Selection and characterization of monoclonal antibodies developed to interferon-induced dsRNA-binding proteins.
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Antibodies, Monoclonal↗
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Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase.
The P68 protein kinase (referred to as P68 based on its M(r) of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by dsRNAs. The kinase is under tight controls in virus-infected cells since once activated, it phosphorylates its natural substrate eukaryotic initiation factor 2 (elF-2), leading to potential limitations in functional elF-2 and decreases in protein synthesis initiation. To further delineate the molecular mechanisms underlying kinase regulation, we attempted to express the P68 protein kinase in insect cells using a baculovirus vector. Repeated efforts to isolate recombinant baculoviruses containing a wild-type kinase failed, whereas recombinants expressing a nonfunctional kinase with a catalytic domain II mutation were readily isolated. When used to infect Spodoptera frugiperda cells, the recombinant virus expressed the exogenous mutant protein at almost 5-10% of the total proteins synthesized. We then purified the kinase by immunoaffinity chromatography to raise monospecific antiserum which recognized not only the human native wild-type P68, but also kinase homologues in murine, bovine, and monkey cells as determined by immunoblot and immunoprecipitation analysis. Fortunately, kinase function also could be assayed using this antibody since the human and nonhuman kinase homologues, present in immunoprecipitates, were autophosphorylated and phosphorylated the natural substrate, elF-2 alpha. Further, this antiserum recognized epitopes throughout the molecule including the amino and carboxyl termini in contrast to the available monoclonal antibody. In vitro assays using the polyclonal antibody revealed the importance of the amino terminus, especially amino acids 1-97, in the binding of the kinase to viral RNA activators and inhibitors. Finally, we determined that the P68 amino terminus was both necessary and sufficient for binding dsRNA as we were able to transfer dsRNA-binding properties to a reporter gene product previously unable to bind RNA.
Baculoviridae↗
Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.
By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.
Amino Acid Sequence↗
Proteolytic cleavage of the reovirus sigma 3 protein results in enhanced double-stranded RNA-binding activity: identification of a repeated basic amino acid motif within the C-terminal binding region.
The reovirus capsid protein sigma 3 was examined for double-stranded RNA (dsRNA)-binding activity by Northwestern (RNA-protein) blot analysis. Treatment of virion-derived sigma 3 protein with Staphylococcus aureus V8 protease led to an increase in the dsRNA-binding activity associated with the C-terminal fragment of the protein. Recombinant C-terminal fragments of the sigma 3 protein were expressed in Escherichia coli from the S4 cDNA of reovirus serotype 1. These truncated sigma 3 proteins displayed proteolytic processing and dsRNA-binding activity similar to those observed for native, virion-derived sigma 3 protein as measured by Northwestern blot analysis. Construction of a modified pET3c vector, pET3Exo, allowed the production of 3'-terminal deletions of the S4 cDNA by using exonuclease III and rapid screening of the induced truncated sigma 3 proteins. An 85-amino-acid domain within the C-terminal portion of the sigma 3 protein which was responsible for dsRNA-binding activity was identified. The 85-amino-acid domain possessed a repeated basic amino acid motif which was conserved in all three serotypes of reovirus. Deletion of one of the basic motifs, predicted to be an amphipathic alpha-helix, destroyed dsRNA-binding activity.
Amino Acid Sequence↗
A conserved double-stranded RNA-binding domain.
We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.
Amino Acid Sequence↗
Identification of the double-stranded RNA-binding domain of the human interferon-inducible protein kinase.
The interferon-inducible double-stranded (ds) RNA-activated protein kinase (p68 kinase) is a physiologically important enzyme that regulates the rate of cellular and viral protein synthesis by phosphorylating and thereby inactivating the peptide chain initiation factor 2. We have generated a cDNA clone of the human p68 kinase by polymerase chain reaction cloning using the recently published sequence of this enzyme. Active enzyme was synthesized by in vitro transcription-translation of the cDNA clone. This system was used for mapping the dsRNA-binding domain of the enzyme. Progressive deletions from the carboxyl terminus were introduced by digesting the cDNA with suitable restriction enzymes. Expression of proteins harboring deletions from the amino terminus was achieved by cloning DNA fragments into appropriately constructed expression vectors. Affinity of the truncated proteins for dsRNA was examined by testing their capacity to bind to dsRNA-agarose beads. Our results demonstrated that the dsRNA-binding domain lies at the amino terminus of the protein. A truncated protein containing the first 170 amino acid residues from the amino terminus could bind to dsRNA. However, deletion of 34 residues from the amino terminus or 41 residues from the carboxyl terminus of this truncated protein eliminated its dsRNA-binding activity. Comparison of the primary structure and the secondary structure of this region of p68 kinase and the corresponding region of 2'-5'-oligoadenylate synthetase revealed no apparent similarity.
