A conserved domain of herpes simplex virus ICP34.5 regulates protein phosphatase complex in mammalian cells

ICP34.5, encoded by herpes simplex virus 1, is a protein phosphatase 1 (PP1) regulatory subunit that mediates dephosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha). However, the mechanism of its action remains poorly understood. Here, we show that amino acid substitutions in the arginine-rich motif have differential effects on ICP34.5 activity. The phenotypes parallel with viral protein synthesis and cytopathic effects in virus infected cells. Besides the consensus PP1 binding motif, the Arg-motif appears to enhance the interaction between ICP34.5 and PP1. These results suggest that concerted action between the PP1 binding domain and the effector domain of ICP34.5 is crucial for eIF2alpha dephosphorylation and viral protein synthesis.     

The IE180 protein of pseudorabies virus suppresses phosphorylation of translation initiation factor eIF2α

We have previously shown that the porcine alphaherpesvirus pseudorabies virus (PRV) efficiently interferes with phosphorylation of the eukaryotic translation initiation factor eIF2α. Inhibition of phosphorylation of eIF2α has been reported earlier for the closely related alphaherpesvirus herpes simplex virus 1 (HSV-1) through its ICP34.5 and US11 proteins. PRV, however, does not encode an ICP34.5 or US11 orthologue. Assays using cycloheximide, UV-inactivated PRV, or phosphonoacetic acid (PAA) showed that de novo expression of one or more (immediate) early viral protein(s) is required for interference with eIF2α phosphorylation. In line with this, a time course assay showed that eIF2α phosphorylation was abolished within 2 h after PRV inoculation. PRV encodes only one immediate-early protein, IE180, the orthologue of HSV-1 ICP4. As reported earlier, a combinational treatment of cells with cycloheximide and actinomycin D allowed expression of IE180 without detectable expression of the US3 early protein in PRV-infected cells. This led to a substantial reduction in eIF2α phosphorylation levels, indicative for an involvement of IE180. In support of this, transfection of IE180 also potently reduced eIF2α phosphorylation. IE180-mediated interference with eIF2α phosphorylation was not cell type dependent, as it occurred both in rat neuronal 50B11 cells and in swine testicle cells. Inhibition of the cellular phosphatase PP1 impaired PRV-mediated interference with eIF2α phosphorylation, indicating that PP1 is involved in this process. In conclusion, the immediate-early IE180 protein of PRV has the previously uncharacterized ability to suppress phosphorylation levels of the eukaryotic translation initiation factor eIF2α.     

Regulation of eIF2alpha phosphorylation by different functions that act during discrete phases in the herpes simplex virus type 1 life cycle

Multiple herpes simplex virus type 1 functions control translation by regulating phosphorylation of the initiation factor eIF2 on its alpha subunit. Both of the two known regulators, the gamma(1)34.5 and Us11 gene products, are produced late in the viral life cycle, although the gamma(1)34.5 gene is expressed prior to the gamma(2) Us11 gene, as gamma(2) genes require viral DNA replication for their expression while gamma(1) genes do not. The gamma(1)34.5 protein, through a GADD34-related domain, binds a cellular phosphatase (PP1alpha), maintaining pools of active, unphosphorylated eIF2. Infection of a variety of cultured cells with a gamma(1)34.5 mutant virus results in the accumulation of phosphorylated eIF2alpha and the inhibition of translation prior to the completion of the viral lytic program. Ectopic, immediate-early Us11 expression prevents eIF2alpha phosphorylation and the inhibition of translation observed in cells infected with a gamma(1)34.5 mutant by inhibiting activation of the cellular kinase PKR and the subsequent phosphorylation of eIF2alpha; however, a requirement for the Us11 protein, produced in its natural context as a gamma(2) polypeptide, remains to be demonstrated. To determine if Us11 regulates late translation, we generated two Us11 null viruses. In cells infected with a Us11 mutant, elevated levels of activated PKR and phosphorylated eIF2alpha were detected, viral translation rates were reduced 6- to 7-fold, and viral replication was reduced 13-fold compared to replication in cells infected with either wild-type virus or a virus in which the Us11 mutation was repaired. This establishes that the Us11 protein is critical for proper late translation rates. Moreover, it demonstrates that the shutoff of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus, previously ascribed solely to the gamma(1)34.5 mutation, actually results from the combined loss of gamma(1)34.5 and Us11 functions, as the gamma(2) Us11 mRNA is not translated in cells infected with a gamma(1)34.5 mutant.     

