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PubMed1994

The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.

Adenosine Deaminase↗
PubMed1988

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↗
PubMed1995

The non-RNase H domain of Saccharomyces cerevisiae RNase H1 binds double-stranded RNA: magnesium modulates the switch between double-stranded RNA binding and RNase H activity.

Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function. The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus. Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain. Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus. When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity. Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration. These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities. Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate. Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA.

Amino Acid Sequence↗
PubMed1997

Double-stranded RNA-dependent protein kinase (PKR) is regulated by reovirus structural proteins.

Reovirus sigma3 is a virion outer shell protein that also binds dsRNA and stimulates translation by blocking activation of the dsRNA-dependent protein kinase, PKR. Purified sigma3 was shown by gel shift assay to bind specifically to RNA duplexes of minimal length 32-45 base pairs. PKR binding to dsRNA was prevented by sigma3, and translation inhibition of luciferase reporter by PKR expression in transfected cells was reversed by sigma3. Association of sigma3 with its outer capsid partner mu1/mu1C eliminated dsRNA binding and prevented restoration of protein synthesis. Analyses of sigma3 mutants demonstrated a direct correlation between dsRNA binding and reversal of the down-regulation of translation by PKR. In infected cells, sigma3 was stable but dsRNA binding decreased, presumably due to mu1/mu1C complex formation. The results suggest a functional transition from early inhibition of PKR activation by sigma3 to its association with mu1/mu1C in capsid structures.

Animals↗
PubMed1993

Identification of a conserved motif that is necessary for binding of the vaccinia virus E3L gene products to double-stranded RNA.

The E3L gene of vaccinia virus encodes the double-stranded (ds) RNA binding proteins p20 and p25 that exhibit inhibitory activity for the IFN-induced, P1/elF-2 alpha protein kinase. A region in the E3L encoded proteins (residues 156-180) shares a high degree of similarity with several proteins that bind double-helical RNA including the P1/elF-1 alpha kinase, bacterial and yeast RNase III, and a human transactivator response element/Rev response element binding protein. In this study, mutants of E3L proteins were constructed in order to determine the region of the proteins required for dsRNA binding and kinase inhibitory activity. Our data indicate that both the region necessary for dsRNA binding and for kinase inhibitory activity are located at the carboxyl terminus of the protein. The E3L proteins with 7 amino acids deleted from the carboxyl terminus (184-190) could bind to dsRNA, but with lower affinity than could the full-length protein. This protein did not detectably inhibit kinase in vitro. Deletion of 26 amino acids from the carboxyl terminus of the E3L proteins (165-190) abolished dsRNA binding activity and kinase inhibitory activity. In addition, mutations at amino acid 164, 167, or 174 severely inhibited binding to dsRNA. On the other hand, deletion of 83 amino acids from the amino terminus did not affect the proteins' ability to bind dsRNA or inhibit kinase. These results suggest that a region of sequence between amino acids 164 and 183 is necessary for E3L proteins' dsRNA binding activity. This region lies within the homologous domain that the E3L proteins share with other dsRNA binding proteins.

Amino Acid Sequence↗
PubMed1996

Specific mutations near the amino terminus of double-stranded RNA-dependent protein kinase (PKR) differentially affect its double-stranded RNA binding and dimerization properties.

The amino-terminal region of the double-stranded (ds) RNA-dependent protein kinase, PKR, has been shown to mediate both dsRNA binding and protein dimerization. To critically examine if PKR dimerization is dependent on dsRNA binding, we generated a series of mutants that are incapable of binding dsRNA. Some, but not all, of these mutants retained the ability to dimerize, as shown by a two-hybrid transcriptional activation assay in vivo and a chemical cross-linking assay in vitro. These mutants were used further to demonstrate that the translational inhibitory activity of PKR in vivo requires dsRNA binding; PKR mutants that dimerized but did not bind dsRNA could not inhibit the translation of a transfected reporter gene.

Amino Acid Sequence↗
PubMed1997

Complementation of deletion of the vaccinia virus E3L gene by the Escherichia coli RNase III gene.