Base Sequence↗
Characterization of a vaccinia virus-encoded double-stranded RNA-binding protein that may be involved in inhibition of the double-stranded RNA-dependent protein kinase.
The work described in this article identifies a vaccinia virus-encoded protein that may be involved in inhibition of the interferon-induced, double-stranded RNA-dependent protein kinase. Extracts prepared from vaccinia virus (WR strain)-infected cells contain an inhibitor of this kinase. Inhibition was reduced in extracts from which dsRNA-binding proteins had been removed by preadsorption to poly(rI).poly(rC)-Sepharose, suggesting that a dsRNA-binding protein may be involved in kinase inhibition. A single major virus-specific polypeptide of Mr = 25,000 (p25) bound to the poly(rI).poly(rC)-Sepharose. p25 was synthesized in a coupled in vitro transcription/translation system programmed with vaccinia cores, indicating that it is a vaccinia-encoded protein. Synthesis of p25 was detected at early times, by 2 hr post infection, peaked at 5 hours postinfection, and decreased during the late phase of virus replication. In the presence of cytosine arabinoside p25 synthesis did not decrease at late times postinfection. Kinase inhibitory activity accumulated with similar kinetics to p25, both in the presence and absence of cytosine arabinoside. Kinase inhibitory activity copurified with p25, through gel filtration, and Cibacron blue-affinity chromatography. Removal of p25 by precipitation with antiserum to p25 decreased kinase inhibitory activity in extracts prepared from vaccinia virus-infected cells. These results suggest that p25 may be necessary for the specific kinase inhibitory activity detected in vaccinia virus-infected cells.
Animals↗
Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase.
The double-stranded RNA (dsRNA)-binding domain of the human p68 kinase has been localized to the N-terminal half of the enzyme by using progressive deletion analysis and in vitro binding assays. To further define the domains responsible for binding to dsRNA, we cloned the mouse dsRNA-activated p65 kinase and used sequence alignment to identify conserved domains in the N-terminal region. Deletions in either of two 12-amino-acid-long and arginine- or lysine-rich regions abrogated binding to dsRNA. Moreover, in an in vivo growth inhibition assay in the yeast Saccharomyces cerevisiae, these mutants failed to exhibit a slow-growth phenotype.
Amino Acid Sequence↗
Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation.
The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a potent cellular antiviral enzyme which is activated by autophosphorylation in response to double-stranded RNA (dsRNA). Activated dsI has also been implicated as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN). We have shown previously that introduction of a transforming ras gene into BALB/c-3T3 fibroblasts blocks induction of responsive genes by PDGF and IFN. We therefore investigated the effect of transforming ras genes on dsI activity in these cells. We report here that dsRNA-mediated activation of dsI is blocked in v-ras-containing cells in a manner specific to ras and not attributable to the transformed phenotype since: 1) a dexamethasone-inducible v-Ha-ras gene produced the effect within 18 h of induction; 2) morphologic reversion of ras-transformed cells with cAMP or the Krev-1 gene restored potential for dsI activation; and 3) transformation by v-mos or v-abl had no effect on dsI activation. Latent dsI levels were unaffected by v-ras. A heat-sensitive dsI inhibitory activity could be demonstrated in v-ras-containing cells which functioned in trans when mixed with untransformed cell extracts prior to stimulation with dsRNA. The inhibitory activity, which was destroyed by phenol-chloroform extraction, did not bind dsRNA.
3T3 Cells↗
Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.
COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.
Animals↗
Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.
The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.
Amino Acid Sequence↗
Translational effects and sequence comparisons of the three serotypes of the reovirus S4 gene.