AlaArg motif in the carboxyl terminus of the gamma(1)34.5 protein of herpes simplex virus type 1 is required for the formation of a high-molecular-weight complex that dephosphorylates eIF-2alpha

The gamma(1)34.5 protein of herpes simplex virus (HSV) type 1 functions to prevent the shutoff of protein synthesis mediated by the double-stranded-RNA-dependent protein kinase PKR. This is because gamma(1)34.5 associates with protein phosphatase 1 (PP1) through its carboxyl terminus, forming a high-molecular-weight complex that dephosphorylates the alpha subunit of translation initiation factor eIF-2 (eIF-2alpha). Here we show that Val193Glu and Phe195Leu substitutions in the PP1 signature motif of the gamma(1)34.5 protein abolished its ability to redirect PP1 to dephosphorylate eIF-2alpha and replication of mutant viruses was severely impaired. The gamma(1)34.5 protein, when expressed in Sf9 cells using a recombinant baculovirus, was capable of directing specific eIF-2alpha dephosphorylation. Deletions of amino acids 258 to 263 had no effect on activity of gamma(1)34.5. However, deletions of amino acids 238 to 258 abolished eIF-2alpha phosphatase activity but not PP1 binding activity. Interestingly, deletions in the AlaArg motif of the carboxyl terminus disrupted the high-molecular-weight complex that is required for dephosphorylation of eIF-2alpha. These results demonstrate that gamma(1)34.5 is functionally active in the absence of any other HSV proteins. In addition to a PP1 binding domain, the carboxyl terminus of gamma(1)34.5 contains an effector domain that is required to form a functional complex.     

Effect of γ34.5 deletions on oncolytic herpes simplex virus activity in brain tumors

The ICP34.5 protein of herpes simplex virus (HSV) is involved in many aspects of viral pathogenesis; promoting neurovirulence, inhibiting interferon-induced shutoff of protein synthesis, interacting with PCNA and TBK1, inhibiting dendritic cell (DC) maturation, and binding to Beclin 1 to interfere with autophagy. Because of its key role in neuropathogenicity, the γ34.5 gene is deleted in all oncolytic HSVs (oHSVs) currently in clinical trial for treating malignant gliomas. Unfortunately, deletion of γ34.5 attenuates virus replication in cancer cells, especially human glioblastoma stem cells (GSCs). To develop new oHSVs for use in the brain and that replicate in GSCs, we explored the effect of deleting the γ34.5 Beclin 1 binding domain (BBD). To ensure cancer selectivity and safety, we inactivated the ICP6 gene (UL39, large subunit of ribonucleotide reductase), constructing ICP6 mutants with different γ34.5 genotypes: Δ68HR-6, intact γ34.5; Δ68H-6, γ34.5 BBD deleted; and 1716-6, γ34.5 deleted. Multimutated Δ68H-6 exhibited minimal neuropathogenicity in HSV-1-susceptible mice, as opposed to Δ68H and Δ68HR-6. It replicated well in human glioma cell lines and GSCs, effectively killing cells in vitro and prolonging survival of mice bearing orthotopic brain tumors. In contrast, 1716 and 1716-6 barely replicated in GSCs. Infection of glioma cells with Δ68H-6 and 1716-6 induced autophagy and increased phosphorylation of eIF2α, while inhibition of autophagy, by Beclin 1 short hairpin RNA (shRNA) knockdown or pharmacological inhibition, had no effect on virus replication or phosphorylated eIF2α (p-eIF2α) levels. Thus, Δ68H-6 represents a new oHSV vector that is safe and effective against a variety of brain tumor models.     

An N-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type 1 ICP34.5 protein and its ligand, protein phosphatase 1.

The ICP34.5 protein facilitates herpes simplex virus replication by binding and activating protein phosphatase 1 (PP1) by means of a very conserved C-terminal GADD34-like region. Natural variants of the ICP34.5 differing in the number of arginines in an Arg-rich cluster at the N terminus and the number of Pro-Ala-Thr repeats in the central bridge region of the protein were cloned as fusion proteins with a reporter peptide (c-Myc or hrGFP) at the C terminus. The natural variants were obtained from strains differing in passage history, tissue culture behavior, and neuroinvasive disease potential. In transfected cells, these variants localized to different subcellular compartments. The N-terminal Arg-rich cluster acted as a cellular localization signal for discrete regions of the nucleus and cytoplasm, but the ultimate location of ICP34.5 was determined by the number of Pro-Ala-Thr repeats in the central bridge region. PP1 colocalized with the ICP34.5 variant in cells expressing the ICP34.5. The ICP34.5-mediated, herpes simplex virus strain-dependent differences in the modulation of PP1 location and function may be responsible for the strain-associated differences in tissue culture behavior and virulence of the virus.     

