Hepatitis C Virus Envelope Protein E2 Does Not Inhibit PKR by Simple Competition with Autophosphorylation Sites in the RNA-Binding Domain

Generation of radiolabeled peptides.

PKR, purified to the mono-S stage from IFN-α-treated 293 cells (26), was activated in vitro in the presence of reovirus dsRNA (provided by A. Shatkin) and [γ-³²P]ATP under conditions described previously (51). Radiolabeled PKR was immunoprecipitated with a polyclonal antibody (21) and was eluted from protein A-Sepharose beads with formic acid, digested with cyanogen bromide (CNBr) as described previously (51), and lyophilized. The CNBr digestion products were separated by high-performance liquid chromatography (HPLC) (51), fractions were collected and counted by Cerenkov radiation, and the radioactive peaks were pooled. Radiolabeled peptides were resolved in Tris-Tricine gels (47); the gels were fixed, dried, and exposed to film for autoradiography.

Secondary peptidase digestion.

Secondary digestion of radiolabeled PKR peptides was performed with the following enzymes: endoproteinases Lys-C, Asp-N, and Arg-C (sequencing grade; Boehringer Mannheim); trypsin (tolylsulfonyl phenylalanyl chlomethyl ketone treated; Sigma); chymotrypsin (TLCK [Nα-p-tosyl-l-lysine chloromethyl ketone] treated; Sigma); and Staphylococcus aureus V8 (Sigma). Digestion with Arg-C was conducted in accordance with the manufacturer's specifications, and digestion with Lys-C, Asp-N, trypsin, and chymotrypsin was performed as described previously for Lys-C (51).

Radioactive sequence analysis.

Secondary digests were lyophilized, resuspended in 60% acetonitrile, and coupled to arylamine-derivatized polyvinylidene difluoride (PVDF) membranes (Sequalon AA; Millipore) with carbodiimide. PVDF-bound peptides were subjected to repetitive Edman degradation reactions on a protein sequencer (Applied Biosystems; 473A) (45), and radioactivity was monitored as described above.

Kinase assays.

For peptide phosphorylation kinetics and phosphoamino acid analysis, mono S-purified PKR (26) was added to a kinase reaction mixture (51) and the mixture was incubated with dsRNA and [γ-³²P]ATP at 30°C for 20 min. For preincubation assays, peptides were incubated with PKR before addition of dsRNA and ATP. Peptide P1 consists of amino acids 77 to 113 of PKR: H-KEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKR-OH. Peptide P2 consists of amino acids 230 to 254 of PKR: H-NGLRNNQRKAKRSLAPRFDLPDMKE-OH. Incubations were conducted at 30°C for 20 min unless otherwise specified, and peptides were analyzed in sodium dodecyl sulfate (SDS)–20% polyacrylamide gels, which were fixed, dried, and exposed to Kodak XAR-5 film for autoradiography. Phosphoamino acid analysis was performed as described previously (51). For assays with PKR from yeast, extracts were prepared from H1817 cultures induced with synthetic minimal medium containing 10% galactose and 2% raffinose as described previously (51) and protein concentrations were determined using a Coomassie blue dye-binding assay (Bio-Rad). Yeast extracts (50 μg of protein) were immunoprecipitated with a polyclonal anti-PKR antibody (21) and protein A-Sepharose. The immunoprecipitates were washed and then incubated as described above. To assay eIF2 phosphorylation, eIF2 was added after the initial incubation (as described in reference 33) and incubation resumed for another 20 min at 30°C.

Mutagenesis.

Sites of autophosphorylation were mutated by the oligonucleotide-directed site-specific mutagenesis procedure (1, 60). Serine and threonine residues were changed to alanine to generate RBD mutant S83A/T88A/T89A/T90A, triple mutant S242A/T255A/T258A (51), and combination mutant Tri-RBD, which has all seven sites changed to alanine. Mutations were made in pUC19D (21), which contains the full-length PKR cDNA.

PKR expression in S. cerevisiae.

PKR mutants were subcloned into pEMBLyex4 as described previously (43). Plasmids bearing the autophosphorylation site mutations were introduced into yeast strains H1816 and H1817 (13), which both have GCN2 deleted; in addition, H1817 has mutant eIF2α (S51A).

Phosphatase treatment.

Total cellular protein extracts (15 μg) from transformed H1817 cells were treated with lambda phosphatase in accordance with the manufacturer's specifications (New England Biolabs) or were incubated in phosphatase buffer alone and were resolved in SDS–10% polyacrylamide gels. The proteins were then transferred to nitrocellulose for Western blotting. The membrane was incubated in 5% nonfat milk in Tris-buffered saline–Tween 20 (51) and probed with a monoclonal anti-PKR antibody (30), and the proteins were visualized by chemiluminescence (ECL; Amersham).

PKR expression in mammalian cells.

PKR mutants were subcloned into pcDNA3 (Invitrogen). Monolayers of COS1 cells in 10-cm-diameter plates were transfected in duplicate with plasmids (15 μg) bearing the autophosphorylation site mutations, as well as a transfection efficiency control, by the calcium phosphate method (46). Cytoplasmic extracts were prepared at 48 h posttransfection, and total protein concentration was measured by a Coomassie blue assay (Bio-Rad). To assess the amount of PKR protein that was present in each sample, 100 μg of total protein was examined by Western blotting as described above. The upper half of the blot was probed with the PKR monoclonal antibody (30), and the lower half of the blot was probed with an actin polyclonal antibody (Santa Cruz Biotechnology).

HCV E2 peptide phosphorylation inhibition assay.

PKR from yeast H1817 strain was autophosphorylated as in the kinase assay described above. Glutathione-coated Sepharose beads were used to purify bacterially expressed glutathione S-transferase (GST) protein or GST fused to the HCV E2 protein as described previously (52). The protein was eluted with glutathione, and 0.5 μg was incubated with the immunoprecipitated PKR initially for 20 min at 30°C. Poly(I:C) and [γ-³²P]ATP were added, and the incubation continued for another 10 min. Then peptide (3 μg) P1 or P2 was added, and incubation continued for an additional 10 min at 30°C. The reactions were analyzed as described above.

HCV E2 binding and peptide competition assay.

Histidine-tagged PKR was bound to nickel-Sepharose beads as described previously (52). HCV E2 was synthesized in vitro with the T7 TNT-quick (Promega) coupled transcription-translation system in the presence of [³⁵S]methionine (New England Nuclear, DuPont). Increasing amounts of synthetic peptide P1 or P2 were added to the PKR-bound beads in 0.25 M Tris (pH 8.0)–0.1% NP-40–10 mM imidazole. Radiolabeled E2 (5 μl of the in vitro translation reaction mixture) was then added, and the mixture was incubated on ice for 2 h. The beads were washed five times in the incubation buffer (increased to contain 0.3% NP-40). The last wash was removed, and adherent labeled E2 protein was analyzed by gel electrophoresis and autoradiography.

Identification of PKR autophosphorylation sites.

To identify the sites that are autophosphorylated when PKR is activated in vitro, we labeled PKR with [γ-³²P]ATP in the presence of reovirus dsRNA and then fragmented the protein with cyanogen bromide as described previously (51). The predicted pattern of PKR cleavage at methionine residues with cyanogen bromide is depicted in Fig. 1A. Nine peptides ranging from 1.0 to 11.6 kDa are expected if cleavage is complete. The products were separated by HPLC. Fractions were collected, counted by Cerenkov radiation, and pooled as shown in Fig. 1B. Four major peaks, peaks A to D, were obtained. Analysis of radioactive peaks A and B was reported previously (51). When pool C, acquired from the most prominent peak, was resolved in a Tris-Tricine gel, phosphorylated peptides migrating with apparent molecular masses of 10.3 and 15.5 kDa were observed together with a minor peptide at ∼21 kDa (Fig. 1C, lane 2). Two of these peptides (15.5 and 21 kDa) probably represent partial digestion products, as the largest peptide predicted after cyanogen bromide digestion is 11.6 kDa (Fig. 1A) and secondary digestion of the 15.5-kDa peptide with cyanogen bromide yielded only a 10.3-kDa radioactive peptide (data not shown).

Phosphoamino acid analysis demonstrated that both phosphoserine and phosphothreonine were present in the 15.5- and 10.3-kDa bands (51). Pool D contained similar peptides, although the 10.3-kDa peptide was relatively less abundant (51), and pools C and D gave identical results upon subsequent analysis. A 10.3-kDa peptide was also released by cyanogen bromide treatment of phosphorylated p20, a subfragment of PKR containing residues 1 to 184 (data not shown). As reported previously, recombinant p20 can be phosphorylated by PKR (48). This finding suggested that the 10.3-kDa peptide comes from the dsRNA-binding domain of PKR.