Reovirus S4 RNA codes for the dsRNA-binding polypeptide sigma 3, a major virion outer capsid component that also has translational effects in both infected and transfected mammalian cells. To compare the composition and properties of the three different serotypes of sigma 3, a DNA copy of the type 2 gene was cloned and sequenced. The total lengths (1196) and the sequences of leader (33 nucleotides) and trailer (66 nucleotides) regions are highly conserved among the three S4 serotypes. The type 1 and 3 S4 genes are highly related (77 mismatches). However, the type 2 gene contains many mismatches relative to the type 1 and 3 genes (260 and 270 positions, respectively). Most of the mismatches are third position changes, resulting in sigma 3 polypeptides that are 90% or more identical. Transient expression vectors, constructed by replacing the chloramphenicol acetyltransferase (CAT) gene in pRSVCAT with S4 DNA, were used to test the effects of polypeptide sigma 3 on CAT expression in cotransfected COS cells. Transfection with the correctly oriented DNAs resulted in synthesis of the corresponding sigma 3 polypeptides which enhanced CAT expression. The type 2 and type 3 S4 genes were considerably more stimulatory than type 1 when compared to CAT DNA alone. However, with all three serotypes the CAT activity was significantly higher in cells cotransfected with S4 DNA in the correct orientation as compared to the reverse arrangement.
Amino Acid Sequence↗
dsRNAscan maps human dsRNAome, revealing conservation, intermolecular dsRNA, and correlates of ADAR dependency.
The human transcriptome contains millions of A-to-I editing sites arising from an unclear number of poorly characterized dsRNAs. Editing sites reveal the presence of dsRNA, but this method is limited by transcription levels, read depth, and ADAR expression and cannot identify unedited dsRNA. To address these limitations, we developed dsRNAscan. Applying dsRNAscan to the human genome predicted 5 million dsRNAs, mostly in repetitive and intergenic regions. Machine learning models trained on A-to-I editing and RNA structure-probing data identified ∼2.4 million high-confidence predictions, which were enriched at dsRNA-binding protein binding sites. Additionally, we predicted hundreds of dsRNAs conserved across vertebrates and observed thousands of editing-enriched regions suspected to arise from intermolecular dsRNAs formed with sense-antisense transcripts. Quantifying expression of intramolecular and intermolecular dsRNAs accessible to cytoplasmic immune sensors revealed that their ratio correlated with ADAR dependency across cancer cell lines. The human dsRNAome is available as a resource at https://dsrna.chpc.utah.edu/.
A-to-I RNA editing↗
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.
Animals↗
Double stranded RNA and the nuclear matrix--implications for the 2-5A system.
RNA species present on rat liver nuclear matrices were investigated. Nuclear matrices prepared by extensive digestion of isolated nucleii with DNase and RNaseA followed by low and high (2M NaC1) salt washes were labelled in vitro with T4 RNA ligase and [5'-32P]pCp and the labelled RNA analysed by gel electrophoresis. Despite the extensive RNaseA treatment, a prominent RNA species migrating as a heterodisperse band of 220-300 nucleotides (termed MX220-300), was observed--only minor amounts of other RNA molecules were seen. A comparison of RNA isolated from in-vitro labelled nuclear matrices with isolated matrix RNA that was subsequently labelled, indicated that part of MX220-300 was preferentially exposed on the nuclear matrix structure. Analysis of MX220-300 indicated that it was composed of a polyadenylic acid moiety hydrogen bonded to a smaller molecule of polyuridylic acid. No evidence was found for the presence of guanosine or cytosine residues. Control experiments in which labelled polyuridylic acid was added to nucleii prior to the preparation of MX220-300, virtually excluded the possibility that the partial double stranded RNA structure was an artefact of matrix preparation. An analysis of proteins in the nuclear matrix structure that interact with double stranded (ds)RNA showed at least 2 proteins having molecular weights of 62K and 66K daltons that recognized and bound polyadenylic/polyuridylic acid. Competition experiments with unlabelled polyinosinic/polycytidylic acid indicated that these proteins specifically recognized the dsRNA structure. The 62K and 66K dalton matrix proteins that specifically bound dsRNA were observed in nuclear matrices prepared from HeLa, Ehrlich ascites tumor and rat liver cells. It is not known whether these matrix located dsRNA binding proteins have 2-5A synthetase activity. The relevance of the above findings to the 2-5A system will be discussed.
