PKR Activation Research Articles

PKR inhibits the DNA damage response, and is associated with poor survival in AML and accelerated leukemia in NHD13 mice

AbstractIncreased expression of the interferon-inducible double-stranded RNA-activated protein kinase (PKR) has been reported in acute leukemia and solid tumors, but the role of PKR has been unclear. Now, our results indicate that high PKR expression in CD34+ cells of acute myeloid leukemia (AML) patients correlates with worse survival and shortened remission duration. Significantly, we find that PKR has a novel and previously unrecognized nuclear function to inhibit DNA damage response signaling and double-strand break repair. Nuclear PKR antagonizes ataxia-telangiectasia mutated (ATM) activation by a mechanism dependent on protein phosphatase 2A activity. Thus, inhibition of PKR expression or activity promotes ATM activation, γ-H2AX formation, and phosphorylation of NBS1 following ionizing irradiation. PKR transgenic but not PKR null mice demonstrate a mutator phenotype characterized by radiation-induced and age-associated genomic instability that was partially reversed by short-term pharmacologic PKR inhibition. Furthermore, the age-associated accumulation of somatic mutations that occurs in the Nup98-HOXD13 (NHD13) mouse model of leukemia progression was significantly elevated by co-expression of a PKR transgene, whereas knockout of PKR expression or pharmacologic inhibition of PKR activity reduced the frequency of spontaneous mutations in vivo. Thus, PKR cooperated with the NHD13 transgene to accelerate leukemia progression and shorten survival. Taken together, these results indicate that increased nuclear PKR has an oncogenic function that promotes the accumulation of potentially deleterious mutations. Thus, PKR inhibition may be a therapeutically useful strategy to prevent leukemia progression or relapse, and improve clinical outcomes.

ID: 32: Roles of A-to-I editing by RNA adenosine deaminase ADAR1 in innate immunity RNA sensing

ADAR1, an interferon inducible adenosine deaminase acting on RNA, catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in RNA with double-stranded (ds) character, leading to genetic recoding and altered RNA structure as I pairs as G instead of A. Adar1 expression occurs from multiple promoters and encodes an IFN-inducible p150 cytoplasmic protein and a constitutively expressed N-terminally truncated p110 nuclear protein. The roles of ADAR1 p150 and p110 in uninfected and virus-infected cells were examined using human cell clones stably deficient in ADAR1, mouse cells genetically deficient in ADAR1, and by complementation strategies. Exemplified by measles virus (MV) infected cells, ADAR1 suppressed activation of PKR and IRF3, suppressed IFN β induction, and protected cells from stress granule formation and cytopathic damage. RNA expression profiles and A-to-I editing were characterized by qPCR and RNA deep sequencing, with differences found between CKO mutant and parental MV infections, and between untreated and IFN-treated uninfected cells. Both non-self (viral) and self (cellular) RNAs with ds character were identified and their editing characterized. Results obtained with mouse cells genetically deficient in either adar1 or the related deaminase adar2, or stat1 or stat2, suggest a central role of ADAR1 in overall A-to-I editing and p150 in the IFN-induced editing and innate immunity RNA sensing. In summary, ADAR1 behaved as an anti-apoptotic host factor that suppressed innate immune activities and was pro-viral. Our findings furthermore implicate a balanced interplay between PKR and ADAR1, two IFN inducible dsRNA binding enzymes, in modulating IFN β protein production and stress responses.

ID: 104: Opposing roles of two dsRNA binding proteins PACT and TRBP on RIG-I induced interferon production

An integral aspect of innate immunity is the ability to detect non-self molecules to initiate antiviral signaling via pattern recognition receptors (PRRs). One such receptor is the RNA helicase RIG-I, which has the ability to detect and be activated by 5’triphosphate uncapped double stranded RNA (dsRNA) as well as the viral mimic dsRNA polyI:C. Once activated, RIG-I’s CARD domains oligomerize and initiate downstream MAVS signaling ultimately inducing interferon (IFN) production. Another dsRNA binding protein PACT, originally identified as the cellular protein activator of PKR, has recently been shown to stimulate RIG-I signaling in response to polyI:C treatment, in part by stimulating RIG-I’s ATPase and helicase activities and resulting in an enhanced induction of IFN. TRBP (TAR-RNA-binding protein), which is about 45% homologous to PACT is known to inhibit PKR signaling by sequestration of PKR and its’ activators, dsRNA and PACT. Despite the domain homology and similar structure of PACT and TRBP, the role of TRBP is yet to be explored in RIG-I like receptor (RLR) signaling. This work focuses on the effect of TRBP on RIG-I signaling and IFN production. Our results indicate that TRBP acts as an inhibitor of RIG-I signaling in a PACT- and PKR-independent manner. This work has major implications on viral susceptibility, disease progression, and antiviral immunity as it demonstrates the regulatory interplay between two dsRNA binding proteins PACT and TRBP on RIG-I mediated IFN production.

Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus.

Double-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from the Poxviridae family, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKR in vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV.IMPORTANCE Our previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR, leading to an inhibition of PKR activation (Y. Y. Tseng, F. Y. Lin, S. F. Cheng, D. Tscharke, S. Chulakasian, C. C. Chou, Y. F. Liu, W. S. Chang, M. L. Wong, and W. L. Hsu, J Virol 89:4966-4979, 2015, doi:10.1128/JVI.03714-14). In the current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to bind PKR and its two activators, dsRNA and PACT. In addition, OV20.0 binds directly to the RNA binding domains (RBDs) of PKR, and this interaction does not require dsRNA. Moreover, OV20.0 interacts with or occupies the RBD2 and the kinase domain of PKR, which then prevents PACT binding to PKR. Finally, OV20.0 associates with PACT via the RBDs, which may reduce the ability of PACT to induce PKR activation. The findings in this study provide new concepts in relation to how ORFV modulates PKR activation.

Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT

PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis.

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser51 phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells.

Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells

Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, and PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV.

IL-24 Induces Apoptosis via Upregulation of RNA-Activated Protein Kinase and Enhances Temozolomide-Induced Apoptosis in Glioma Cells.

Human interleukin-24 (IL-24) has been found recently to play a tumor-suppressor role in a variety of tumors, including gliomas. However, the exact mechanism of glioma tumor suppression by IL-24 remains unclear. We collected by surgery 30 gliomas at different grades and evaluated IL-24 and double-stranded RNA-activated protein kinase (PKR) expression using fluorescence quantitative real-time PCR and immunohistochemical techniques. Two human glioma cell lines, U87 and U251, were transfected with Ad5F35-IL24 via recombinant adenovirus-mediated gene transfer and apoptosis, as well as PKR and eIF-2α expression analyzed. The results showed that IL-24 and PKR expression decreased with increasing tumor grade. Compared with cells of the control groups, Ad5F35-IL24-infected U87 and U251 cells exhibited a significantly increased apoptosis and elevated PKR, eIF-2α, p-PKR, and p-eIF-2α levels, while the expression of Bcl-2 was decreased. Finally, IL-24 also sensitized apoptosis of glioma cells to temozolomide (TMZ). This study indicates that IL-24 upregulates expression and activation of PKR, further increasing expression and activation of eIF-2α, and decreasing Bcl-2 to promote apoptosis. IL-24 also increases chemosensitivity of glioma cells to TMZ.

Effects of Ethanol on the Cerebellum: Advances and Prospects.

Alcohol abuse causes cerebellar dysfunction and cerebellar ataxia is a common feature in alcoholics. Alcohol exposure during development also impacts the cerebellum. Children with fetal alcohol spectrum disorder (FASD) show many symptoms associated specifically with cerebellar deficits. However, the cellular and molecular mechanisms are unclear. This special issue discusses the most recent advances in the study of mechanisms underlying alcoholinduced cerebellar deficits. The alteration in GABAA receptor-dependent neurotransmission is a potential mechanism for ethanol-induced cerebellar dysfunction. Recent advances indicate ethanol-induced increases in GABA release are not only in Purkinje cells (PCs), but also in molecular layer interneurons and granule cells. Ethanol is shown to disrupt the molecular events at the mossy fiber - granule cell - Golgi cell (MGG) synaptic site and granule cell parallel fibers - PCs (GPP) synaptic site, which may be responsible for ethanol-induced cerebellar ataxia. Aging and ethanol may affect the smooth endoplasmic reticulum (SER) of PC dendrites and cause dendritic regression. Ethanol withdrawal causes mitochondrial damage and aberrant gene modifications in the cerebellum. The interaction between these events may result in neuronal degeneration, thereby contributing to motoric deficit. Ethanol activates doublestranded RNA (dsRNA)-activated protein kinase (PKR) and PKR activation is involved ethanolinduced neuroinflammation and neurotoxicity in the developing cerebellum. Ethanol alters the development of cerebellar circuitry following the loss of PCs, which could result in modifications of the structure and function of other brain regions that receive cerebellar inputs. Lastly, choline, an essential nutrient is evaluated for its potential protection against ethanol-induced cerebellar damages. Choline is shown to ameliorate ethanol-induced cerebellar dysfunction when given before ethanol exposure.