This work investigated whether the Escherichia coli RNase III gene, rnc+, could complement vp1080, a mutant vaccinia virus that is deleted of its E3L gene. Like E3L, rnc+ codes for a dsRNA binding protein that contains an additional nucleolytic activity. Rnc genes were cloned into the eukaryotic expression vector pMTVa-, expressed in COS-1 cells, and shown to be functional. Transient rescue experiments in HeLa cells demonstrated that the cleavage function of the rnc+ gene was necessary for full rescue of vp1080. The rnc 70 gene, which encodes a product deficient in catalytic activity but still capable of binding to dsRNA, rescued vp1080 weakly. The rnc 105 gene, which encodes a product that cannot bind or cleave dsRNA, was unable to rescue vp1080. The rnc genes were also inserted into the E3L locus of vp1080. While recombinants containing the rnc+ gene or the rnc 70 gene regained the IFN resistance phenotype in RK13 cells, full host range of vaccinia virus was only restored in the recombinant containing the rnc+ gene. Thus, the ability of RNase III to process dsRNA appears to be necessary to restore the host range phenotype. The vp-rnc 105 recombinant behaved similarly to vp1080.

Amino Acid Sequence↗
PubMed1992

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↗
PubMed1998

Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation.

Protein kinase PKR is an interferon-induced enzyme that plays a key role in the control of viral infections and cellular homeostasis. Compared with other known kinases, PKR is activated by a distinct mechanism that involves double-stranded RNA (dsRNA) binding in its N-terminal region in an RNA sequence-independent fashion. We report here the solution structure of the 20 kDa dsRNA-binding domain (dsRBD) of human PKR, which provides the first three-dimensional insight into the mechanism of its dsRNA-mediated activation. The structure of dsRBD exhibits a dumb-bell shape comprising two tandem linked dsRNA-binding motifs (dsRBMs) both with an alpha-beta-beta-beta-alpha fold. The structure, combined with previous mutational and biochemical data, reveals a highly conserved RNA-binding site on each dsRBM and suggests a novel mode of protein-RNA recognition. The central linker is highly flexible, which may enable the two dsRBMs to wrap around the RNA duplex for cooperative and high-affinity binding, leading to the overall change of PKR conformation and its activation.

Amino Acid Sequence↗
PubMed1997

Complementation of vaccinia virus deleted of the E3L gene by mutants of E3L.

Vaccinia virus devoid of its E3L gene is sensitive to treatment of RK-13 cells with interferon-alpha and fails to replicate or form plaques in HeLa cells. In order to determine function of the E3L gene, vaccinia virus recombinants were constructed by inserting mutant E3L genes or a gene coding for an alternative dsRNA-binding protein into virus deleted of its wild type E3L gene. Those viruses that expressed proteins that retained dsRNA binding activity were resistant to the effects of interferon in RK-13 cells and could replicate in HeLa cells. Recombinant viruses that expressed E3L mutant proteins which were unable to bind to dsRNA were interferon sensitive in RK-13 cells and could not replicate in HeLa cells. In addition, a virus that expressed a mutant E3L protein previously characterized as having a low binding affinity for dsRNA exhibited an intermediate phenotype: it was interferon resistant in RK-13 cells but could not replicate in HeLa cells. This work suggests that the E3L gene of vaccinia virus functions primarily as a dsRNA-binding protein in allowing resistance to interferon and in promoting replication in HeLa cells.

Antiviral Agents↗
PubMed1996

Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR.

The human double-stranded RNA- (dsRNA) activated protein kinase (PKR) has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). The minimal-length polypeptide required to bind dsRNA contains both dsRBMs, as determined by mobility-shift and filter-binding assays. Mobility-shift experiments indicate binding requires a minimum of 16 base pairs of dsRNA, while a minimal-length site for saturation of longer RNAs is 11 base pairs. Bulge defects in the helix disfavor binding, and single-stranded tails do not strongly influence the dsRNA length requirement. These polypeptides do not bind an RNA-DNA hybrid duplex or dsDNA as judged by either mobility-shift or competition experiments, suggesting 2'-OH contacts on both strands of the duplex stabilize binding. Related experiments on chimeric duplexes in which specific sets of 2'-OHs are substituted with 2'-H or 2'-OCH3 reveal that the 2'-OHs required for binding are located along the entire 11 basepair site. These results are supported by Fe(II) EDTA footprinting experiments that show protein-dependent protection of the minor groove of dsRNA. The dependence of dsRNA-protein binding on salt concentration suggests that only one ionic contact is made between the protein and dsRNA phosphate backbone and that at physiological salt concentrations 90% of the free energy of binding is nonelectrostatic. Thus, the specificity of PKR for dsRNA over RNA-DNA hybrids and dsDNA is largely due to molecular recognition of a network of 2'-OHs involving both strands of dsRNA and present along the entire 11 base-pair site.

