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.     

Induction of protein kinase PKR-dependent activation of interferon regulatory factor 3 by vaccinia virus occurs through adapter IPS-1 signaling

Interferon regulatory factor 3 (IRF-3) undergoes phosphorylation-induced activation in virus-infected cells and plays an important role in the antiviral innate immune response. The E3L protein encoded by vaccinia virus is known to impair phosphorylation and activation of IRF-3. Kinases in addition to I kappaB kinase-related kinases are implicated in the IRF-3-dependent antiviral response. To test in human cells the role of the protein kinase regulated by RNA (PKR) in IRF-3 activation, HeLa cells made stably deficient in PKR using an RNA interference strategy were compared with PKR-sufficient cells. Rapid phosphorylation and nuclear accumulation of IRF-3 were detected in PKR-sufficient cells following infection with E3L deletion mutant (DeltaE3L) virus. By contrast, the full IRF-3 activation response was largely abolished in PKR-deficient cells. The DeltaE3L virus-induced IRF-3 activation seen in PKR-sufficient cells was diminished by treatment with cytosine beta-D-arabinofuranoside. Furthermore, the vaccinia mutant ts23, which displays increased viral double-stranded RNA production at 39 degrees C, induced PKR-dependent IRF-3 phosphorylation at 39 degrees C but not at 31 degrees C. Both IRF-3 phosphorylation and cell apoptosis induced by infection with DeltaE3L virus were dependent upon RIG-I-like receptor signal transduction components, including the adapter IPS-1. These data suggest that PKR facilitates the host innate immune response and apoptosis in virus-infected cells by mediating IRF-3 activation through the mitochondrial IPS-1 signal transduction pathway.     

Paramyxovirus-induced shutoff of host and viral protein synthesis: role of the P and V proteins in limiting PKR activation

The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI⁻ mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI⁻ mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI⁻ virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2α. WT SV5 was a poor activator of the eIF-2α kinase protein kinase R (PKR). By contrast, the P/V-CPI⁻ mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI⁻ mutant was restored to ~50% that of control HeLa cells. By contrast, the rates of P/V-CPI⁻ viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI⁻ mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI⁻ mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.     

Protein Kinase PKR Mediates the Apoptosis Induction and Growth Restriction Phenotypes of C Protein-Deficient Measles Virus

The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKR^kd cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (C^ko) virus but had no effect on the growth of either wild-type (WT) or V knockout (V^ko) virus. Increased growth of the C^ko virus in PKR^kd cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, V^ko, or especially C^ko virus caused significantly less apoptosis in PKR^kd cells than in PKR-sufficient cells. Although apoptosis induced by C^ko virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of C^ko virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.     

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

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

Protein Kinase PKR Amplification of Interferon β Induction Occurs through Initiation Factor eIF-2α-mediated Translational Control

The protein kinase PKR is activated by RNA with double-stranded (ds) structure and subsequently impairs translation through phosphorylation of protein synthesis initiation factor eIF-2α. PKR also mediates activation of signal transduction pathways leading to interferon beta (IFN-β) gene induction following virus-infection or RNA transfection. We previously demonstrated in measles virus-infected cells that PKR is required for the maximal induction of IFN-β gene expression by the interferon promoter stimulator gene 1 (IPS-1) adaptor-dependent cytosolic RNA sensor pathway. While both IPS-1 and PKR are important mediators of IFN-β induction, with PKR contributing to an enhanced NF-κB activation, the mechanism by which PKR enhances NF-κB activity and amplifies IFN-β induction is unresolved. Herein we tested the possibility that PKR could activate signal transduction pathways indirectly through translational control responses. Following transfection with synthetic or natural dsRNAs or infection with measles virus, we observed increased mRNA but decreased protein levels for the inhibitor of NF-κB signaling, IκB-α, that correlated with PKR activation and eIF-2α phosphorylation. Importantly, knockdown of PKR increased IκB-α protein levels and impaired IFN-β induction. Additionally, inhibition of translation by cycloheximide treatment rescued IFN-β induction following PKR knockdown but not IPS-1 knockdown. Mutation of eIF-2α to prevent phosphorylation also impaired IFN-β induction in PKR-sufficient virus-infected cells. These results suggest that an eIF-2α-dependent translation inhibition mechanism is sufficient to explain the PKR-mediated amplification of IPS-1-dependent IFN-β induction by foreign RNA.     

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

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

Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells.

