Inhibition of PKR by RNA and DNA viruses

Virus Research 119 (2006) 100–110

Inhibition of PKR by RNA and DNA viruses Jeffrey O. Langland a , Jason M. Cameron a,b , Michael C. Heck a , James K. Jancovich a,b , Bertram L. Jacobs a,b,∗ b

a Center for Infectious Disease and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-5401, USA School of Life Sciences, Graduate Program in Molecular and Cellular Biology, Arizona State University, Tempe, AZ 85287-4501, USA

Abstract Interferons were the first of the anti-viral innate immune modulators to be characterized, initially characterized solely as anti-viral proteins [reviewed in Le Page, C., Genin, P., Baines, M.G., Hiscott, J., 2000. Inteferon activation and innate immunity. Rev. Immunogenet. 2, 374–386]. As we have progressed in our understanding of the interferons they have taken a more central role in our understanding of innate immunity and its interplay with the adaptive immune response. One of the key players in function of interferon is the interferon-inducible enzyme, protein kinase (PKR, activatable by RNA). The key role played by PKR in the innate response to virus infection is emphasized by the large number of viruses, DNA viruses as well as RNA viruses, whose hosts range from insects to humans, that code for PKR inhibitors. In this review we will first describe activation of PKR and then describe the myriad of ways that viruses inhibit function of PKR. © 2005 Elsevier B.V. All rights reserved. Keywords: PKR; Interferon; Interferon-resistance; RNA viruses; DNA viruses; Innate immune evasion

1. Introduction Interferons were the first of the anti-viral innate immune modulators to be characterized, initially characterized solely as anti-viral proteins (reviewed in Le Page et al., 2000). As we have progressed in our understanding of the interferons they have taken a more central role in our understanding of innate immunity and its interplay with the adaptive immune response. One of the key players in function of interferon is the interferoninducible enzyme, protein kinase (PKR, activatable by RNA). The key role played by PKR in the innate response to virus infection is emphasized by the large number of viruses, DNA viruses as well as RNA viruses, whose hosts range from insects to humans, that code for PKR inhibitors. In this review we will first describe activation of PKR and then describe the myriad of ways that viruses inhibit function of PKR. Activation of PKR appears to occur concomitantly with protein homodimerization and intermolecular phosphorylation (Kostura and Mathews, 1989; Langland et al., 1994; Patel et al., 1995; Thomis and Samuel, 1995). The PKR enzyme is composed of two well characterized domains consisting of an N-

∗

Corresponding author. Tel.: +1 480 965 4684; fax: +1 480 727 7615. E-mail address: [email protected] (B.L. Jacobs).

0168-1702/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2005.10.014

terminal regulatory domain that consists of consensus dsRNAbinding motifs and a C-terminal catalytic domain that contains conserved motifs for protein kinase activity (Meurs et al., 1990). Activation of PKR by RNA is dependent on the dsRNA structure, rather than the nucleotide sequence. Approximately 50 bp of duplexed RNA are required for full activation (Robertson and Mathews, 1996), although shorter stretches of secondary structure on predominantly single stranded RNA can activate PKR (Ben-Asouli et al., 2002). The role of binding to dsRNA may be to induce dimerization which then leads to intermolecular autophosphorylation and activation (Romano et al., 1998a). PKR can also be activated by a related dsRNA-binding protein, PACT (Patel and Sen, 1998). Activation of PKR is likely associated with phosphorylation of T446 and T451 with-in the so-called activation loop of PKR (Galabru and Hovanessian, 1987; Kostura and Mathews, 1989). Activated PKR is involved in a number of cellular regulatory roles. Most well characterized is PKR’s involvement in the phosphorylation of eukaryotic translation initiation factor, eIF2 (Clemens and Elia, 1997; Meurs et al., 1992). eIF2 containing an ␣ subunit phosphorylated on S51 becomes a potent inhibitor of eIF2B, the nucleotide exchange factor necessary for recycling of eIF2. Therefore, the phosphorylation of eIF2 by PKR during virus infection ultimately leads to an inhibition in protein synthesis and a block in viral replication.

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Fig. 1.

PKR also plays a role in regulating several signal transduction cascades in the cell. The transcription factor NF-␬B, which leads to expression of many pro-inflammatory genes, can be activated indirectly by PKR via association with TRAF and activation of the I kappa B kinase (IKK) complex (Gil et al., 2004). PKR has also been shown to play a role in the activation of p38 MAP kinases and the stress-activated protein kinase (SAPK)/c-Jun amino-terminal kinases (JNKs) (Goh et al., 2000). Interestingly, the activation of transcription factors IRF-3 and IRF-7, which lead to the expression of interferon-␤, can occur in the presence of dsRNA, but this induction does not appear to require PKR suggesting the presence of additional dsRNA-responsive enzymes present in the cell (Smith et al., 2001). Many viruses have evolved elaborate mechanisms to inhibit the PKR response. These viral countermeasures have been shown to block the PKR response at virtually every step in the pathway, from activation through substrate phosphorylation (Fig. 1; Table 1). For many viruses, multiple mechanisms are encoded to evade PKR activity (Fig. 1; Table 1). For most of these viruses it is unclear if these multiple PKR inhibitory mechanisms are redundant roles or are necessary functions to regulate PKR activity at different stages in the virus life cycle. Such viruses include vaccinia virus, herpes simplex virus, Epstein-Barr virus, influenza virus, and hepatitis C virus.

