Induction of interferon in HeLa cells of a protein kinase activated by double-stranded RNA

Treatment of HeLa cells with human fibroblast interferon results in increased levels of latent protein kinase activity that can be activated by double-stranded RNA (dsRNA). The protein kinase activity in extracts of interferon-treated cells is assayed by two methods: (a) inhibition of protein synthesis in rabbit reticulocyte lysates and (b) phosphorylation of two polypeptides of Mr 72000 (P1) and 38000 (the eIF-2 alpha subunit of initiation factor 2). When extracts of interferon-treated cells are fractionated by centrifugation at 150000 x g, the protein kinase activity is found in the pellet fraction. The kinase is maximally activated by 0.1 micrograms/ml poly(I) . poly(C). An increase in protein kinase activity is detected after 8 h of treatment with 100 units interferon/ml or after a 17-h treatment with 12.5 units/ml or greater interferon concentrations. Therefore, the kinase activity increases as a function of both time of treatment and interferon concentration. Addition of actinomycin D to cells during interferon treatment prevents this increase. The protein kinase activity decreases gradually over three days when interferon-treated cells are subsequently grown in the absence of interferon.     

Protein kinase PKR plays a stimulus- and virus-dependent role in apoptotic death and virus multiplication in human cells

The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-alpha)-induced apoptosis was comparable between the HeLa lines. NF-kappaB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-alpha. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype.     

A kinase able to phosphorylate exogenous protein synthesis initiation factor eIF-2 alpha is present in lysates of mengovirus-infected L cells.

Infection of mouse L929 cells by mengovirus resulted in the expression of a kinase activity that selectively phosphorylated the small, 38,000-molecular-weight subunit of eucaryotic initiation factor 2 and histone H2. This kinase activity was independent of host cell RNA synthesis and was located in the postribosomal supernatant (S-100 fraction) early after infection (up to 3 h). At later times after infection (5 h), kinase activity was also associated with the polysome fraction. The kinase present in the S-100 fraction bound strongly to DEAE-cellulose, its peak activity eluting at 0.5 M KCl. Kinase activity was independent of the presence of exogenous double-stranded RNA, and KCl at concentrations greater than 0.1 M inhibited eucaryotic initiation factor 2 phosphorylation. The 67,000-molecular-weight phosphoprotein activated in interferon-treated cells by double-stranded RNA was not detected by standard phosphorylation assays in lysates from mengovirus-infected cells. Labeling of this protein in vivo during 5 h of infection was also not detected. The DEAE-cellulose-purified mengovirus kinase inhibited protein synthesis in reticulocyte lysates, and the inhibition was not reversible by high concentrations of poly(I).poly(C).     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

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.     

A heat-sensitive inhibitor in poliovirus-infected cells which selectively blocks phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase.

We partially purified an inhibitor from poliovirus-infected HeLa cells which specifically blocked phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase. The inhibitory activity eluted from a sizing column with an approximate molecular weight of 80,000 to 100,000 and was sensitive to heat, suggesting a protein nature for the inhibitor. No specific virus-encoded protein purified with the inhibitor. The inhibition of phosphorylation of the alpha subunit of eucaryotic initiation factor 2 was not due to a protein phosphatase associated with the inhibitor. The inhibitor did not seem to prevent phosphorylation of the double-stranded RNA-activated protein kinase but inhibited the phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the activated kinase. Double-stranded RNA-induced inhibition of in vitro protein synthesis in reticulocyte lysates could be prevented by the addition of the partially purified inhibitor during preincubation of lysate with double-stranded RNA.     

Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.     

Possible involvement of the 90-kDa heat shock protein in the regulation of protein synthesis

The heme-sensitive eukaryotic initiation factor (eIF)-2 alpha kinase regulates translational activity in reticulocytes by phosphorylation of the smallest subunit of eukaryotic peptide initiation factor 2, eIF-2. Highly purified preparations of the kinase contain an abundant 90-kDa polypeptide which appears to modulate the activity of the enzyme. The physical properties and structural characteristics of the reticulocyte 90-kDa peptide are similar to those of the 90-kDa heat shock protein (hsp 90) from HeLa and other mammalian cells. The reticulocyte and HeLa cell proteins are shown to be immunologically cross-reactive. A direct comparison of the two proteins by one-dimensional peptide mapping of large peptides generated by limited proteolysis and by reversed-phase high performance liquid chromatography analysis of tryptic peptides indicates that they represent the same protein species. Like the 90-kDa reticulocyte protein, HeLa cell hsp 90 causes increased eIF-2 alpha phosphorylation by the heme-sensitive kinase and is a potent inhibitor of protein synthesis in the reticulocyte lysate system. A potential mechanism for the latter inhibition is inferred. These results implicate hsp 90 in the regulation of protein synthesis via its interaction with and perhaps regulation of the heme-sensitive kinase and phosphorylation of eIF-2 alpha.     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.     

