Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2α (eIF2α). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2α, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and λ repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

The major antigenic protein of infectious bursal disease virus, VP2, is an apoptotic inducer.

Infectious bursal disease virus (IBDV) is the causative agent of an economically important poultry disease. Vaccinia virus recombinants expressing the IBDV mature structural capsid proteins VP2 and VP3 were generated by using vectors for inducible gene expression. Characterization of these recombinant viruses demonstrated that expression of VP2 leads to induction of apoptosis in a variety of mammalian cell lines. Transfection of cell cultures with a expression vector containing the VP2 coding region under the control of the immediate-early promoter-enhancer region of human cytomegalovirus also triggers programmed cell death. The apoptotic effect of VP2 is efficiently counteracted by coexpression of the proto-oncogene bcl-2. The results presented demonstrate that VP2 is a bona fide apoptotic inducer. Evaluation of the significance of this finding for the virus life cycle must await further research.     

Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch

Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T=13 lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an amphipathic alpha-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal amphipathic alpha-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the amphipathic alpha-helix in the precursor capsid, as a five-alpha-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.     

Variola virus E3L Zα domain,but not its Z-DNA binding activity,is required for PKR inhibition

Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)–activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.     

Double-Stranded RNA-Activated Protein Kinase (PKR) Is Negatively Regulated by 60S Ribosomal Subunit Protein L18

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2α), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2α in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2α phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.     

Specific Inhibition of the PKR-Mediated Antiviral Response by the Murine Cytomegalovirus Proteins m142 and m143

Double-stranded RNA (dsRNA) produced during viral infection activates several cellular antiviral responses. Among the best characterized is the shutoff of protein synthesis mediated by the dsRNA-dependent protein kinase (PKR) and the oligoadenylate synthetase (OAS)/RNase L system. As viral replication depends on protein synthesis, many viruses have evolved mechanisms for counteracting the PKR and OAS/RNase L pathways. The murine cytomegalovirus (MCMV) proteins m142 and m143 have been characterized as dsRNA binding proteins that inhibit PKR activation, phosphorylation of the translation initiation factor eIF2α, and a subsequent protein synthesis shutoff. In the present study we analyzed the contribution of the PKR- and the OAS-dependent pathways to the control of MCMV replication in the absence or presence of m142 and m143. We show that the induction of eIF2α phosphorylation during infection with an m142- and m143-deficient MCMV is specifically mediated by PKR, not by the related eIF2α kinases PERK or GCN2. PKR antagonists of vaccinia virus (E3L) or herpes simplex virus (γ34.5) rescued the replication defect of an MCMV strain with deletions of both m142 and m143. Moreover, m142 and m143 bound to each other and interacted with PKR. By contrast, an activation of the OAS/RNase L pathway by MCMV was not detected in the presence or absence of m142 and m143, suggesting that these viral proteins have little or no influence on this pathway. Consistently, an m142- and m143-deficient MCMV strain replicated to high titers in fibroblasts lacking PKR but did not replicate in cells lacking RNase L. Hence, the PKR-mediated antiviral response is responsible for the essentiality of m142 and m143.     

Host proteolytic activity is necessary for infectious bursal disease virus capsid protein assembly

In many viruses, a precursor particle, or procapsid, is assembled and undergoes massive chemical and physical modification to produce the infectious capsid. Capsid assembly and maturation are finely tuned processes in which viral and host factors participate. We show that the precursor of the VP2 capsid protein (pVP2) of the infectious bursal disease virus (IBDV), a double-stranded RNA virus, is processed at the C-terminal domain (CTD) by a host protease, the puromycin-sensitive aminopeptidase (PurSA). The pVP2 CTD (71 residues) has an important role in determining the various conformations of VP2 (441 residues) that build the T = 13 complex capsid. pVP2 CTD activity is controlled by co- and posttranslational proteolytic modifications of different targets by the VP4 viral protease and by VP2 itself to yield the mature VP2-441 species. Puromycin-sensitive aminopeptidase is responsible for the peptidase activity that cleaves the Arg-452-Arg-453 bond to generate the intermediate pVP2-452 polypeptide. A pVP2 R453A substitution abrogates PurSA activity. We used a baculovirus-based system to express the IBDV polyprotein in insect cells and found inefficient formation of virus-like particles similar to IBDV virions, which correlates with the absence of puromycin-sensitive aminopeptidase in these cells. Virus-like particle assembly was nonetheless rescued efficiently by coexpression of chicken PurSA or pVP2-452 protein. Silencing or pharmacological inhibition of puromycin-sensitive aminopeptidase activity in cell lines permissive for IBDV replication caused a major blockade in assembly and/or maturation of infectious IBDV particles, as virus yields were reduced markedly. PurSA activity is thus essential for IBDV replication.     

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.     

A new double-stranded RNA-binding protein that interacts with PKR

We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein. p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA. Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members. Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas heterodimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity. We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R). While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression. These transformants could be rescued by overexpression of the α-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2α), one of the known substrates for PKR. We hypothesize that competing heterodimers between p74–K296R PKR and eIF2α–K296R PKR may control cell growth such that stabilization of the p74–K296R PKR heterodimer induces abnormal morphology and cell death.     