Dephosphorylation of eIF-2alpha mediated by the gamma(1)34.5 protein of herpes simplex virus type 1 is required for viral response to interferon but is not sufficient for efficient viral replication

The gamma(1)34.5 protein of herpes simplex virus type 1 (HSV-1) functions to block the shutoff of protein synthesis involving double-stranded RNA-dependent protein kinase (PKR). In this process, the gamma(1)34.5 protein recruits cellular protein phosphatase 1 (PP1) to form a high-molecular-weight complex that dephosphorylates eIF-2alpha. Here we show that the gamma(1)34.5 protein is capable of mediating eIF-2alpha dephosphorylation without any other viral proteins. While deletion of amino acids 1 to 52 from the gamma(1)34.5 protein has no effect on eIF-2alpha dephosphorylation, further truncations up to amino acid 146 dramatically reduce the activity of the gamma(1)34.5 protein. An additional truncation up to amino acid 188 is deleterious, indicating that the carboxyl-terminal domain alone is not functional. Like wild-type HSV-1, the gamma(1)34.5 mutant with a truncation of amino acids 1 to 52 is resistant to interferon, and resistance to interferon is coupled to eIF-2alpha dephosphorylation. Intriguingly, this mutant exhibits a similar growth defect seen for the gamma(1)34.5 null mutant in infected cells. Restoration of the wild-type gamma(1)34.5 gene in the recombinant completely reverses the phenotype. These results indicate that eIF-2alpha dephosphorylation mediated by the gamma(1)34.5 protein is required for HSV response to interferon but is not sufficient for viral replication. Additional functions or activities of the gamma(1)34.5 protein contribute to efficient viral infection.     

In vivo replication of an ICP34.5 second-site suppressor mutant following corneal infection correlates with in vitro regulation of eIF2 alpha phosphorylation

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2 alpha. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2 alpha following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2 alpha phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2 alpha, while the wild-type virus substantially reduced eIF2 alpha phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation.     

Analysis of the role of autophagy in replication of herpes simplex virus in cell culture

The herpes simplex virus type 1 (HSV-1) neurovirulence gene encoding ICP34.5 controls the autophagy pathway. HSV-1 strains lacking ICP34.5 are attenuated in growth and pathogenesis in animal models and in primary cultured cells. While this growth defect has been attributed to the inability of an ICP34.5-null virus to counteract the induction of translational arrest through the PKR antiviral pathway, the role of autophagy in the regulation of HSV-1 replication is unknown. Here we show that HSV-1 infection induces autophagy in primary murine embryonic fibroblasts and that autophagosome formation is increased to a greater extent following infection with an ICP34.5-deficient virus. Elimination of the autophagic pathway did not significantly alter the replication of wild-type HSV-1 or ICP34.5 mutants. The phosphorylation state of eIF2alpha and viral protein accumulation were unchanged in HSV-1-infected cells unable to undergo autophagy. These data show that while ICP34.5 regulates autophagy, it is the prevention of translational arrest by ICP34.5 rather than its control of autophagy that is the pivotal determinant of efficient HSV-1 replication in primary cell culture.     

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.     

Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1

The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.     

The translation initiation factor eIF2beta is an interactor of protein phosphatase-1

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2alpha, eIF2Bepsilon and eIF4E. Other initiation factors, including eIF2beta, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2beta contains a putative PP1-(protein phosphatase-1) binding RVxF-motif. The predicted eIF2beta-PP1 interaction was confirmed by PP1 binding and co-immunoprecipitation assays on cell lysates as well as with the purified components. Site-directed mutagenesis showed that eIF2beta contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2beta functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and Ser51 of eIF2alpha by PP1, but did not affect the dephosphorylation of Ser464 of eIF2Bepsilon by this phosphatase. Strikingly, eIF2beta emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2beta was decreased by the mere mutation of its RVxF-motif. These results make eIF2beta an attractive candidate substrate for associated PP1 in vivo. The overexpression of wild-type eIF2beta or eIF2beta with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal conditions.     