PKR activation enhances replication of classical swine fever virus in PK-15 cells

Classical swine fever (CSF) is a highly contagious swine disease that is responsible for economic losses worldwide. Protein kinase R (PK)R is an important protein in the host viral response; however, the role of PKR in CSFV infection remains unknown. This issue was addressed in the present study using the PK-15 swine kidney cell line. We found that CSFV infection increased the phosphorylation of eukaryotic translation initiation factor (eIF)2α and its kinase PKR. However, the expression of viral proteins continued to increase. Furthermore, PKR overexpression enhanced CSFV replication, while PKR inhibition resulted in reduced CSFV replication and an increase in interferon (IFN) induction. In addition, PKR was responsible for eIF2α phosphorylation in CSFV-infected cells. These results suggest that the activation of PKR during CSFV infection is beneficial to the virus. The virus is able to commandeer the host cell's translation machinery for viral protein synthesis while evading innate immune defenses.

An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions.

During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes invitro. The structure of the NPBP/ΔNPNTD complex, solved to3.7Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.

A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2α phosphorylation in obesity.

Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.

Stress granules regulate double-stranded RNA-dependent protein kinase activation through a complex containing G3BP1 and Caprin1.

ABSTRACTStress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition.

Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.

P5.03 - Control of cell fate by the mammalian target of rapamycin (mTOR) under stress depends on eIF2α serine 51 phosphorylation

ABSTRACT The mammalian/mechanist target of rapamycin (mTOR) plays an essential role in cell growth and metabolism by regulating protein and RNA stability, transcription as well as mRNA translation. Phosphorylation of the a subunit of translation initiation factor eIF2 at serine 51 (eIF2aS51P) is a key regulator of mRNA translation and an important determinant of cell fate under stress. Some studies have provided evidence for a cross-talk between mTOR and eIF2aS51P under stress through poorly defined mechanisms. Herein, we demonstrate that genetic as well as pharmacological inhibition of mTOR complex 2 (mTORC2) induces eIF2aS51P via the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK as a result of Akt inactivation. Analysis of the functional interplay between eIF2aS51P and mTOR in tuberous sclerosis complex (TSC)-deficient cells showed that mTORC2 inactivation induces the PERK-eIF2aS51P arm to substitute for the loss of Akt and promote survival in response to ER stress (see Figure above). On the other hand, TSC-deficient cells decrease PERK activity and increase the activity of the double-stranded RNA-activated protein kinase PKR to promote death via eIF2aS51P, JNK activation and increased autophagy in response to oxidative stress (see attached Figure). The balance between PERK inactivation and PKR activation in TSC-mutant cells under oxidative stress is controlled by mTORC1 and ribosomal S6 kinase 1, 2 (S6K1, 2) activity. Impaired eIF2aS51P accelerates TSC-mutant tumor initiation in mice, which is associated with an impaired production of reactive oxygen species (ROS) compared to eIF2aS51P-proficient TSC-mutant tumors. Our study reveals that eIF2aS51P acts as a double-edged sword downstream of mTOR to promote either survival or death in response to distinct forms of stress with implications in tumor development and treatment with chemotherapeutic drugs. Download : Download full-size image P5.03 FIGURES .

Functional analysis of the short isoform of orf virus protein OV20.0.

Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant.

P53-derived host restriction of HIV-1 replication by protein kinase R-mediated Tat phosphorylation and inactivation.

Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53 silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR silencing. In addition, ectopic expression of PKR abolished Tat activity in p53(-/-) and eIF2α(CA) cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62, and S68), which inhibits Tat-mediated provirus transcription in three critical steps: (i) phosphorylation near the arginine-rich motif (ARM) inhibits Tat translocation into the nucleus, (ii) accumulation of Tat phosphorylation abolishes Tat-Tat-responsive region (TAR) binding, and (iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United States. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributable, at least in part, to a noncanonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis. HIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However, the detailed molecular mechanism is still unclear. In the present study, we found that the p53-mediated HIV-1 restriction is attributed to a p53/PKR/Tat-inactivation pathway. HIV-1 infection activated p53, which subsequently induced PKR expression and activation. PKR directly phosphorylated Tat exclusively at five specific Ser/Thr residues, which was accompanied by significant suppression of HIV-1 replication. Accumulation of Tat phosphorylation at these sites inhibited Tat function by blocking Tat nuclear localization, Tat binding to TAR, and Tat-cyclin T1 interaction. Our findings provide a better understanding of the p53-derived host restriction mechanism against HIV-1 replication in AIDS pathogenesis and may contribute to further research focusing on the investigation of potential therapeutic targets for HIV-1.

Dissecting the roles of TRBP and PACT in double-stranded RNA recognition and processing of noncoding RNAs.

HIV TAR RNA‐binding protein (TRBP) and Protein Activator of PKR (PACT) are double‐stranded (ds) RNA‐binding proteins that participate in both small regulatory RNA biogenesis and the response to viral dsRNA. Despite considerable progress toward understanding the structure–function relationship of TRBP and PACT, their specific roles in these seemingly distinct cellular pathways remain unclear. Both proteins are composed of three copies of the double‐stranded RNA‐binding domain, two of which interact with dsRNA, while the C‐terminal copy mediates protein–protein interactions. PACT and TRBP are found in a complex with the endonuclease Dicer and facilitate processing of immature microRNAs. Their precise contribution to the Dicing step has not yet been defined: possibilities include precursor recruitment, rearrangement of dsRNA within the complex, loading the processed microRNA into the RNA‐induced silencing complex, and distinguishing different classes of small dsRNA. TRBP and PACT also interact with the viral dsRNA sensors retinoic acid‐inducible gene I (RIG‐I) and double‐stranded RNA‐activated protein kinase (PKR). Current models suggest that PACT enables RIG‐I to detect a wider range of viral dsRNAs, while TRBP and PACT exert opposing regulatory effects on PKR. Here, the evidence that implicates TRBP and PACT in regulatory RNA processing and viral dsRNA sensing is reviewed and discussed in the context of their molecular structure. The broader implications of a link between microRNA biogenesis and the innate antiviral response pathway are also considered. WIREs RNA 2015, 6:271–289. doi: 10.1002/wrna.1272This article is categorized under: 1RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications2RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms3Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs

Deficient PKR in RAX/PKR Association Ameliorates Ethanol-Induced Neurotoxicity in the Developing Cerebellum.

Ethanol-induced neuronal loss is closely related to the pathogenesis of fetal alcohol spectrum disorders. The cerebellum is one of the brain areas that are most sensitive to ethanol. The mechanism underlying ethanol neurotoxicity remains unclear. Our previous in vitro studies have shown that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) regulates neuronal apoptosis upon ethanol exposure and ethanol activates PKR through association with its intracellular activator RAX. However, the role of PKR and its interaction with RAX in vivo have not been investigated. In the current study, by utilizing N-PKR-/- mice, C57BL/6J mice with a deficient RAX-binding domain in PKR, we determined the critical role of RAX/PKR association in PKR-regulated ethanol neurotoxicity in the developing cerebellum. Our data indicate that while N-PKR-/- mice have a similar BAC profile as wild-type mice, ethanol induces less brain/body mass reduction as well as cerebellar neuronal loss. In addition, ethanol promotes interleukin-1β (IL-1β) secretion, and IL-1β is a master cytokine regulating inflammatory response. Importantly, ethanol-promoted IL-1β secretion is inhibited in the developing cerebellum of N-PKR-/- mice. Thus, RAX/PKR interaction and PKR activation regulate ethanol neurotoxicity in the developing cerebellum, which may involve ethanol-induced neuroinflammation. Further, PKR could be a possible target for pharmacological intervention to prevent or treat fetal alcohol spectrum disorder (FASD).

Direct activation of ribosome-associated double-stranded RNA-dependent protein kinase (PKR) by deoxynivalenol, anisomycin and ricin: a new model for ribotoxic stress response induction.

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR’s interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR.