Semliki forest virus capsid protein inhibits the initiation of translation by upregulating the double-stranded RNA-activated protein kinase (PKR)

We investigated the possible translational role which elevated concentrations of highly purified Semliki Forest virus (SFV) capsid (C)-protein molecules may play in a cell-free translation system. Here we decomonstrate that in the absence of double-stranded RNA high concentrations of C protein triggered the phosphorylation of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. Activated PKR in turn phosphorylated its natural substrate, the subunit of eukaryotic initiation factor 2 (eIF-2), thereby inhibiting initiation of host cell translation. These findings were further strengthened by experiments showing that during natural infection with SFV the maximum phosphorylation of PKR coincided with the maximum synthesis of C protein 4–9 hours post infection. Thus, our results demonstrate that high concentrations of C-protein molecules may act in a hitherto novel mechanism on PKR to inhibit host cell protein synthesis during viral 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.     

Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

Background  

Double-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far.     

Three leucine-rich sequences and the N-terminal region of double-stranded RNA-activated protein kinase (PKR) are responsible for its cytoplasmic localization

The double-stranded RNA-activated-protein kinase PKR was originally identified as a ribosomal protein that regulates protein synthesis at the translational level. While PKR locates predominantly to the cytoplasm, nuclear or nucleolar species of PKR have been detected. Here, we demonstrate that PKR possesses three leucine-rich sequences resembling nuclear export signals (NESs). Enhanced green fluorescent protein (EGFP) fused to one of these sequences and transfected in COS-1 cells exhibited predominant cytoplasmic staining, which was abrogated by a leucine to alanine substitution. In addition, Leptomycin B (LMB), an inhibitor of NES-mediated nuclear export, inhibited the cytoplasmic localization of EGFP-NES, indicating the potential activity of these stretches as NESs. Although EGFP fused to a PKR with three NES mutations still located to the cytoplasm, an additional N-terminal deletion impaired the cytoplasmic predominance, suggesting that the N-terminal region is also required for localization. These results suggest that the cytoplasmic localization of PKR is regulated by NESs as well as the N-terminal sequence.     

Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation.

Recently we reported that introduction of catalytically inactive PKR molecules into NIH 3T3 cells causes malignant transformation and the development of tumors in nude mice. We have proposed that PKR may be a tumor suppressor gene possibly because of its translational inhibitory properties. We have now designed and characterized a number of PKR mutants encoding proteins that retain their catalytic competence but are mutated in their regulatory double-stranded RNA (dsRNA) binding domains (RBDs). RNA binding analysis revealed that PKR proteins either lacking or with point mutations in the first RBD (RBD-1) bound negligible amounts of dsRNA activator or adenovirus VAI RNA inhibitor. Despite the lack of binding, such variants remained functionally competent but were much less active than wild-type PKR. PKR variants completely lacking RBD-1 were largely unresponsive to dsRNA in activation assays but could be activated by heparin. To complement these studies, we evaluated the effects of point mutations in RBD-1 or the removal of either RBD-1 or RBD-2 on the proliferation rate of mouse 3T3 cells. We were unsuccessful at isolating stably transformed cells expressing RBD-1 point mutants or RBD-2-minus mutants. In contrast, NIH 3T3 cells, which constitutively expressed PKR proteins that lacked RBD-1, were selected. These cells displayed a transformed phenotype and caused tumors after inoculation in nude mice. Further, levels of endogenous eIF-2 alpha phosphorylation in RBD-1-minus cell lines were reduced, suggesting that such mutants act in a dominant negative manner to inhibit the function of endogenous PKR. These results emphasize the importance of RBD-1 in PKR control of cell growth and provide additional evidence for the critical role played by PKR in the regulation of malignant transformation.     

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.     

The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins.

PKR is a serine/threonine protein kinase induced by interferon treatment and activated by double-stranded RNAs. As a result of activation, PKR becomes autophosphorylated and catalyzes phosphorylation of the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2). While studying the regulation of PKR in virus-infected cells, we found that a cellular 58-kDa protein (P58) was recruited by influenza virus to downregulate PKR and thus avoid the kinase's deleterious effects on viral protein synthesis and replication. We now report on the cloning, sequencing, expression, and structural analysis of the P58 PKR inhibitor, a 504-amino-acid hydrophilic protein. P58, expressed as a histidine fusion protein in Escherichia coli, blocked both the autophosphorylation of PKR and phosphorylation of the alpha subunit of eIF-2. Western blot (immunoblot) analysis showed that P58 is present not only in bovine cells but also in human, monkey, and mouse cells, suggesting the protein is highly conserved. Computer analysis revealed that P58 contains regions of homology to the DnaJ family of proteins and a much lesser degree of similarity to the PKR natural substrate, eIF-2 alpha. Finally, P58 contains nine tandemly arranged 34-amino-acid repeats, demonstrating that the PKR inhibitor is a member of the tetratricopeptide repeat family of proteins, the only member identified thus far with a known biochemical function.     

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.     

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.     

Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA.

The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1. These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system. Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR. A second Epstein-Barr virus RNA, EBER-2, also regulates PKR. EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by U.V. crosslinking experiments and filter binding assays. The affinities of all three RNAs for PKR in vitro are similar (Kd = ca. 0.3 nM). Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus.     

Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s).     

Mechanism of interaction of the double-stranded RNA (dsRNA) binding domain of protein kinase R with short dsRNA sequences

The dsRNA-activated protein kinase (PKR) plays a major role in the cellular response to viral infection. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. The dsRBD consists of two tandem copies of a conserved double-stranded RNA binding motif, dsRBM1 and dsRBM2. dsRNA binding is believed to activate PKR by inducing dimerization and subsequent autophosphorylation reactions. We have characterized the function of the dsRBD by assessing the binding of dsRBM1 and dsRBD to a series of dsRNA sequences ranging from 15 to 45 bp. For dsRBM1, the binding stoichiometries agree with an overlapping ligand binding model where the motif binds to multiple faces of the dsRNA duplex and overlaps along the helical axis. Similar behavior is observed for a dsRBD containing both dsRBM1 and dsRBM2 for sequences up to 30 bp; however, the binding affinity is enhanced 30-fold. Longer dsRNA sequences exhibit lower-than-expected stoichiometries, indicating a change in binding mode. NMR spectroscopy was used to define the regions of the dsRBD that interact with dsRNA. dsRNA binding induces exchange broadening of cross-peaks in 1H-15N HSQC spectra. For a 20 bp dsRNA, the resonances most affected map to the known dsRNA binding regions of dsRBM1 as well as the N-terminus of dsRBM2. For a longer 40 bp sequence, additional regions of dsRBM2 exhibit enhanced broadening. These data support a model in which dsRBM1 plays the dominant role in binding short dsRNA sequences and dsRBM2 makes additional interactions with the longer sequences capable of activating PKR.     

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.     

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.