Mechanism of interferon action. Effect of double-stranded RNA and the 5'-O-monophosphate form of 2',5'-oligoadenylate on the inhibition of reovirus mRNA translation in vitro

The effect of reovirus double-stranded RNA (dsRNA) and 5'-O-monophosphate form of 2',5'-oligoadenylate (pA(2'p5'A)2) on the translation and degradation of reovirus messenger RNA and on protein phosphorylation was examined in extracts prepared from interferon-treated mouse L fibroblasts. The following results were obtained. 1) The enhanced degradation of reovirus [3H]mRNA observed in the presence of either dsRNA or the 5'-O-triphosphate form of 2',5'-oligoadenylate (pppA(2'p5'A)3) was completely blocked by pA(2'p5'A)2. 2) The dsRNA-dependent phosphorylation of protein P1 and the alpha subunit of eukaryotic initiation factor (eIF-2) depended in a similar manner upon the concentration of dsRNA and was optimal at low dsRNA concentrations (0.1 to 1 microgram/ml). However, high concentrations of dsRNA (greater than 100 micrograms/ml) drastically reduced the phosphorylation of both P1 and eIF-2 alpha. Neither P1 nor eIF-2 alpha phosphorylation was affected by either pA(2'p5'A)2 or pppA(2'p5'A)3. 3) The translation of reovirus mRNA in vitro was inhibited by the addition of either low concentrations of dsRNA or pppA(2'p5'A)3. Whereas pA(2'p5'A)2 completely reversed the pppA(2'p5'A)3-mediated inhibition of translation, the inhibition mediated by low concentrations of dsRNA was only partially reversed by pA(2'p5'A)2. Under conditions where the pppA-(2'p5'A)3mediated degradation of reovirus mRNA was blocked, the translation of reovirus mRNA was still inhibited by low but not by high concentrations of dsRNA in a manner that correlated with the activation of P1 and eIF-2 alpha phosphorylation. These results suggest that the pppA(2'p5'A)n-dependent ribonuclease is not required and that protein phosphorylation may indeed be sufficient for the dsRNA-dependent inhibition of reovirus mRNA translation in cell-free systems derived from interferon-treated mouse fibroblasts.     

Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2: Further evidence for a common mechanism of inhibition of protein synthesis

The role of reversing factor (RF) in the regulation of protein synthesis by inhibitory protein kinases that phosphorylate the 38,000-dalton subunit of initiation factor eIF-2 has been examined. Results show that as with the heme-regulated protein kinase (HRI), RF restores protein synthesis in reticulocyte lysates inhibited by translational inhibitors from rat liver, wheat germ, Krebs ascites cell, by oxidized glutathione, the protein kinase activated by double stranded RNA (dRI), and the interferon-induced double stranded RNA activated protein kinase from Ehrlich ascites and Hela cells. These findings suggest that RF plays an important role in eukaryotic protein chain initiation cycle.     

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.     

An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.     

Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity.

We have investigated the interaction of VAI RNA with the interferon-induced, double-stranded (ds) RNA-activated protein kinase, P68, both of which regulate protein synthesis in adenovirus-infected cells. Previous work has shown that during infection by the VAI RNA-negative mutant, dl331, both viral and cellular protein synthesis are inhibited due to phosphorylation of the alpha-subunit of the eukaryotic initiation factor, eIF-2, by the P68 protein kinase. Utilizing monoclonal antibodies specific for P68, we demonstrated that the physical levels of P68 in dl331-infected, wild-type Ad2-infected and uninfected cells were all comparable suggesting that the elevated kinase activity detected during mutant infection was not due to increased P68 synthesis. To examine the basis of the increased activity of P68, the protein kinase was purified from infected-cell extracts using the monoclonal antibody. We found that P68 was heavily autophosphorylated during dl331 infection but not during wild-type or mock infection. The extent of autophosphorylation correlated with elevated P68 activity and the loss of the dsRNA requirements to phosphorylate the exogenous substrates, eIF-1 alpha and histones. We also analyzed VAI RNA function in vitro and present evidence that purified VAI RNA can block the autophosphorylation of P68 in the ribosomal salt wash fraction of interferon-treated cells. Finally we suggest VAI RNA functions through a direct interaction with the P68 protein kinase, since we demonstrated that VAI RNA forms a complex with P68 both in vitro and in vivo.     

