Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

ISG15 is one of the most strongly induced genes upon viral infection, interferon (IFN) stimulation, and lipopolysaccharide (LPS) stimulation, and only one copy has been found in mammals so far. Here two fish ISG15 genes, termed CaISG15-1 and CaISG15-2, have been cloned and sequenced from UV-inactivated GCHV (grass carp haemorrhagic virus)-infected and IFN-produced CAB cells (crucian carp Carassius auratus blastulae embryonic cells) by suppression subtractive hybridization. The full-length cDNA sequences of two crucian carp ISG15 encode a 155-amino-acid protein and a 161-amino-acid protein, both of which show 78.9% identity overall and possess the characteristic structures of mammalian ISG15 proteins including two tandem ubiquitin-like domains and the C-terminal canonical LRLRGG motif. In CAB cells treated with different stimuli including active virus, UV-inactivated GCHV and IFN containing supernatant (ICS), the expression of both CaISG15-1 and CaISG15-2 was upregulated but displayed different kinetics. Poly I:C and LPS were also able to induce an increase in mRNA for both genes. In CAB cells responsive to active GCHV, UV-inactivated GCHV, CAB ICS, Poly I:C and LPS, CaISG15-1 was upregulated more significantly than CaISG15-2. These results suggest that there are two ISG15 homologues in crucian carp, both of which might play distinct roles in innate immunity against viral and bacterial infection.     

CaPKR-like在鲫鱼与草鱼组织中的表达特性分析

摘要：PKR是由干扰素诱导的最重要的抗病毒蛋白之一,能抑制细胞和病毒蛋白的合成。鲫鱼PKR基因(CaPKR-like)是鱼类第一个,也是非哺乳类脊椎动物第一个报道的PKR全长cDNA序列。本研究制备了CaPKR-like N-端包含Za结构域的多克隆抗体,采用Western blots检验PKR-like在鲫鱼和草鱼组织中的表达特性。Western blots显示在未诱导的鲫鱼和草鱼中该基因表达水平非常低,但是经Poly I:C免疫一周后,在鲫鱼和草鱼等8种组织中有较强的PKR-like蛋白产生。结果暗示：CaPKR-like与哺乳类PKR有相同的组织表达特性。。     

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2alpha. The interaction between PoPKR and eIF2alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.     

Cooperative roles of fish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2α kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2α. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.     

鲫Nub1基因的克隆及特征分析

Nub1(NEDD8 Ultimate Buster-1)是近年来发现的一种干扰素诱导表达基因,过量表达该基因能抑制细胞生长。研究通过RACE-PCR方法从产生干扰素的鲫囊胚培养细胞(Carassius auratus blastulae embryonic cells,CAB)中克隆出鲫Nub1基因。鲫Nub1全长cDNA为2298bp,编码一个由589个氨基酸残基组成的蛋白。推导的鲫NUB1蛋白具有哺乳类同源蛋白保守的结构域,包括N端的UBL结构域和C端两个UBA结构域,与已知的哺乳类包括人和小鼠、鸟类和两栖类的同源蛋白具有41%-45%的相似性。诱导表达分析显示PolyI:C和LPS均能诱导CAB细胞Nub1mRNA的上调,表明Nub1基因在鱼类的抗病免疫反应中发挥某种重要作用。       

Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR.

The protein kinase PKR (dsRNA-dependent protein kinase) phosphorylates the eukaryotic translation initiation factor eIF2alpha to downregulate protein synthesis in virus-infected cells. Two double-stranded RNA binding domains (dsRBDs) in the N-terminal half of PKR are thought to bind the activator double-stranded RNA, mediate dimerization of the protein and target PKR to the ribosome. To investigate further the importance of dimerization for PKR activity, fusion proteins were generated linking the PKR kinase domain to heterologous dimerization domains. Whereas the isolated PKR kinase domain (KD) was non-functional in vivo, expression of a glutathione S-transferase-KD fusion, or co-expression of KD fusions containing the heterodimerization domains of the Xlim-1 and Ldb1 proteins, restored PKR activity in yeast cells. Finally, coumermycin-mediated dimerization of a GyrB-KD fusion protein increased eIF2alpha phosphorylation and inhibited reporter gene translation in mammalian cells. These results demonstrate the critical importance of dimerization for PKR activity in vivo, and suggest that a primary function of double-stranded RNA binding to the dsRBDs of native PKR is to promote dimerization and activation of the kinase domain.     

Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha).

During viral infection, phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) by the interferon-induced RNA-dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double-stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N-terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild-type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2alpha phosphorylation by our purified PKR was increased in response to dsRNA, but not single-stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2alpha kinase activity. Removal of as few as 25 residues at the C-terminal junction of dsRBD2 dramatically increased eIF2alpha kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative-acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2alpha. Heparin, a known in vitro activator of PKR, enhanced eIF2alpha phosphorylation by PKR mutants lacking their entire N-terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative-acting sequences in PKR.     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.     

Conserved intermolecular salt bridge required for activation of protein kinases PKR, GCN2, and PERK

The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2alpha to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg(262) from one protomer and Asp(266) from the second protomer. Interestingly, these two residues are conserved in all eIF2alpha kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.     