Table 1 Regulation of PKR by viral products Mechanism

Virus

Gene product

I. dsRNA binding proteins

Vaccinia vims Reovirus Influenza virus Rotavirus group C Rotavirus group A Herpes simplex virus Epstein-Barr virus

E3L ␴3 NS1 NSP3 NSP5? Us11

II. RNA inhibitors

Epstein-Barr virus Adenovirus Hepatitis C virus

EBER RNA VAI RNA, VAII RNA? IRES

III. PKR interaction

Hepatitis C virus Influenza virus Vaccinia virus Baculovirus Herpes simplex virus-1 Epstein-Barr virus Human herpes virus-8 Vaccinia virus Ambystoma tigrinum virus HIV

NS5A, E2 p58 E3L PK2 Us11

IV. Competitive inhibitor

2. DsRNA binding proteins DsRNA is the most well characterized danger signal that the cell uses to recognize the presence of viral infection. Therefore, it is not surprising that many viruses synthesize excessive amounts of dsRNA-binding proteins which function to bind to and sequester any free dsRNA molecules. Such well charac-

SM

SM vIRF-2 K3L ReIF2H Tat

V. PKR degradation

Polio virus

Protease

VI. eIF2␣ phosphatase

Herpes virus Papilloma virus SV-40

␥34.5 E6 (GADD34/PP1␣) Large-T antigen

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terized proteins include the ␴3 protein of reovirus, the NSP3 protein of rotaviruses, the NS1 protein of influenza virus, and the E3L protein of vaccinia virus (Chien et al., 2004; Jacobs and Langland, 1998; Langland et al., 1994). In the early 1980s vaccinia virus was shown to produce an inhibitor of PKR (Paez and Esteban, 1984; Rice and Kerr, 1984; Whitaker-Dowling and Youngner, 1983). This inhibitor was reported to be proteinaceous and to interact with dsRNA. Nearly 10 years later, the viral gene encoding this inhibitor was identified as the vaccinia virus E3L gene (Chang et al., 1992). The E3L gene encodes a dsRNA-binding protein containing one copy of a highly conserved dsRNA-binding motif in the C-terminus. This dsRNA-binding domain is essential for the replication of vaccinia virus in a wide range of host cells and necessary for the interferon-resistance phenotype of the virus (Beattie et al., 1995; Chang et al., 1995). Deletion of the E3L gene from vaccinia virus leads to loss of kinase inhibitory activity (Beattie et al., 1995). The full-length protein exists as a dimer in solution and this protein–protein interaction appears to contribute to high affinity binding to dsRNA (Romano et al., 1998b). Vaccinia virus constructs expressing dsRNA-binding proteins in place of the E3L gene maintain the phenotypic characteristics of the wild-type virus in cells-in-culture (Beattie et al., 1995; Langland et al., 1994; Shors et al., 1997). This rescue phenotype was observed even with the expression of a functional dsRNA-binding protein that has no apparent sequence homology to E3L (Beattie et al., 1995). More than 20 functionally distinct proteins, viral, and cellular, containing the conserved dsRNA-binding motif have been identified (Saunders and Barber, 2003). Over the years, mutational analysis of E3L and other proteins containing this conserved motif of 65–68 amino acids has revealed many of the residues required for high affinity binding to dsRNA. The E3L protein binds dsRNA molecules in a sequence independent manner with a KD ∼ 7–9 nm (Ho and Shuman, 1996). The dsRNA-binding domain of E3L shares significant homology to dsRNA domains identified on many cellular proteins, with the greatest homology with the ADAR protein, followed by the testis nuclear RNA binding protein and PKR. In recent years, the structure of the dsRNA-binding motif complexed with dsRNA has been resolved. The structure reveals that an RNA duplex of 12–16 bp is necessary for binding (Ramos et al., 2001; Ryter and Schultz, 1998). Interaction involves recognition of two successive minor grooves and spanning across the intervening major groove on one face of the RNA duplex. This manner of interaction explains the non-sequence specific recognition of dsRNA and lack of binding to ssRNA or dsDNA. The porcine group C rotavirus NSP3 gene encodes a fulllength polypeptide with a Mr of 45,000 (p45) which is subsequently proteolytically cleaved into polypeptides with Mr ’s of 38,000 (p38) and 8000 (p8) (Langland et al., 1994). Compared with group A rotaviruses, this additional p8 protein is not present and only a single polypeptide, p34 protein (Mr = 34,600), is observed. The p8 protein contains the conserved dsRNAbinding domain and is capable of specific dsRNA interaction. Therefore, evolutionarily, group C rotaviruses appear to have added a dsRNA-binding domain tag on the end of the NSP3

