense-strand RNA. The RNA preparation was free from significant levels of antisense RNA. HIV-1 leader RNA was shown to activate dsRNA-dependent protein kinase in a cell-free system from interferon-treated HeLa cells. Affinity resins, consisting of HIV-1 leader RNA covalently attached to cellulose, immobilized and activated dsRNA-dependent protein kinase and 2-5A-synthetase. HIV-1 leader RNA, therefore, may be a contributing factor in the mechanism by which interferon inhibits HIV replication.

Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein.

In this report we demonstrate that reovirus serotype 1-infected cells contain an inhibitor of the interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase. We provide evidence that suggests that the virus-encoded sigma 3 protein is likely responsible for this kinase inhibitory activity. We could not detect activation of the dsRNA-dependent protein kinase in extracts prepared from either interferon-treated or untreated reovirus serotype 1-infected mouse L cells under conditions that led to activation of the kinase in extracts prepared from either interferon-treated or untreated, uninfected cells. Extracts from reovirus-infected cells blocked activation of kinase in extracts from interferon-treated cells when the two were mixed prior to assay. The kinase inhibitory activity in extracts of reovirus-infected cells could be overcome by adding approximately 100-fold excess of dsRNA over the amount required to activate kinase in extracts of uninfected cells. Kinase inhibitory activity in extracts of interferon-treated, virus-infected cells could be overcome with somewhat less dsRNA (approximately 10-fold excess). Most of the inhibitory activity in the extracts could be removed by adsorption with immobilized anti-reovirus sigma 3 serum or immobilized dsRNA, suggesting that the dsRNA-binding sigma 3 protein is necessary for kinase inhibitory activity. Purified sigma 3 protein, when added to reaction mixtures containing partially purified kinase, inhibited enzyme activation. Control of activation of this kinase, which can modify eukaryotic protein synthesis initiation factor 2, may be relevant to the sensitivity of reovirus replication to treatment of cells with interferon and to the shutoff of host protein synthesis in reovirus-infected cells.

Tobacco mosaic virus infection stimulates the phosphorylation of a plant protein associated with double-stranded RNA-dependent protein kinase activity.

The influence of tobacco mosaic virus (TMV) infection on nucleotide binding and phosphorylation of an Mr 68,000 host-encoded protein (p68) was examined. The phosphorylation of p68 in homogenates from TMV-infected tissues was 4-fold greater than in homogenates from mock inoculated tissues. Phosphorylation of p68 in extracts from mock inoculated tissues was enhanced by the addition of double-stranded (ds) RNA. Nucleotide photoaffinity labeling experiments indicate that p68 contains an ATP binding site with characteristics consistent with protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase from interferon-treated human cells immunoprecipitated p68 from extracts of TMV-infected tissue, and p68-containing immunocomplexes catalyzed the phosphorylation of endogenous p68. These data suggest that p68 may be an autophosphorylating, dsRNA-dependent protein kinase involved in viral pathogenesis. Based upon analogous functions demonstrated for dsRNA-dependent protein kinases in mammalian systems, p68 may have a role in the regulation of protein synthesis and viral replication in infected cells.

Viroid-induced phosphorylation of a host protein related to a dsRNA-dependent protein kinase.

Viroids are very small, unencapsidated RNAs that replicate and induce severe disease in plants without encoding for any proteins. The mechanisms by which the viroid RNA regulates these events and interacts with host factors are unknown. An Mr 68,000 host-encoded protein has been identified that is differentially phosphorylated in extracts from viroid-infected and mock-inoculated tissues. This phosphoprotein is immunologically related to a double-stranded (ds) RNA-dependent protein kinase from virus-infected, interferon-treated human cells. Further, nucleotide photoaffinity labeling indicates that the protein has an ATP binding site. This protein is similar to dsRNA-dependent protein kinases implicated in mammalian systems in the regulation of protein synthesis and virus replication.