Activation of the Double-stranded RNA-dependent Protein Kinase PKR by Small Ubiquitin-like Modifier (SUMO)

The dsRNA-dependent kinase PKR is an interferon-inducible protein with ability to phosphorylate the α subunit of the eukaryotic initiation factor (eIF)-2 complex, resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. Here we analyzed the modification of PKR by the small ubiquitin-like modifiers SUMO1 and SUMO2 and evaluated the consequences of PKR SUMOylation. Our results indicate that PKR is modified by both SUMO1 and SUMO2, in vitro and in vivo. We identified lysine residues Lys-60, Lys-150, and Lys-440 as SUMOylation sites in PKR. We show that SUMO is required for efficient PKR-dsRNA binding, PKR dimerization, and eIF2α phosphorylation. Furthermore, we demonstrate that SUMO potentiates the inhibition of protein synthesis induced by PKR in response to dsRNA, whereas a PKR SUMOylation mutant is impaired in its ability to inhibit protein synthesis and shows reduced capability to control vesicular stomatitis virus replication and to induce apoptosis in response to vesicular stomatitis virus infection. In summary, our data demonstrate the important role of SUMO in processes mediated by the activation of PKR.     

Mutant Influenza Viruses with a Defective NS1 Protein Cannot Block the Activation of PKR in Infected Cells

A short model genome RNA and also the genome RNA of influenza A virus bearing both 5′- and 3′-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2α). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2α phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2α.     

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.     

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.     

Nck-1 interacts with PKR and modulates its activation by dsRNA

Activation of the double-stranded RNA (dsRNA)-activated protein kinase PKR results in inhibition of general translation through phosphorylation of the eukaryotic initiation factor 2 alpha-subunit on serine 51 (eIF2αSer51). Previously, we have reported that the adaptor protein Nck-1 modulates eIF2αSer51 phosphorylation by a subset of eIF2α kinases, including PKR. Herein, we demonstrate that Nck-1 prevents efficient activation of PKR by dsRNA, revealing that Nck-1 acts at the level of PKR. In agreement, Nck-1 impairs p38MAPK activation and attenuates cell death induced by dsRNA, in addition to diminish eIF2αSer51 phosphorylation. Our data show that the inhibitory effect of Nck-1 on PKR is reversible, as it could be overcome by increasing levels of dsRNA. Interestingly, we found that Nck-1 interacts with the inactive form of PKR, independently of its Src homology domains. Furthermore, we uncovered that Nck-1 is substrate of PKR in vitro. All together, our data provide the first evidence identifying Nck-1 as a novel endogenous regulator of PKR and support the notion that Nck-1-PKR interaction could be a way to limit PKR activation.     

Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L⁻C7L⁻) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L⁻C7L⁻ at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L⁻C7L⁻ fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.Vaccinia virus (VACV) is the prototypical member of the poxvirus family of large, complex, double-stranded DNA viruses (21). VACV has a very broad host range and is capable of infecting many vertebrate animal species. Its host range, however, can be significantly narrowed by deleting from its genome some of the so-called host range genes, the most important of which are E3L, K1L, and C7L (17). VACV mutants deleted of E3L (ΔE3L) or both K1L and C7L (ΔK1LΔC7L) replicate abortively and express only a subset of viral genes in most mammalian cell lines (3, 24). These mutants are highly attenuated in animal hosts but are capable of eliciting immune responses, making them attractive vaccine vectors for infectious diseases and cancers (27, 28). NYVAC, a VACV strain derived through deletion of 18 genes, including both K1L and C7L (27), has been used as the vector for an AIDS vaccine (2).The functions of E3L and the host factors that restrict the replication of the ΔE3L mutant have been studied extensively. E3L encodes a 20-kDa and a 25-kDa protein that bind double-stranded RNA (dsRNA) and Z form DNA (6, 15). The E3 proteins antagonize the dsRNA-dependent protein kinase PKR (5), which exists as an inactive form in the cells and undergoes autophosphorylation and activation upon binding to dsRNA. The activated PKR phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in a block in protein translation at the initiation step. The infection of most mammalian cells by the ΔE3L mutant leads to the activation of PKR and a block in translating viral mRNAs (16). The replication of the ΔE3L mutant in nonpermissive HeLa cells can be rescued by silencing PKR expression (32), while its replication in permissive Huh7 (human hepatoma) cells can be blocked by upregulating PKR expression with interferon (IFN) treatment (1). In addition to affecting PKR, E3 has also been shown to inactivate IFN-stimulated gene 15 (ISG15) (14), another IFN effector that plays a role in host defense against VACV.Like the replication of the ΔE3L mutant, the replication of the ΔK1LΔC7L mutant in nonpermissive HeLa cells is blocked at the translation of viral mRNA. However, the host factors that restrict the replication of the ΔK1LΔC7L mutant and the molecular functions of K1L or C7L remain a mystery. K1 and C7 share no amino acid sequence homology, but either K1L or C7L can complement the replication defect of the ΔK1LΔC7L mutant in most cell lines. The exception is rabbit RK13 cells, where K1L but not C7L can complement (24). K1L is present in only a few orthopoxviruses, while C7L or a functional homologue of C7L is present in almost all mammalian poxviruses (18). K1 comprises multiple ankyrin repeats, a protein motif that is involved in protein-ligand interaction. It was shown to prevent the degradation of IκBα and to thus inhibit host NF-κB activation in RK13 cells (25). C7L has no homologue outside the poxvirus family, and its molecular function remains unknown. It may play a role in inhibiting cellular apoptosis in response to VACV infection (23).As E3L supports VACV replication by antagonizing innate antiviral pathways that are inducible by IFNs, we hypothesize that K1L and C7L might function similarly by antagonizing IFN effectors. In the current study, we tested the hypothesis and found that both K1L and C7L can antagonize antiviral activities induced by type I IFNs. We found that K1L and C7L do not antagonize IFN by inhibiting IFN signaling or PKR activation, demonstrating that K1L and C7L are novel IFN antagonists functioning differently than previously identified IFN antagonists in VACV. Furthermore, we tested a panel of K1L mutant viruses and showed that K1L''s functions of regulating VACV host range and of antagonizing IFN are genetically nonsegregable, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.