coding sequence permitting multiple physiological functions of the final protein products. The p38 polypeptide is likely involved in genomic replicase and assembly activity, whereas the p8 proteolytic product is likely involved in sequestering dsRNA to prevent activation of PKR. The rotavirus NSP5 protein has also been shown to bind dsRNA, however the role of this binding has not been determined and may involve destabilization of RNA secondary structures, RNA packaging, and/or regulation of PKR activity (Vende et al., 2002). One of the first viral dsRNA binding proteins identified was the reovirus ␴3 protein. This protein shares no sequence similarity to the conserved dsRNA-binding proteins discussed above. The identification of ␴3 protein as an inhibitor or PKR stemmed from several experiments (reviewed in Jacobs and Langland, 1998). In 1976, Huismans and Joklik identified ␴3 as the only reovirus protein in extracts from infected cells that bound specifically to dsRNA followed by Imani and Jacobs (1988) showing that extracts from reovirus T1 infected cells contained a PKR-inhibitory activity that copurified with ␴3. All three serotypes of reovirus induce the synthesis of an inhibitor of PKR, although with different kinetics and to different extents (reviewed in Jacobs and Langland, 1998). Inhibition of PKR activity was demonstrated as being due to binding and sequestering of dsRNA since inhibition of PKR activity could be overcome by the addition of excess dsRNA, since ␴3 acted in a noncatalytic manner, and since PKR inhibition by mutants of ␴3 correlated with binding to dsRNA (Denzler and Jacobs, 1994; Imani and Jacobs, 1988; Yue and Shatkin, 1997). Recently, avian reovirus has been shown to encode a dsRNA-binding protein, ␴A, which blocks activation of PKR suggesting the broad nature of PKR inhibition by viruses in divergent species (GonzalezLopez et al., 2003). The influenza virus NS1 protein is a multifunctional protein involved in both protein–protein and protein–RNA interactions (Lu et al., 1995). NS1 is capable of binding to poly(A) and a stem-bulge region in U6 small nuclear RNA as well as nonspecifically to dsRNA (Lu et al., 1995). As with many viral dsRNAbinding proteins, binding of NS1 to RNA requires dimerization and binding leads to a block in the activation of PKR (Lu et al., 1995; Wang et al., 1999). Like the reovirus ␴3 protein, NS1 does not share any sequence homology with the consensus dsRNAbinding domain and binding to dsRNA differs in that NS1 sits astride the minor groove of dsRNA with a few amino acids in the helix 2-helix 2 face forming an electrostatically stabilized interaction with the phophodiester backbone (Chien et al., 2004). Recently, Epstein-Barr virus (EBV) and herpes simplex virus (HSV) have been shown to encode proteins which bind to dsRNA and interact directly with PKR (Khoo et al., 2002; Poppers et al., 2003). The EBV SM protein RNA-binding domain and the RNA-binding domain of HSV Us11 protein both contain multiple copies of the amino acid sequence RXP (Poppers et al., 2003). This RXP domain has been demonstrated to be a dsRNA recognition motif for both Us11 and SM proteins (Khoo et al., 2002; Poppers et al., 2003). Furthermore, this domain is also involved in direct binding of each protein to PKR (Cassady and Gross, 2002; Poppers et al., 2003). It has been suggested that interaction of these proteins with PKR may occur through

