Interferon Action and the Double‐Stranded RNA‐Dependent Enzymes ADAR1 Adenosine Deaminase and PKR Protein Kinase

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

Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA

We studied the involvement of interferon-regulated, PKR on 2-ME-mediated actions in human osteosarcoma cells. Our results show that PKR is activated by 2-ME treatment and is necessary for 2-ME-mediated induction of osteosarcoma cell death. Osteosarcoma is the most common primary bone tumor and most frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a metabolite of 17beta-estradiol, induces interferon gene expression and apoptosis in human osteosarcoma cells. In this report, we studied the role of interferon-regulated double-stranded (ds)RNA-dependent protein kinase (PKR) protein on 2-ME-mediated cell death in human osteosarcoma cells. Western blot analyses were used to measure PKR protein and phosphorylation levels. Cell survival and apoptosis assays were measured using trypan blue exclusion and Hoechst dye methods, respectively. A transient transfection protocol was used to express the dominant negative PKR mutants. PKR was increased in 2-ME-treated MG63 cells, whereas 17beta-estradiol, 4-hydroxyestradiol, and 16alpha-hydroxyestradiol, which do not induce cell death, had no effect on PKR protein levels. Also, 2-ME treatment induced PKR kinase activity as indicated by increased autophosphorylation and phosphorylation of the endogenous substrate, eukaryotic initiation factor (eIF)-2alpha. dsRNA poly (I).poly (C), an activator of PKR protein, increased cell death when osteosarcoma cells were treated with a submaximal concentration of 2-ME. In contrast, a serine-threonine kinase inhibitor SB203580 and a specific PKR inhibitor 2-aminopurine (2-AP) blocked the 2-ME-induced cell death in MG63 cells. A dominant negative PKR mutant protein conferred resistance to 2-ME-induced cell death to MG63 osteosarcoma and 2-ME-mediated PKR regulation did not require interferon gene expression. PKR protein is activated in cell free extracts by 2-ME treatment, resulting in autophosphorylation and in the phosphorylation of the substrate eIF-2alpha. We conclude from these results that PKR is regulated by 2-ME independently of interferon and is essential for 2-ME-mediated cell death in MG63 osteosarcoma cells.

METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection.

Double-stranded RNA-dependent protein kinase R (PKR) has been implicated in anti-viral (antitumor) and apoptotic responses. PKR is activated by extracellular stresses and phosphorylates the alpha subunit of protein synthesis initiation factor eIF2, thereby inhibiting protein synthesis and impeding virus multiplication. Phosphorylation of eIF2alpha in mammalian cells has been shown to be increased after ultraviolet (UV) stress and to be required for UV-induced repression of protein translation. UVA is an important etiological factor in skin carcinogenesis and we observed that UVA induced phosphorylation of PKR (Thr(451)) and eIF2alpha (Ser(51)) in mouse skin epidermal JB6 Cl41 cells. The induction was suppressed by the MEK1 inhibitor, PD 98059. UVA stimulation of PKR and eIF2alpha phosphorylation was also inhibited by a dominant-negative mutant (DNM) of ERK2- or RSK2-deficient cells (RSK2(-)). An inhibitor of p38, SB 202190 or a DNM of p38alpha kinase (DNM-p38alpha) suppressed UVA-induced phosphorylation of eIF2alpha (Ser(51)) but had no effect on phosphorylation of PKR (Thr(451)). Our data indicated that phosphorylation of PKR at Thr(451) is mediated through ERK2 and RSK2, but not through p38 kinase, and is involved in the regulation of Ser(51) phosphorylation of eIF2alpha in UVA-irradiated JB6 cells. In vitro and in vivo kinase assays indicated that phosphorylation of eIF2alpha at Ser(51) occurred indirectly through ERK2, RSK2 or p38 kinase in the cellular response to UVA. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer.

The murine 200 family proteins p202a, p202b, and p204, and also RNA-dependent protein kinase (PKR) are inducible by interferons (IFNs). p202a, p202b, and p204 modulate the activity of a large variety of transcription factors and also are involved in muscle differentiation. PKR is a multifunctional serine/threonine kinase, which is involved in antiviral defense and cell growth control and in the response to various stress signals. We reported earlier that the level of p204 increases during cultured C2C12 myoblast differentiation to myotubes in consequence of transactivation by the skeletal muscle-specific MyoD protein. The levels of p202a, p202b, and PKR also increase during the differentiation. We report here that these increased protein levels also are due to the transactivation of their genes by MyoD. This is made possible by the occurrence in each of these genes of at least six E boxes, which are recognition sites for MyoD. We also show that the distribution of the p204, p202a, p202b, and PKR proteins in five tissues of adult C129 mice is the same in wild-type mice and mice lacking the IFN-alpha, IFN-beta, and IFN-gamma receptors. This indicates that the synthesis and distribution of these proteins in uninfected adult mice are not affected by endogenous IFNs.

While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.

Interaction of the Human Protein Kinase PKR with the Mouse PKR Homolog Occurs via the N-Terminal Region of PKR and Does Not Inactivate Autophosphorylation Activity of Mouse PKR

Role of the RNA-dependent protein kinase in the regulated expression of genes in transfected cells

RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

The protein kinase activity dependent on double-stranded (ds)RNA is enhanced in mouse and human cells following treatment with interferon. This kinase activity is manifested by the phosphorylation of (a) a 65,000–67,000-Mr protein (p65) in mouse cells or a 68,000–72,000-Mr protein (p68) in human cells; (b) a 35,000-Mr protein which is the α -subunit of protein synthesis initiation factor eIF2; and (c) added calf thymus histones (HIIA). This kinase activity is independent of cyclic AMP or cyclic GMP and is markedly stimulated by Mn. It phosphorylates p65 and p68 by their serine and threonine residues. The role of such protein kinase activity is considered to be the phosphorylation of the α -subunit of eIF2, thus mediating inhibition of the initiation of protein synthesis in cell-free system (for references see 1). The phosphorylation of p65 and p68 has been shown during virus infection in mouse and human cells, respectively (2–4). The enhanced phosphorylation of eIF2 has also been reported in interferon-treated cells infected with reovirus or encephalomyocarditis virus (4,5). Recent data in cells infected with adenovirus suggests that the dsRNA-dependent protein kinase plays a decisive role in the control of cellular and viral Referencesprotein synthesis (6).

Activation of the interferon-inducible, double-stranded RNA-dependent protein kinase was monitored in monolayer cultures of control and interferon-treated HeLa cells infected with encephalomyocarditis virus. The extent of phosphorylation in the intact cell of the alpha-subunit of eucaryotic protein synthesis initiation factor eIF2 by the kinase was determined for the first time in this type of system, using a two-dimensional immunoblot technique. Virus protein synthesis and the kinetics of activation of the ppp(A2'p)nA (n greater than or equal to 2) system were analyzed in parallel. Enhanced phosphorylation of eIF2-alpha was obvious at 9 h and increased by 12 h postinfection. ppp(A2'p)nA and ppp(A2'p)nA-mediated rRNA cleavage were observed from 6 h. No viral protein synthesis was detected in cells in which a general inhibition of protein synthesis developed with time. It can be concluded that both the kinase and ppp(A2'p)nA system are active in interferon-treated, encephalomyocarditis virus-infected HeLa cells.

Characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.