No peaks of radioactivity were released upon the direct sequencing of the material in pools C or D. Radioactivity did remain bound to the filter, however, indicating that the N terminus of this peptide was blocked or that the phosphoamino acids were located past the point of efficient sequencing (∼25 to 30 amino acids). To test the latter possibility, material in the two pools was subjected to secondary digestion with one or two of six specific proteases. The positions of labeled amino acids were identified by counting the radioactivity of the products of sequential Edman degradation reactions. Radioactive peaks were obtained, and the results are tabulated in Fig. 1D. A summary of the sequencing data is shown in Fig. 1E. Comparison with the PKR sequence indicated that serine 83 and one or more of threonines 88, 89, and 90 were phosphorylated.

Several factors make it difficult to discriminate unambiguously among the three threonine residues, most notably the occurrence of multiple neighboring cleavage sites and the propensity of the proteases for partial cleavage. Two of the proteases used for secondary digestion recognize lysine (trypsin, Lys-C) or leucine (chymotrypsin), and incomplete digestion products were obtained because of the presence of two lysines and three leucines just upstream of the phosphorylation site (Fig. 1E). These proteases cleave at the C-terminal side of amino acids in a peptide, and in a run of identical residues they tend to cleave preferentially between the residues rather than after them. Thus the C-terminal residue in the run may not be recognized by the protease, leaving that amino acid on the N terminus and causing a shift or “stuttering” in the profiles of radioactivity obtained after Edman degradation reactions. Nevertheless, detailed examination of the profiles suggested that threonines 88, 89, and 90 are all phosphorylated. In radioactive sequencing experiments of this kind, a declining trail of radioactivity is generally observed in the Edman degradation cycles that follow the position of the radiolabeled amino acid. The pattern of increased yield followed by some carryover or “lag” derivatives (due to incomplete release of the phosphoamino acid derivative from the filter) is often used as a criterion to assess whether the yield of the phosphoamino acid is significantly greater than background. In the sequencing reactions of pools C and D with chymotrypsin, V8, and Lys-C (Fig. 1D), however, the radioactive peaks corresponding to the first threonine position were followed by two higher peaks, corresponding to the second and third threonine, and then a trail of radioactivity, indicating that all three threonines are phosphorylated. Thus we conclude that four residues, serine 83 and threonines 88, 89, and 90, located in the spacer between the two dsRBMs, are probably all phosphorylated.

Phosphorylation of PKR peptides in vitro.

To corroborate the sequencing data, we tested two synthetic peptides corresponding to the RBD spacer region (P1; amino acids 77 to 113) and the central region of PKR surrounding serine 242 (P2; amino acids 230 to 254). Both P1 and P2 were phosphorylated efficiently by PKR (Fig. 2A and B), verifying that they are substrates for phosphorylation by PKR and that these sites can be recognized by intermolecular phosphorylation. Phosphoamino acid analysis (Fig. 2C) revealed that P1 is labeled on both the serine and the threonine residues whereas P2 is labeled on serine only, as expected. The phosphorylation of P1 and P2 continued after PKR autophosphorylation reached saturation (Fig. 2B); under these conditions, neither of the peptides interfered with PKR autophosphorylation (data not shown), suggesting that they do not compete effectively with the activation reaction.

Autophosphorylation sites in the RNA-binding domain do not affect PKR function in yeast.

To examine the significance of the four autophosphorylation sites in the RBD spacer, the serine and three threonine residues were all changed to alanine (Fig. 3A). This PKR RBD mutant (S83A/T88A/T89A/T90A) was tested in yeast together with wild-type PKR and three other mutants: K296R, which is inactive as a result of a mutation in the ATP-binding/phosphotransfer site (23); Triple mutant S242A/T255A/T258A, which is partially inactivated by three mutations in the central region (51); and Tri-RBD, which contains all seven of the mutations in the Triple and RBD mutants.