Respiratory Syncytial Virus Limits α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation to Maintain Translation and Viral Replication

The impact of respiratory syncytial virus (RSV) on morbidity and mortality is significant in that it causes bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and pneumonia in immunocompromised hosts. RSV activates protein kinase R (PKR), a cellular kinase relevant to limiting viral replication (Groskreutz, D. J., Monick, M. M., Powers, L. S., Yarovinsky, T. O., Look, D. C., and Hunninghake, G. W. (2006) J. Immunol. 176, 1733–1740). It is activated by autophosphorylation, likely triggered by a double-stranded RNA intermediate during replication of the virus. In most instances, ph-PKR targets the α subunit of eukaryotic translation initiation factor 2 (eIF2α) protein via phosphorylation, leading to an inhibition of translation of cellular and viral protein. However, we found that although ph-PKR increases in RSV infection, significant eIF2α phosphorylation is not observed, and inhibition of protein translation does not occur. RSV infection attenuates eIF2α phosphorylation by favoring phosphatase rather than kinase activity. Although PKR is activated, RSV sequesters PKR away from eIF2α by binding of the kinase to the RSV N protein. This occurs in conjunction with an increase in the association of the phosphatase, PP2A, with eIF2α following PKR activation. The result is limited phosphorylation of eIF2α and continued translation of cellular and viral proteins.     

The alpha subunit of eukaryotic initiation factor 2B (eIF2B) is required for eIF2-mediated translational suppression of vesicular stomatitis virus

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.     

A herpesvirus virulence factor inhibits dendritic cell maturation through protein phosphatase 1 and Ikappa B kinase

Dendritic cells are sentinels in innate and adaptive immunity. Upon virus infection, a complex program is in operation, which activates IκB kinase (IKK), a key regulator of inflammatory cytokines and costimulatory molecules. Here we show that the γ(1)34.5 protein, a virulence factor of herpes simplex viruses, blocks Toll-like receptor-mediated dendritic cell maturation. While the wild-type virus inhibits the induction of major histocompatibility complex (MHC) class II, CD86, interleukin-6 (IL-6), and IL-12, the γ(1)34.5-null mutant does not. Notably, γ(1)34.5 works in the absence of any other viral proteins. When expressed in mammalian cells, including dendritic cells, γ(1)34.5 associates with IKKα/β and inhibits NF-κB activation. This is mirrored by the inhibition of IKKα/β phosphorylation, p65/RelA phosphorylation, and nuclear translocation in response to lipopolysaccharide or poly(I:C) stimulation. Importantly, γ(1)34.5 recruits both IKKα/β and protein phosphatase 1, forming a complex that dephosphorylates two serine residues within the catalytic domains of IκB kinase. The amino-terminal domain of γ(1)34.5 interacts with IKKα/β, whereas the carboxyl-terminal domain binds to protein phosphatase 1. Deletions or mutations in either domain abolish the activity of γ(1)34.5. These results suggest that the control of IκB kinase dephosphorylation by γ(1)34.5 represents a critical viral mechanism to disrupt dendritic cell functions.     

Herpes simplex virus 2 infection impacts stress granule accumulation

Interference with stress granule (SG) accumulation is gaining increased appreciation as a common strategy used by diverse viruses to facilitate their replication and to cope with translational arrest. Here, we examined the impact of infection by herpes simplex virus 2 (HSV-2) on SG accumulation by monitoring the localization of the SG components T cell internal antigen 1 (TIA-1), Ras-GTPase-activating SH3-domain-binding protein (G3BP), and poly(A)-binding protein (PABP). Our results indicate that SGs do not accumulate in HSV-2-infected cells and that HSV-2 can interfere with arsenite-induced SG accumulation early after infection. Surprisingly, SG accumulation was inhibited despite increased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), implying that HSV-2 encodes previously unrecognized activities designed to maintain translation initiation downstream of eIF2α. SG accumulation was not inhibited in HSV-2-infected cells treated with pateamine A, an inducer that works independently of eIF2α phosphorylation. The SGs that accumulated following pateamine A treatment of infected cells contained G3BP and PABP but were largely devoid of TIA-1. We also identified novel nuclear structures containing TIA-1 that form late in infection. These structures contain the RNA binding protein 68-kDa Src-associated in mitosis (Sam68) and were noticeably absent in infected cells treated with inhibitors of viral DNA replication, suggesting that they arise as a result of late events in the virus replicative cycle.     

Translation of viral mRNA without active eIF2: the case of picornaviruses

Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.