Interferons induce tyrosine phosphorylation of the eIF2alpha kinase PKR through activation of Jak1 and Tyk2

The interferon (IFN)-inducible, double-stranded RNA activated protein kinase (PKR) is a dual-specificity kinase, which has an essential role in the regulation of protein synthesis by phosphorylating the translation eukaryotic initiation factor 2 (eIF2). Here, we show the tyrosine (Tyr) phosphorylation of PKR in response to type I or type II IFNs. We show that PKR physically interacts with either Jak1 or Tyk2 in unstimulated cells and that these interactions are increased in IFN-treated cells. We also show that PKR acts as a substrate of activated Jaks, and is phosphorylated at Tyr 101 and Tyr 293 both in vitro and in vivo. Moreover, we provide strong evidence that both the induction of eIF2alpha phosphorylation and inhibition of protein synthesis by IFN are impaired in cells lacking Jak1 or Tyk2, which corresponds to a lack of induction of PKR tyrosine phosphorylation. We conclude that PKR tyrosine phosphorylation provides an important link between IFN signalling and translational control through the regulation of eIF2alpha phosphorylation.     

PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR

The interferon (IFN)-induced, double-stranded (ds)RNA-activated serine-threonine protein kinase, PKR, is a key mediator of the antiviral activities of IFNs. In addition, PKR activity is also involved in regulation of cell proliferation, apoptosis, and signal transduction. In virally infected cells, dsRNA has been shown to bind and activate PKR kinase function. Implication of PKR activity in normal cellular processes has invoked activators other than dsRNA because RNAs with perfectly duplexed regions of sufficient length that are able to activate PKR are absent in cellular RNAs. We have recently reported cloning of PACT, a novel protein activator of PKR. PACT heterodimerizes with PKR and activates it by direct protein-protein interaction. Overexpression of PACT in mammalian cells leads to phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha), the cellular substrate for PKR, and leads to inhibition of protein synthesis. Here, we present evidence that endogenous PACT acts as a protein activator of PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. Following exposure of cells to these stress agents, PACT is phosphorylated and associates with PKR with increased affinity. PACT-mediated activation of PKR leads to enhanced eIF2alpha phosphorylation followed by apoptosis. Based on the results presented here, we propose that PACT is a novel stress-modulated physiological activator of PKR.     

RNase L mediates transient control of the interferon response through modulation of the double-stranded RNA-dependent protein kinase PKR

The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2alpha, eIF2alpha, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2alpha appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses.     

The double-stranded RNA-activated protein kinase PKR is dispensable for regulation of translation initiation in response to either calcium mobilization from the endoplasmic reticulum or essential amino acid starvation

The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR. Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR). However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation. However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity. Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha. Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells). In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells. Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation.     

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.     

Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR

PKR, an interferon (IFN)-inducible protein kinase activated by double-stranded RNA, inhibits translation by phosphorylating the initiation factor eIF2alpha chain. We show that human IFN-gamma mRNA uses local activation of PKR in the cell to control its own translation yield. IFN-gamma mRNA activates PKR through a pseudoknot in its 5' untranslated region. Mutations that impair pseudoknot stability reduce the ability to activate PKR and strongly increase the translation efficiency of IFN-gamma mRNA. Nonphosphorylatable mutant eIF2alpha, knockout of PKR and PKR inhibitors 2-aminopurine, transdominant-negative PKR, or vaccinia E3L correspondingly enhances translation of IFN-gamma mRNA. The potential to form the pseudoknot is phylogenetically conserved. We propose that the RNA pseudoknot acts to adjust translation of IFN-gamma mRNA to the PKR level expressed in the cell.     

Interaction between RAX and PKR modulates the effect of ethanol on protein synthesis and survival of neurons

Ethanol exposure inhibits protein synthesis and causes cell death in the developing central nervous system. The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival. PKR has been well known for its anti-viral response. Upon activation by viral infection or dsRNA, PKR phosphorylates its substrate, the alpha-subunit of eukaryotic translation initiation factor-2 (eIF2alpha) leading to inhibition of translation initiation. It has recently been shown that, in the absence of a virus or dsRNA, PKR can be activated by direct interactions with its protein activators, PACT, or its mouse homologue, RAX. We have demonstrated that exposure to ethanol increased the phosphorylation of PKR and eIF2alpha in the developing cerebellum. The effect of ethanol on PKR/eIF2alpha phosphorylation positively correlated to the expression of PACT/RAX in cultured neuronal cells. Using PKR inhibitors and PKR null mouse fibroblasts, we verified that ethanol-induced eIF2alpha phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2alpha phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2alpha activation. Ethanol promoted PKR and RAX association in cells expressing wild-type RAX but not in cells expressing S18A RAX. S18A RAX functioned as a dominant negative protein and blocked ethanol-induced inhibition of protein synthesis and cell death. Our results suggest that the interactions between PKR and PACT/RAX modulate the effect of ethanol on protein synthesis and cell survival in the central nervous system.