The vaccinia virus E3L gene codes for double-stranded RNA (dsRNA) binding proteins which can prevent activation of the dsRNA-dependent, interferon-induced protein kinase PKR. Activated PKR has been shown to induce apoptosis in HeLa cells. HeLa cells infected with vaccinia virus with the E3L gene deleted have also been shown to undergo apoptosis, whereas HeLa cells infected with wild-type vaccinia virus do not. In this report, using virus recombinants expressing mutant E3L products or alternative dsRNA binding proteins, we show that suppression of induction of apoptosis correlates with functional binding of proteins to dsRNA. Infection of HeLa cells with ts23, which leads to synthesis of increased dsRNA at restrictive temperature, induced apoptosis at restrictive but not permissive temperatures. Treatment of cells with cytosine arabinoside, which blocks the late buildup of dsRNA in vaccinia virus-infected cells, prevented induction of apoptosis by vaccinia virus with E3L deleted. Cells transfected with dsRNA in the absence of virus infection also underwent apoptosis. These results suggest that dsRNA is a trigger that can initiate a suicide response in virus-infected and perhaps uninfected cells.     

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

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

The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.     

The Second-Site Mutation in the Herpes Simplex Virus Recombinants Lacking the γ134.5 Genes Precludes Shutoff of Protein Synthesis by Blocking the Phosphorylation of eIF-2α

In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the γ₁34.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2α is phosphorylated, and protein synthesis is shut off. Although PKR is also activated in cells infected with the wild-type virus, the product of the γ₁34.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1α and redirects it to dephosphorylate eIF-2α, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the γ₁34.5 gene yields second-site, compensatory mutants lacking various domains of the α47 gene situated next to the U_S11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759–4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the γ₁34.5, U_S8, -9, -10, and -11, and α47 (U_S12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the U_S10 gene and the promoter of the α47 gene fused to the coding domain of the U_S11 gene. R5104 exhibited a protein synthesis profile similar to that of wild-type virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed the following: (i) PKR was activated in cells infected with parent or mutant virus but not in mock-infected cells, consistent with earlier studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells lacked the phosphatase activity specific for eIF-2α characteristic of wild-type virus-infected cells; and (iii) lysates of R3616 and R5103, which lacked the second-site compensatory mutation, contained an activity which phosphorylated eIF-2α in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2α. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2α, in cells infected with γ₁34.5⁻ virus carrying the compensatory mutation, eIF-2α is not phosphorylated. The activity made apparent by the second-site mutation may represent a more ancient mechanism evolved to preclude the shutoff of protein synthesis.     

The Infectious Bursal Disease Virus RNA-Binding VP3 Polypeptide Inhibits PKR-Mediated Apoptosis

Infectious bursal disease virus (IBDV) is an avian pathogen responsible for an acute immunosuppressive disease that causes major losses to the poultry industry. Despite having a bipartite dsRNA genome, IBDV, as well as other members of the Birnaviridae family, possesses a single capsid layer formed by trimers of the VP2 capsid protein. The capsid encloses a ribonucleoprotein complex formed by the genome associated to the RNA-dependent RNA polymerase and the RNA-binding polypeptide VP3. A previous report evidenced that expression of the mature VP2 IBDV capsid polypeptide triggers a swift programmed cell death response in a wide variety of cell lines. The mechanism(s) underlying this effect remained unknown. Here, we show that VP2 expression in HeLa cells activates the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), which in turn triggers the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). This results in a strong blockade of protein synthesis and the activation of an apoptotic response which is efficiently blocked by coexpression of a dominant negative PKR polypeptide. Our results demonstrate that coexpression of the VP3 polypeptide precludes phosphorylation of both PKR and eIF2α and the onset of programmed cell death induced by VP2 expression. A mutation blocking the capacity of VP3 to bind dsRNA also abolishes its capacity to prevent PKR activation and apoptosis. Further experiments showed that VP3 functionally replaces the host-range vaccinia virus (VACV) E3 protein, thus allowing the E3 deficient VACV deletion mutant WRΔE3L to grow in non-permissive cell lines. According to results presented here, VP3 can be categorized along with other well characterized proteins such us VACV E3, avian reovirus sigmaA, and influenza virus NS1 as a virus-encoded dsRNA-binding polypeptide with antiapoptotic properties. Our results suggest that VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing programmed cell death.     

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

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

The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms.

Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2 alpha [eIF-2 alpha]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2 alpha phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50- to 100-fold more efficiently than the K3L gene product. However, further studies demonstrated that the mechanism of action of these two inhibitors was different. Whereas the E3L inhibitor interfered with the binding of the kinase to double-stranded RNA, the K3L inhibitor did not. We propose that the K3L inhibitor acts through its homology to eIF-2 alpha to interfere with the interaction of eIF-2 alpha with PKR. The two inhibitors did not display a synergistic effect on translation or eIF-2 alpha phosphorylation. In addition, neither K3L nor E3L expression detectably altered cellular protein synthesis.     

Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection

The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoprotein's association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the ΔYI, ΔIK, and ΔLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal α-helical heptad repeats, respectively, inhibited WT and ΔLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.     

Adaptive Gene Amplification As an Intermediate Step in the Expansion of Virus Host Range

The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.     

TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress

The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTΔ13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTΔ13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTΔ13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2^−/− murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.