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dsRNA-dependent and -independent associations (Poppers et al., 2003). These activities may allow the Us11 and SM proteins to inhibit PKR activity by sequestering dsRNA and by direct PKR interaction (see Section 4). Indeed, the Us11 RXP domain can prevent PKR activation by PACT, a cellular protein that activates PKR in the absence of dsRNA (Peters et al., 2002). This supports the prospect that Us11 can inhibit PKR activation in a dsRNA-independent manner. 3. RNA inhibitors Several viruses have evolved specialized mechanisms designed to inhibit the activity of PKR by making RNA with secondary structures that bind directly to PKR, but do not lead to the activation of the kinase domain. Viruses that make these RNAs include adenovirus, Epstein-Barr virus, and hepatitis C virus. Adenovirus encodes two RNA polymerase III directed small RNAs, called virus-associated RNAs I and II (VAI and VAII). VAI RNA is required for efficient translation of viral and cellular mRNAs at late times during infection. VAII RNA is synthesized in lower amounts in infected cells. Virus deleted for the VAI gene replicates poorly, while virus deleted for VAII does not have a detectable phenotype (Thimmappaya et al., 1982). VAI RNA is highly structured in solution (Furtado et al., 1989; Mellitis et al., 1990), and binds to PKR in competition with dsRNA (Galabru et al., 1989; Katze et al., 1987; Kostura and Mathews, 1989; Mellitis et al., 1990) but fails to efficiently lead to PKR autophosphorylation or activation, enabling protein synthesis to proceed normally (Ghadge et al., 1994). The ability to bind to PKR in competition with dsRNA is not sufficient for VAI function, since several mutants of VAI that bind PKR in vitro do not effectively support adenovirus replication (Mellitis et al., 1990). Deletion of the VAI gene leads to increased eIF2␣ phosphorylation, and changes a normally interferon-resistant virus into a virus that is sensitive to the effects of interferon. A deletion of the VAI gene can be complemented by over-expression of a non-phosphorylatable variant of eIF2␣ (Davies et al., 1989). VAI is 160 nucleotides in size and is present at late times after infection at approximately 108 molecules per cell. There are three main structural elements to VAI RNA, a terminal stem, an apical stem-loop, and a central domain. The ends of the RNA are imperfectly base-paired, and the central domain consists of two loops and a short stem-loop (Furtado et al., 1989; Mellitis and Mathews, 1988). The specific area of VAI that interacts with PKR is located in the apical stem of the RNA, and a second site exists in the central domain. PKR contains an Nterminal double-stranded RNA-binding domain that consists of two copies of a conserved dsRNA-binding motif, dsRBMI, and dsRBMII (Fierro-Monti and Mathews, 2000; Meurs et al., 1990). The apical stem can interact with either dsRBMI or dsRBMII of PKR, but only binding one domain at a time. The central domain interacts weakly with dsRBMI (Spanggord et al., 2002). The central domain of VAI contains a pair of tetranucleotides, GGGU and ACCC. These tetranucleotides are complementary and phylogenetically conserved. Mutational analysis revealed that substitutions that disrupt the stem structure abolish the func-

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tion of VAI RNA. This tetranucleotide stem comprises a critical element in the structure of the central domain as seen by nuclease sensitivity analysis (Ma and Mathews, 1996). During the protein–RNA interaction, PKR protects the proximal area of the apical stem structure, and an adjacent region in the central domain as determined by nuclease degradation analysis. From footprinting with probes specific for the RNA, PKR interacts directly with the apical stem and central domain of VAI RNA, strongly with the minor groove of the apical stemloop and a weaker interaction with the central domain (Clarke and Mathews, 1995). The Epstein-Barr virus encodes analogous RNAs that seem to have similar function. EBER-1 is 167 nucleotides in length and EBER-2 is 172 nucleotides in size. EBV is distinctly different virus than adenovirus, in terms of life cycle and gene expression, however, there are some commonalities between EBER RNAs and VA RNAs. EBER-1 and EBER-2 RNA were found to bind PKR in vitro (Clarke et al., 1991; Sharp et al., 1993). The Kd for VAI and the EBERs are similar, in the nanomolar range. Like VA RNA, there is significant secondary structure that is involved in this binding, mainly double-stranded stem and stem-loop regions (Glickman et al., 1988). Also, like VA RNAs, EBERs are not capped at their 5 ends, nor are they polyadenylated on their 3 ends, but end in stretches of oligo-(U) (Clemens, 1993; Clemens et al., 1994). EBER RNAs are found in the nucleus and cytoplasm of infected cells (Schwemmle et al., 1992), as is VAI RNA (Jimenez Garcia et al., 1993). EBERs are thought to have a similar function as VA RNAs because of the observation that in adenovirus mutants lacking the VAI gene, expression of the EBER genes could at least partially complement (Bhat and Thimmappaya, 1985). There is evidence that shows EBER-1 has a direct effect on PKR in vitro, since EBER-1 can reverse the inhibitory effects of dsRNA on protein synthesis (Clarke et al., 1990a,b). EBERs have been shown to bind to the ribosomal protein L22 (Toczyski et al., 1994). However, the role of at least the Epstein-Barr virus encoded small RNAs in infected cells is unclear since deletion of the gene for these RNAs has no effect on sensitivity of the virus to interferon-treatment (Swaminathan et al., 1992). Hepatitis C virus (HCV) has numerous mechanisms to escape the host interferon system, one of which is the inhibition of PKR by the internal ribosome entry site (IRES) on the genomic RNA. The IRES mediates translation of the genome into a large polyprotein. The IRES has an extensive secondary structure, which has been shown to bind to many cellular factors such as the La antigen, eukaryotic initiation factor 3, polyprimidine tract binding protein, and the 40s ribosomal subunit (Ali et al., 2000; Anwar et al., 2000; Fukushi et al., 1999, 2001; Lytle et al., 2002; Pestova et al., 1998; Sizova et al., 1998). The HCV IRES binds to PKR in competition with dsRNA and prevents the autophosphorylation and activation of PKR in vitro (Vyas et al., 2003). It is also worth discussing that viral and cellular RNAs exist with secondary structures that can bind to, and activate PKR. The human immunodeficiency virus type 1 (HIV-1) TAR stem-loop structure present at the 5 end of all HIV-1 mRNA transcripts is able to bind to PKR (Gatignol et al., 1991; Gunnery et al., 1992,