Activation of hemin-regulated initiation factor-2 kinase in heat-shocked HeLa cells

Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 degrees C, but it resumed after 20 min at a rate about 50% that of control cells. After 10 min of heat shock, the binding of Met-tRNAf to 40 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the alpha subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and heat-shocked cells were assayed for in vitro protein synthesis. Both extracts were active when supplemented with hemin, but the extract from heat-shocked cells had little initiation activity without this addition. A Mr 90,000 polypeptide and eIF-2 alpha were phosphorylated in this extract, but hemin or an antibody which inhibits the protein kinase designated heme-controlled repressor reduced this phosphorylation. These findings implicated heme-controlled repressor as the kinase at least in part responsible for eIF-2 alpha phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2 alpha phosphorylation after heat shock were reduced by adding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of Met-tRNAf to 40 S ribosomal subunits was inhibited by about 50% in extracts prepared from cells heat-shocked for 40 min, and eIF-2 alpha phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2 alpha phosphorylation limits mRNA translation even after partial recovery of protein synthesis.     

Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2.

A poliovirus type 2 Lansing mutant was constructed by inserting 6 base pairs into the 2Apro region of an infectious cDNA clone, resulting in the addition of a leucine and threonine into the polypeptide sequence. The resulting small-plaque mutant, 2A-2, had a reduced viral yield in HeLa cells and synthesized viral proteins inefficiently. Infection with the mutant did not lead to specific inhibition of host cell protein synthesis early in infection, and this defect was attributed to a failure to induce cleavage of the cap-binding complex protein p220. At late times after infection with the mutant virus, both cellular and viral protein syntheses were severely inhibited. To explain this global inhibition of protein synthesis, the phosphorylation state of the alpha subunit of eucaryotic initiation factor 2 (eIF-2 alpha) was examined. eIF-2 alpha was phosphorylated in both R2-2A-2- and wild-type-virus-infected cells, indicating that poliovirus does not encode a function that blocks phosphorylation of eIF-2 alpha. The kinetics and extent of eIF-2 alpha phosphorylation correlated with the production of double-stranded RNA in infected cells, suggesting that eIF-2 alpha is phosphorylated by P1/eIF-2 alpha kinase. When HeLa cells were infected with R2-2A-2 in the presence of 2-aminopurine, a protein kinase inhibitor, much higher virus titers were produced, cleavage of p220 occurred, and host cell protein synthesis was specifically inhibited. Since phosphorylation of eIF-2 alpha was not inhibited by 2-aminopurine, we propose that 2-aminopurine rescues the ability of R2-2A-2 to induce cleavage of p220 by inhibition of a second as yet unidentified kinase.     

Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus.

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.     

The interferon-induced protein kinase PK-i from mouse L cells.

Interferon-treated L cells are characterized by an increased protein kinase activity that can selectively phosphorylate the small subunit of eukaryotic initiation factor 2. This protein kinase, PK-i, has been extensively purified and shown to be a potent inhibitor of mRNA translation. The purified PK-i contains the endogenously phosphorylated 67,000 Mr protein characteristic of interferon-treated cell extracts. PK-i can also phosphorylate arginine-rich histones. Purified PK-i can be activated by preincubation with ATP (but not adenylyl imidodiphosphate) and low concentrations of double-stranded RNA. The activation results in an increase in the first rate of eIF-2 phosphorylation. Activated PK-i becomes resistant to high concentrations of double-stranded RNA and more thermostable. A stimulator of PK-i activity, factor A, was isolated, as well as a specific phosphoprotein phosphatase that dephosphorylates the 67,000 Mr protein and eIF-2. These two factors, which are present in untreated L cells, may regulate the translation inhibitory activity of the interferon-induced and double-stranded RNA-activated protein kinase PK-i.     

Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure

Exposure to arsenite inhibits protein synthesis and activates multiple stress signaling pathways. Although arsenite has diverse effects on cell metabolism, we demonstrated that phosphorylation of eukaryotic translation initiation factor 2 at Ser-51 on the alpha subunit was necessary to inhibit protein synthesis initiation in arsenite-treated cells and was essential for stress granule formation. Of the four protein kinases known to phosphorylate eukaryotic translation initiation factor 2alpha, only the heme-regulated inhibitor kinase (HRI) was required for the translational inhibition in response to arsenite treatment in mouse embryonic fibroblasts. In addition, HRI expression was required for stress granule formation and cellular survival after arsenite treatment. In vivo studies elucidated a fundamental requirement for HRI in murine survival upon acute arsenite exposure. The results demonstrated an essential role for HRI in mediating arsenite stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha, inhibition of protein synthesis, stress granule formation, and survival.