Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA.

The protein kinase DAI, the double-stranded RNA activated inhibitor of translation (also known as PKR), regulates cell growth, virus infection, and other processes. DAI represents a class of proteins containing a recently recognized RNA binding motif, the dsRBM, but little is known about the contacts between these proteins and their RNA ligands. In adenovirus-infected cells, DAI activation is prevented by VA RNAI, a highly structured RNA that binds to the kinase. VA RNA contains three chief structural features: a terminal stem, an apical stem-loop, and a complex central domain. We used enzymatic and chemical footprinting to identify the interactions between DAI and VA RNAI. DAI protects the proximal part of the apical stem structure, an adjacent region in the central domain, and a region surrounding a conserved stem in the central domain from nuclease attack. During binding the RNA undergoes a conformational change that is mainly restricted to the central domain. A similar change is induced by magnesium ions alone. Footprinting and interference binding assays using base-specific chemical probes suggest that the protein does not make major contacts with RNA bases. On the other hand, footprinting with probes specific for the RNA backbone shows that DAI engages in a strong interaction with the minor groove of the apical stem and a weaker interaction in the central domain. A truncated form of DAI, p20, containing only the RNA binding domain, gives a similar protection pattern in the apical stem but protects the central domain less effectively. We conclude that the RNA binding domain of DAI interacts directly with the apical stem and central domain of VA RNA, and that other regions of the protein contribute to interactions with the central domain.     

Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI.

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.     

Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI.

The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.     

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.     

Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI.

The double-stranded RNA activated protein kinase DAI contains an RNA binding domain consisting of two copies of a double-stranded RNA binding motif. We have investigated the role of RNA structure in the interaction between DAI and the structured single-stranded RNA, adenovirus VA RNAI, which inhibits DAI activation. Mutations in the apical stem, terminal stem, and central domain of the RNA were tested to assess the contribution of these elements to DAI binding in vitro. The data demonstrate that over half a turn of intact apical stem is required for the interaction and that there is a correlation between the binding of apical stem mutants and their ability to function both in vivo and in vitro. There was also evidence of preference for GC-rich sequence in the proximal region of the apical stem. In the central domain the correlation between binding and function of mutant RNAs was poor, suggesting that at least some of this region plays no direct role in binding to DAI, despite its functional importance. Exceptionally, central domain mutations that encroached on the phylogenetically conserved stem 4 of VA RNA disrupted binding, and complementary mutations in this sequence partially restored binding. Measurement of the binding of wild-type VA RNAI to DAI and p20, a truncated form of the protein containing the RNA binding domains alone, under various ionic conditions imply that the major interactions are electrostatic and occur via the protein's RNA binding domain. However, differences between full-length DAI and p20 in their binding to mutants in the conserved stem suggest that regions outside the RNA binding domain also participate in the binding. The additional interactions are likely to be non-ionic, and may be important for preventing DAI activation during virus infection.     

The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase

The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.     

Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.

The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.     

Viral evasion of cellular defense mechanisms: regulation of the protein kinase DAI by RNA effectors

The protein kinase DAI (the double-stranded RNA activated inhibitor) plays a critical role in controlling translation in uninfected and infected cells. It is induced by interferon as part of the cellular antiviral defense mechanism. When activated, it leads to a blockade of polypeptide chain initiation, thereby interrupting viral propagation. Many viruses have developed means to neutralize the action of DAI: the sheer profusion of these countermeasures makes it clear that DAI is an important component of the host antiviral defense mechanism and a serious concern to the viruses. This chapter focusses on the mechanism of DAI activation and its inhibition by virus-encoded RNA antidotes.     

Functional expression and characterization of the interferon-induced double-stranded RNA activated P68 protein kinase from Escherichia coli.

The P68 protein (referred to as P68 on the basis of its molecular weight of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by double-stranded (ds) RNAs. Although extensively studied, little is currently known about the regulation of kinase function at the molecular level. What is known is that activation of this enzyme triggers a series of events which lead to an inhibition of protein synthesis initiation and may, in turn, play an integral role in the antiviral response to interferon. To begin to understand P68 and its biological functions in the eukaryotic cell, we have expressed the protein kinase in Escherichia coli under control of the bacteriophage T7 promoter. In rifampicin-treated cells, metabolically labeled with [35S]methionine and induced by IPTG, the P68 kinase was the predominant labeled product. Further, P68 was recovered from extracts as a fully functional enzyme, shown by its ability to become activated and phosphorylate its natural substrate, the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). Moreover, P68 was phosphorylated in vivo in E. coli, providing conclusive evidence that the kinase has the capacity to phosphorylate and activate itself in the absence of other eukaryotic proteins. In contrast, a mutant P68 protein, containing a single amino acid substitution in the invariant lysine in catalytic domain II, was completely inactive. Interestingly, both the mutant and wild-type protein kinases efficiently bound activator dsRNAs despite the fact that only the latter was activated by these RNAs. Finally, the expressed kinase could be isolated from contaminating E. coli proteins in an active form by immunoaffinity chromatography with a monoclonal antibody specific for P68.     

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.     

Homology between double-stranded RNA and nuclear DNA of yeast.

The relationship between mycoviral double-stranded (ds) RNA and host cell DNA was investigated. Radiolabeled ds RNA was denatured and reannealed in the presence and absence of denatured DNA. RNA from killer strains of the yeast Saccharomyces cerevisiae and from nonkiller derivatives was utilized. The above-mentioned strains, as well as one that lacks all ds RNA, were sources for extracted DNA. Net hybridization of ds RNA to DNA occurred regardless of the strains from which the respective nucleic acids were prepared.     

Interaction between the adenovirus DNA-binding protein and double-stranded DNA.

DNA-binding protein was characterized by previous investigators as a single-stranded DNA-binding protein analogous to the gene 32 protein of phage T4 (Van der Vliet &; Levine, 1973; Sugawara et al., 1977). In the studies presented here the interactions between natural and synthetic polynucleotides and the DNA-binding protein of adenovirus 2-infected HeLa cells have been examined. Polynucleotide melting techniques revealed a tight yet dissociable binding to the helix structure of double-stranded DNA. In addition, binding and filter binding competition experiments at high DNA to protein ratios revealed a specific binding to double-stranded DNA termini with a dissociation constant of 1 × 10^?9 to 2 × 10^?9m. The ability of DNA-binding protein to bind to heat-denatured viral DNA was confirmed but the binding to double-stranded DNA termini was more specific on a molar basis. DNA-binding protein can recognize both flush and staggered ends of double-stranded DNA molecules.     

Induction of interferon in HeLa cells of a protein kinase activated by double-stranded RNA

Treatment of HeLa cells with human fibroblast interferon results in increased levels of latent protein kinase activity that can be activated by double-stranded RNA (dsRNA). The protein kinase activity in extracts of interferon-treated cells is assayed by two methods: (a) inhibition of protein synthesis in rabbit reticulocyte lysates and (b) phosphorylation of two polypeptides of Mr 72000 (P1) and 38000 (the eIF-2 alpha subunit of initiation factor 2). When extracts of interferon-treated cells are fractionated by centrifugation at 150000 x g, the protein kinase activity is found in the pellet fraction. The kinase is maximally activated by 0.1 micrograms/ml poly(I) . poly(C). An increase in protein kinase activity is detected after 8 h of treatment with 100 units interferon/ml or after a 17-h treatment with 12.5 units/ml or greater interferon concentrations. Therefore, the kinase activity increases as a function of both time of treatment and interferon concentration. Addition of actinomycin D to cells during interferon treatment prevents this increase. The protein kinase activity decreases gradually over three days when interferon-treated cells are subsequently grown in the absence of interferon.     

Functional characterization of and cooperation between the double-stranded RNA-binding motifs of the protein kinase PKR

The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus. The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1). Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs. dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20. However, dsRBM2 does not enhance the annealing activity of dsRBM1. This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs.     

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.