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1990; Maitra et al., 1994; Park et al., 1994; Schroder et al., 1990; Sengupta and Silverman, 1989; Silverman and Sengupta, 1990). PKR binds to TAR with 100-fold lower affinity than either fully duplexed RNA or VAI RNA (McCormack et al., 1992). The HIV-1 TAR RNA was originally thought to inhibit PKR (Gunnery et al., 1992, 1990), but there was conflicting data that showed it could possibly act as an activator (Sengupta et al., 1990; Sengupta and Silverman, 1989). It was ultimately shown that TAR RNA indeed activates PKR, suggesting a mechanism to control the virus through the interferon system (Gunnery et al., 1992; Maitra et al., 1994). The cellular IFN-␥ mRNA has also been shown to activate PKR in a localized area of the cell (Ben-Asouli et al., 2002). The pseudoknot present in the 5 untranslated region of the mRNA is responsible for this effect. This mechanism seems to function to adjust the translation levels of IFN-␥ in cells. To further highlight the importance of secondary structure, any mutation that disrupted the pseudoknot led to increased levels of IFN-␥ mRNA translation (Ben-Asouli et al., 2002). The ability of the RNA to bind to dsRBMI or dsRBMII of PKR contributes to the ability of the RNA to either activate or inhibit PKR. Biochemical studies suggest that dsRBMII of PKR is acting as an autoinhibitor domain, masking the kinase domain in the inactive conformation (Nanduri et al., 2000; Wu and Kaufman, 1996). The current hypothesis is that PKR activation requires simultaneous binding to both dsRBMs, which relieves the kinase domain of interaction with dsRBMII, leading to kinase activation and autophosphorylation (Nanduri et al., 2000). HIV-1 TAR RNA has binding sites for both dsRBMI and dsRBMII, which can support simulatenous binding of these motifs. Adenovirus VAI RNA has a single major binding site that can be occupied by either dsRBM. These results correlate with the model of simultaneous binding to the dsRBMs leads to activation of PKR, and binding to one of the dsRBM leads to inhibition of PKR (Spanggord et al., 2002). 4. PKR interaction Dimerization of PKR is necessary for trans-autophosphoryation on T446, which leads to activation of the kinase domain responsible for eIF2␣ phosphorylation (Romano et al., 1998a). It is unclear whether dimerization occurs as a result of bridging through dsRNA (Zhang et al., 2001) or through protein–protein interactions via the dsRNA-binding domain (Patel et al., 1995). Both DNA and RNA viruses employ several mechanisms to inhibit PKR dimerization, which inhibits PKR activation in the presence of dsRNA. The interferon sensitivity determining region (ISDR) of NS5 A, from hepatitis C, has been shown to interact with PKR (Gale et al., 1997). Binding studies in yeast have identified that interaction between PKR and NS5A occurs through amino acids 244–296 and amino acids 2209–2274 of PKR and NS5A, respectively (Gale et al., 1998b). Since dsRNA-independent dimerization of PKR is mediated through amino acids 244–296 (Tan et al., 1998), binding of NS5A to PKR directly inhibits PKR dimerization and activation. Recently it has been suggested that NS5A–PKR interaction may be involved in liver carcinoma (De