Adenine Nucleotides↗
The binding of the 69- and 100-kD forms of 2',5'-oligoadenylate synthetase to different polynucleotides.
Three major forms of 2',5' oligoadenylate (2-5A) synthetase are induced in interferon (IFN)-treated human cells: 40-46, 69, and 100 kD. Here we studied the binding and activation of the 69- and 100-kD forms to single-stranded (ss) or double-stranded (ds) RNAs. The 69- and 100-kD form enzymes purified by immunoaffinity chromatography, were shown to be activated by synthetic dsRNAs poly(I).poly(C) or poly(A).poly(U) whereas ssRNAs poly(I), poly(C), poly(A), poly(U), and poly(G) had no effect. Both enzymes were also partially purified by binding to dsRNA or ssRNA-Sepharose. The synthetases bound to dsRNA Sepharose were partially activated but required the addition of soluble poly(I).poly(C) for maximal activity. The synthetases bound to ssRNA-Sepharose manifested no activity but became activated in the presence of soluble dsRNA, poly(I).poly(C). However, activation of such ssRNA-bound enzymes by dsRNA did not result in their dissociation from the ssRNA-Sepharose. These results indicate the presence of different polynucleotide binding sites on the 69- and 100-kD forms of 2-5A synthetase: a specific dsRNA binding site essential for activation and another polynucleotide binding site or sites which, although not specific, might be important for the optimal conformation of these proteins in cells.
2',5'-Oligoadenylate Synthetase↗
Autophosphorylation of the protein kinase dependent on double-stranded RNA.
The double-stranded RNA (dsRNA)-dependent protein kinase (p68 kinase) from interferon-treated human cell is a Mr 68,000 protein induced by interferon. By the use of a specific monoclonal antibody, we have been able to study the two distinct protein kinase activities characteristic of purified p68 kinase. The first activity is functional for endogenous phosphorylation of the enzyme (p68 kinase), whereas the second one is responsible for the phosphorylation of exogenous substrates such as eukaryotic initiation factor 2 and histone. When activated by dsRNA in the presence of Mn2+ and ATP, p68 kinase is autophosphorylated and is then capable of catalyzing phosphorylation of histone in the absence of dsRNA. Whereas binding of 8-azido-[alpha-32P] ATP (8-N3ATP) to p68 kinase is dependent on both dsRNA and Mn2+, phosphorylated p68 kinase binds 8-N3ATP independent of dsRNA. This is consistent with a dsRNA requirement for the autophosphorylation of p68 kinase, but not for the phosphorylation of exogenous substrates. p68 kinase is mainly associated with the ribosomal pellet. It could be recovered efficiently by a buffer containing both high salt and a nonionic detergent. Synthesis of p68 kinase is induced several-fold by interferon in different types of human cells. Partial proteolysis of [35S]methionine and an 8-N3ATP-labeled p68 kinase preparation by Staphylococcus aureus V8 protease indicated the presence of a major Mr 48,000 polypeptide (p48) with a specific ATP-binding site. p48 probably contains the catalytic unit of p68 kinase and is analogous to a similar protein which we have previously described as a distinct protein present in a complexed form with p68 kinase. We now believe that the presence of p48 in previously purified kinase preparations was due to partial degradation of p68 kinase.
Adenosine Triphosphate↗
Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI.
The protein kinase DAI, the double-stranded RNA-activated inhibitor of translation, is an essential component of the interferon-induced cellular antiviral response. The enzyme is regulated by the binding of activator and inhibitor RNAs. We synthesized DAI in vitro and located its RNA-binding domain within the amino-terminal 171 residues. This domain contains two copies of an RNA-binding motif characterized by a high density of basic amino acids, by the presence of conserved residues, and by a probable alpha-helical structure. Deletion of either of the two motifs prevents the binding of dsRNA, but their relative positions can be exchanged, suggesting that they cooperate to interact with dsRNA. Clustered point mutations within the RNA-binding motifs and duplications of the individual motifs indicate that the first copy of the motif plays the more important role. Mutations that impair binding have similar effects on the binding of double-stranded RNAs of various lengths and of adenovirus VA RNAI, implying that discrimination between activator and inhibitory RNAs takes place subsequent to RNA binding.
Amino Acid Sequence↗