Amino Acid Sequence↗
PubMed1992

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↗
PubMed1995

Human PKR transfected into murine cells stimulates expression of genes under control of the HIV1 or HTLV-I LTR.

We have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the beta-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. beta-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, beta-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate beta-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through elF2 phosphorylation, PKR can also positively stimulate gene expression in vivo, most probably through phosphorylation of a substrate distinct from elF2.

3T3 Cells↗
PubMed1995

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Amino Acid Sequence↗
PubMed1997

Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells.

The vaccinia virus E3L gene codes for double-stranded RNA (dsRNA) binding proteins which can prevent activation of the dsRNA-dependent, interferon-induced protein kinase PKR. Activated PKR has been shown to induce apoptosis in HeLa cells. HeLa cells infected with vaccinia virus with the E3L gene deleted have also been shown to undergo apoptosis, whereas HeLa cells infected with wild-type vaccinia virus do not. In this report, using virus recombinants expressing mutant E3L products or alternative dsRNA binding proteins, we show that suppression of induction of apoptosis correlates with functional binding of proteins to dsRNA. Infection of HeLa cells with ts23, which leads to synthesis of increased dsRNA at restrictive temperature, induced apoptosis at restrictive but not permissive temperatures. Treatment of cells with cytosine arabinoside, which blocks the late buildup of dsRNA in vaccinia virus-infected cells, prevented induction of apoptosis by vaccinia virus with E3L deleted. Cells transfected with dsRNA in the absence of virus infection also underwent apoptosis. These results suggest that dsRNA is a trigger that can initiate a suicide response in virus-infected and perhaps uninfected cells.

Animals↗
PubMed1996

Regulated, stable expression and nuclear presence of reovirus double-stranded RNA-binding protein sigma3 in HeLa cells.

Reovirus genome segment S4 codes for polypeptide sigma3, a major outer capsid component of virions and a double-stranded RNA (dsRNA)-binding protein implicated in viral cytopathogenesis. We have constructed a stable HeLa cell line (S4tTA) that produces functional sigma3 under tetracycline transactivator control. In the absence of tetracycline, S4tTA cells synthesized stable dsRNA-binding sigma3 that accumulated in the nucleus as well as in the cytoplasm. However, in induced S4tTA cells also expressing reovirus outer shell polypeptide mu1/mu1C, migration of sigma3 into the nucleus was blocked, probably as a result of formation of a complex with mu1/mu1C which was exclusively in the cytoplasm. Mutant analyses indicated a correlation between dsRNA-binding activity and nuclear entry of sigma3, suggesting an additional role(s) for this capsid protein in virus-cell interactions.

Amino Acid Sequence↗
PubMedDate not supplied

Two basic motifs of reovirus sigma 3 protein are involved in double-stranded RNA binding.

It has been reported that the sigma 3 protein of reovirus can exert an inhibitory effect on the cellular double-stranded RNA (dsRNA) activated protein kinase. Activation of this kinase is thought to be a general mechanism mediating a cellular antiviral response. This enzyme can also be activated upon transfection, resulting in translational inhibition of plasmid-encoded mRNAs. sigma 3 has an affinity for dsRNA postulated to be responsible for antikinase activity. In the present study, site-directed mutagenesis was performed on two basic regions previously suggested as dsRNA-binding motifs and the mutant sigma 3 proteins were then expressed in COS cells. These experiments revealed that both motifs are involved in sigma 3 attachment to RNA. Expression of the mutants lacking RNA-binding capability is stimulated by coexpression of another dsRNA-binding protein, the E3L vaccinia virus protein. These results support a model in which the attachment to dsRNA is directly responsible for the trans-stimulating effect of sigma 3 on expression of cotransfected genes.

Amino Acid Sequence↗
PubMed1998

Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes.

The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA. PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD. RNA ligands were selected by binding to His6-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches (5'AG3'/3'GA5'), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)2 reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch.

Base Sequence↗