Mitri et al., 2002; Gimenez-Barcons et al., 2005). Hepatitis C encodes a second PKR inhibitor, the envelope glycoprotein E2, which contains a 12 amino acid sequence that has homology to the phosphorylation site of eIF2␣ (Taylor et al., 1999). E2 does not function as a pseudosubrate for PKR, but rather leads to inhibition of PKR activation. Glycosylated E2 localizes to the ER while unglycosylated E2 localizes to the cytosol. It is the unglycosylated form of E2 that interacts with PKR within the cytosol of infected cells (Pavio et al., 2002). Additionally, E2 is able to interact with and inhibit the eIF2␣ kinase PERK (PKR-like ER kinase) (Pavio et al., 2003). PERK is localized to the lumen of the ER and activated upon ER stress. Once activated, typically due to ER stress or protein misfolding, the cytoplasmic kinase domain of PERK is able to phosphorylate eIF2␣, thus causing an inhibition of translation (Harding et al., 1999). In addition to binding and sequestering dsRNA, the vaccinia virus protein E3L is capable of binding and inhibiting PKR directly (Romano et al., 1998b). In yeast, PKR inhibition was found to be dependent upon K167 and R168, which are located within the C-terminal dsRNA binding motif of E3L. Immunoprecipitation experiments in cells infected with vaccinia virus revealed that the PKR–E3L interaction is not based upon dsRNA bridging (unpublished observations). The N-terminus of E3L is not required for PKR interaction, but its presence stabilizes the E3L–PKR interaction (Romano et al., 1998b). In cells in culture, the N-terminus of E3L was shown to be required for PKR inhibition at very late times post-infection (Langland and Jacobs, 2004). Interestingly PKR activation and eIF2␣ phosphorylation within cells infected with virus deleted of the N-terminus of E3L did not result in a global shut off of translation and synthesis of viral proteins was unaltered. The N-terminus of E3L is required for pathogenesis in mice and the reduced pathogenesis of virus containing an N-terminal deletion of E3L may be a result of PKR activation within infected tissues (Brandt and Jacobs, 2001; Langland and Jacobs, 2004). Although less well characterized, human herpes virus-8 (HHV-8) encodes four homologues of interferon regulatory factors (IRFs). vIRF-2 is a 163 amino acid protein that is constitutively expressed in HHV-8-positive B-cell lymphomas (Burysek et al., 1999). In vitro binding studies have shown that vIRF-2 is capable of binding to PKR (Burysek and Pitha, 2001). This interaction was sufficient to inhibit PKR autophosphorylation in the presence of dsRNA. As expected, phosphorylation of eIF2␣ was not detected. These results suggest that regulation of PKR may be involved in the formation of latent herpes infections. Inhibition of PKR is not limited to mammalian viruses. The baculovirus, Autographa californica, encodes a kinase-related protein (PK2) with an inactive kinase domain lacking an ATP binding motif. PK2 is capable of blocking eIF2␣ phophorylation in infected insect cells. In yeast, PK2 is able bind to and prevent the activation of human PKR through the formation of inactive heterodimers (Dever et al., 1998). Despite the ability to bind to PKR, PK2 does not act as a pseudosubstrate for PKR (Li and Miller, 1995). Cellular proteins can also be recruited by viruses to inhibit PKR function. Influenza virus uses the cellular chaperone p58 as a PKR inhibitor. p58 is a member of DnaJ family of chaperones

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and encodes nine tetratricopeptide repeat motifs and a J-domain. In uninfected cells, p58 is complexed with Hsp40 (Melville et al., 1997) and p52 (Gale et al., 1998a). Upon infection with influenza virus, p58 becomes activated leading to disassociation from Hsp40 (Melville et al., 1999). The activated p58 is then able to interact with PKR by binding to amino acids 244–296, which prevents dimerization (Tan et al., 1998) and activation of PKR (Lee et al., 1992). 5. eIF2␣ homologues The vaccinia virus K3L gene encodes an eIF2␣ homolog that has been shown to act as a pseudo-substrate competitive inhibitor of PKR and K3L can also likely inhibit PKR autophosphorylation (Beattie et al., 1991; Carroll et al., 1993; Langland and Jacobs, 2002). The K3L gene encodes a relatively small gene product of 88 amino acids in length which has approximately 30% amino acid identity to the N-terminus of eIF2␣. S51, the residue present in eIF2␣ that is phosphorylated by PKR, is not present in K3L. The homology between vaccinia virus K3L and eIF2␣ proteins does not include the 19 residues flanking the S51 phosphorylation site which are perfectly conserved in cellular eIF2␣s from yeast to humans. Instead, the greatest homology between eIF2␣ and K3L residues in a 12 amino acid sequence located approximately 30 amino acids downstream from S51 (residues 72–83). Within this region, a pentapeptide motif, KGYID, has been shown to be important for the interaction of K3L of vaccinia virus with PKR allowing for the continuation of protein synthesis (Dar and Sicheri, 2002). Data by Beattie et al. (1995) suggest that K3L may function very early during vaccinia virus replication, even before the presence of detectable dsRNA in the infected cell. As mentioned previously, PKR is composed of two Nterminal dsRNA-binding domains (amino acids 55–75 and 145–166) and a C-terminal catalytic domain containing eleven conserved kinase subdomains characterizing PKR as a serine/threonine kinase (Meurs et al., 1990). Between subdomains IV and V there is a 24-amino acid kinase insert and a highly conserved LFIQMEFCD motif. This LFIQMEFCD motif is indispensable for kinase activity and is found in all known eIF2␣ kinase family members (Koromilas et al., 1992). As expected, K3L binding to PKR does not require the dsRNAbinding domains present on PKR. Somewhat unexpected, the kinase insert domain of PKR is also dispensable for K3L interaction (Craig et al., 1996). However, K3L does have the ability to inhibit eIF2␣ phosphorylation by all the known eIF2␣ kinases, including GCN2, HRI, and PERK (Carroll et al., 1993; Qian et al., 1996; Sood et al., 2000). Mutational analysis of PKR suggests that amino acids 367–415 of PKR contain the minimal K3L binding site. This region lies between kinase domains V and VI and forms an ␣-helical structure between two ␤-sheets (DeBont et al., 1993). Ranaviruses (genus Ranavirus, family Iridoviridae) are large dsDNA viruses that are particularly pathogenic to amphibians and fish (reviewed in Chinchar, 2002). Replication of ranaviral genomic DNA occurs primarily in the cytoplasm of an infected cell; however, initial rounds of viral DNA replication and early

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viral gene transcription takes place in the nucleus of infected cells (Chinchar, 2002). Several ranavirus contain a gene that encodes an eIF2␣ homologue having a molecular weight of 31 kDa. Alignment of the ranavirus homolog (ReIF2H) with eIF2␣ reveals approximately 35% sequence identity at the Nterminus of the protein and negligible identity at the C-terminal extension region of the protein (Essbauer et al., 2001). A corresponding amino acid to S51 of cellular eIF2␣ is not present in ReIF2H. However, the pentapeptide KGYID motif, which is important for the interaction of K3L of VV with PKR, is modified to KGYVD in all ranavirus eIF2␣ homologue amino acid sequences. Therefore, the ranavirus homolog of eIF2␣ may act as a competitive inhibitor of PKR, binding to PKR and inhibiting eIF2␣ phosphorylation allowing the continuation of protein synthesis in a manner similar to K3L in vaccinia virus infected cells. Alternatively, the ranavirus homolog, given its large size, may also act as an as an alternative eIF2␣-like translation initiation factor that helps recycle eIF2B allowing ternary complex formation to continue. Further molecular characterization of this protein will shed light into the precise mechanism of PKR regulation. HIV encodes a gene expressing a transactivator of transcription, Tat. HIV Tat is a 14 kDa protein that has been shown to bind to HIV TAR RNA forming an important interaction that increases virus replication greater than 100-fold by increasing transcription of HIV mRNA (Baba, 2004; Cullen, 1993). Tat has been shown to bind to cellular PKR and act as a pseudosubstrate to cellular eIF2␣ (Brand et al., 1997; McMillan et al., 1995). HIV Tat is phosphorylated at S62, T64, and S68 (Brand et al., 1997) by PKR thereby inhibiting cellular eIF2␣ phosphorylation and subsequent inhibition of protein synthesis. The Tat-TAR RNA interaction is enhanced as a consequence of HIV Tat phosphorylation by PKR (Endo-Munoz et al., 2005). This suggests that HIV has evolved a mechanism that takes advantage of a cell’s anti-viral defenses, where PKR is primed for action, enhancing rather than inhibiting its replication and survival. 6. PKR degradation Polioviruses encode proteases that help regulate cellular gene expression by a variety of mechanisms (reviewed in Bedard and Semler, 2004). PKR is highly activated in a poliovirusinfected cell resulting in increased levels of eIF2␣ phosphorylation (Black et al., 1989). However, PKR is also rapidly degraded in a poliovirus-infected cell. The degradation of PKR has been shown to require both RNA and protein components, although a detailed understanding of the mechanism of PKR degradation has yet to be determined (Black et al., 1993). Degradation of PKR in infected cells does not require PKR activation; however, viral replication is necessary for PKR degradation. Although polioviruses encode a number of viral proteases necessary for viral protein processing, it is believed that a cellular protease, activated after a poliovirus infection, is responsible for PKR degradation. Two proposed mechanisms include the degradation of PKR after dsRNA has bound to the enzyme or via a dsRNAprotease complex that binds to PKR causing degradation (Black et al., 1993).

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7. eIF2␣ phosphatase As discussed earlier, herpes simplex virus encodes the Us11 gene product which acts as an inhibitor of PKR by interaction with dsRNA and/or PKR. In a wt herpes virus infection, the Us11 protein is a late gene product and is important in countering host defenses and maintaining viral translation at this stage in the virus life cycle (Mulvey et al., 2003). A second viral gene product, ␥1 34.5, appears to be required during earlier stages of the viral life cycle to inhibit PKR activity. The ␥1 34.5 gene product shares homology with the cellular GADD34 protein (Chou and Roizman, 1994). GADD34 has been shown to function as a regulatory subunit for the cellular protein phosphatase 1␣ (PP1␣) forming a GADD34/PP1 holophosphatase complex. GADD34 acts to target the activity of PP1␣ to phosphorylated eIF2␣ thereby maintaining pools of active, unphosphorylated eIF2 (He et al., 1997). During the early life cycle of a herpes virus infection, PKR becomes active leading to the phosphorylation of eIF2␣ (Chou et al., 1995). The herpes virus ␥1 34.5 protein functions in a manner similar to GADD34 in binding to PP1␣ and promoting the dephosphorylation of eIF2␣. In the absence of the viral ␥1 34.5 protein, PKR is activated, eIF2␣ becomes and remains phosphorylated, leading to a block in late viral protein synthesis (Chou et al., 1995). While Us11 and ␥1 34.5 are both capable of inhibiting the translational block induced by PKR, they are not likely redundant functions encoded by herpes simplex viruses. The ␥1 34.5 protein appears to be required to maintain unphosphorylated, active eIF2␣ during the period of virus replication prior to Us11 accumulation. After virus replication has progressed into the later phase, Us11 appears as the major regulator of PKR signaling (Mulvey et al., 2003). The role of these temporally distinct PKR inhibitors in the virus life cycle remain unclear, but may be related to different levels or mechanisms of PKR activation. Indeed, the ␥1 34.5 protein is continuously shuttled between the nucleus, nucleolus, and cytoplasm suggesting differential roles of the protein in virus-infected cells (Cheng et al., 2002). Human papillomavirus (HPV) Type 18 has evolved a similar mechanism for modulating PKR activity. However, instead of encoding a homolog to GADD34, the E6 protein of HPV interacts with both GADD34 and PP1␣ to promote the dephosphorylation of eIF2␣ (Kazemi et al., 2004). The mechanism utilized by E6 to promote eIF2␣ dephosphorylation through GADD34/PP1␣ is unknown, but may be related to localization of high-risk HPV-18 E6 protein to both the nucleus and cytoplasm as opposed to the low-risk HPV-11 E6 which is predominantly localized to the nucleus (Kazemi et al., 2004). The large-T antigen of SV-40 virus, which is functionally related to the HPV E6 protein, also functions to inhibit the PKR response at a step downstream of PKR activation (Rajan et al., 1995). In SV-40 virus infected cells, PKR is highly phosphorylated and active, but does not lead to a block in viral protein synthesis (Rajan et al., 1995). Cells expressing large-T antigen (COS-1) are able to rescue replication of herpes simplex virus deleted of ␥1 34.5 as compared to the parental cells (CV-1) in which translation is blocked following infection with this virus (Randdazzo et al., 1997). This translational block is restored when the herpes

virus infections are performed in the presence of a phosphatase inhibitor, supporting the role of PP1␣ in the large-T antigen regulation of the PKR response (Randdazzo et al., 1997). 8. Conclusions The ability of the cell to recognize viral danger signals, including dsRNA, leading to the initiation of the anti-viral response is very effective at blocking viral replication. Given the diversity of viruses, it is not surprising that many viruses have evolved a multitude of mechanisms to counteract the cellular response. Since most viruses, including those with either RNA or DNA genomes, synthesize dsRNA at some stage during replication, these counterdefenses encompass virtually every type of known virus. The cellular PKR response is one of the most well characterized anti-viral pathways. As discussed, viruses have developed mechanisms to inhibit the PKR response at virtually every stage in the pathway, including recognition of the dsRNA activator, blocking PKR activation, or leading to PKR degradation, or inhibiting substrate phosphorylation. Yet, viral inhibition of the anti-viral response is not limited to PKR alone. Many viruses interact with or inhibit the interferon response at many other points including interferon induction, interferon receptor binding, or interferon signal transduction cascades (reviewed in Goodbourn et al., 2000). It is clear that a constant battle is waged during a viral infection with both the host and virus attempting to keep the upperhand and stay one step ahead. References Ali, N., Pruijn, G.J.M., Kenan, D.J., Keene, J.D., Siddiqui, A., 2000. Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation. J. Biol. Chem. 275, 27531–27540. Anwar, A., Ali, N., Tanveer, R., Siddiqui, A., 2000. Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. J. Biol. Chem. 275, 34231–34235. Baba, M., 2004. Inhibitors of HIV-1 expression and transcription. Curr. Top. Med. Chem. 4, 871–882. Beattie, E., Paoletti, E., Tartaglia, J., 1995. Distinct patterns of IFN sensitivity observed in cells infected with vaccinia K3L- and E3L-mutant viruses. Virology 210, 254–263. Beattie, E., Tartaglia, J., Paoletti, E., 1991. Vaccinia virus-encoded eIF-2 alpha homolog abrogates the antiviral effect of interferon. Virology 183 (1), 419–422. Bedard, K.M., Semler, B.L., 2004. Regulation of picornavirus gene expression. Microbes Infect. 6, 702–713. Ben-Asouli, Y., Banai, Y., Pel-Or, Y., Shir, A., Kaempfer, R., 2002. Human Interferon-␥ mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR. Cell 108, 221–232. Bhat, R.A., Thimmappaya, B., 1985. Construction and analysis of additional adenovirus substitution mutations confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus. J. Virol. 56, 750–756. Black, T.L., Barber, G.N., Katze, M., 1993. Degradation of the interferoninducible 68,000-Mr protein kinase by poliovirus requires RNA. J. Virol. 67, 791–800. Black, T.L., Safer, B., Hovanessian, A., Katze, M., 1989. The cellular 68,000Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation. J. Virol. 63, 2244–2251.

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