Transformants of yeast strain H1816, which carries a wild-type eIF2α allele, were grown on selective media to analyze mutant kinases for their activity (13). Yeast cells that express fully active PKR cannot grow on galactose medium (SGAL) due to overexpression of PKR (driven by the Gal promoter) and consequent phosphorylation of eIF2α. In contrast, expression of PKR at much lower levels on dextrose-containing medium allows cells to grow in the presence of 3-aminotriazole (SD+3AT), an inhibitor of histidine biosynthesis, because a moderate level of eIF2 phosphorylation allows for GCN4 translation (Fig. 3B). Cells transformed with inactive PKR mutants, such as K296R, grow on both synthetic minimal dextrose (SD) and SGAL media but not on SD+3AT (Fig. 3B). The phenotype of yeast cells transformed with the RBD mutant was indistinguishable from that of cells transformed with wild-type PKR, suggesting that the RBD mutant is fully active. Furthermore, the slow-growth phenotype conferred by the Triple mutant in SD+3AT medium (51) was not exacerbated in the Tri-RBD mutant (Fig. 3B). This slow-growth phenotype is attributable to mutations in the central region, most notably T258A, which is important to the activation of PKR (51). These data indicate that phosphorylation of residues in the RBD spacer is not required for PKR function in yeast cells.

Phosphorylation state of PKR in vivo.

To determine specifically whether these residues are required for autophosphorylation of PKR in vivo, yeast strain H1817 was transformed with wild-type or mutant forms of PKR. This strain contains the S51A mutation of eIF2α, allowing for high expression of both active and inactive PKR (13). The yeast was grown under inducing conditions (43), and extracts were treated with lambda phosphatase or were incubated with phosphatase buffer alone. These extracts were then resolved in an SDS gel, transferred to nitrocellulose, and probed with monoclonal anti-PKR antibody.

Wild-type PKR showed a distinct increase in gel mobility upon phosphatase treatment (Fig. 4A), as observed previously (42). Catalytically inactive mutant K296R migrated with the phosphatase-treated form and showed no shift in mobility upon phosphatase treatment, indicating that yeast does not contain endogenous kinases that phosphorylate PKR. When the Tri-RBD mutant, carrying seven alanine substitutions, was treated with phosphatase, it showed a small but reproducible shift in mobility (Fig. 4A), indicating that the mutant kinase is phosphorylated to some extent in vivo. This conclusion was confirmed by labeling with ³²P in vivo and analysis of peptides derived by cyanogen bromide cleavage. Consistent with the removal of the seven sites of phosphorylation in the RBD and central region, phosphopeptides of 3.5, 10.3, and 15.5 kDa were missing. The digest did, however, yield the 21-kDa peptide and a peptide migrating faster than 10 kDa (data not shown), presumably attributable to sites in the activation loop of the kinase domain (42) or to other potential phosphorylation sites reported previously (58).

PKR kinase activity in vitro.

Although these assays suggested that the RBD mutation did not affect kinase activity or autophosphorylation in yeast, we wanted to measure the activities of the mutant kinases directly. PKR was isolated by immunoprecipitation from extracts of H1817 strains carrying mutant or wild-type forms of the enzyme. The amount of PKR protein recovered was normalized by immunoblotting (as shown in Fig. 4A), and its activity was assessed in kinase assays. Figure 4B shows that wild-type PKR was active for autophosphorylation and for phosphorylation of eIF2α, while K296R was inactive in both respects. The Triple mutant was weakly active, and S242A was highly active, as expected from published results (51). Consistent with the in vivo data, the RBD mutations alone did not affect PKR autophosphorylation or the phosphorylation of eIF2α and modestly exacerbated the effects of the Triple mutation when the mutations were present together in Tri-RBD (Fig. 4B). Thus, the RBD sites influence kinase activity modestly in vitro.

In view of their location, we also considered the possibility that the RBD phosphorylation sites might play a role in RNA binding. To address this possibility, we measured the ability of immobilized PKR, isolated by immunoprecipitation from yeast as described above, to interact with ³²P-labeled synthetic dsRNA. Mutations in the RBD did not significantly alter the retention of dsRNA by PKR (data not shown).

Expression of PKR mutants in mammalian cells.

To examine the effect of the RBD mutations in mammalian cells, we measured the levels of mutant PKR during transient expression assays (Fig. 5). Cytoplasmic extracts were prepared from transfected COS1 cells, and equal amounts of total proteins were resolved in a gel; PKR expression was monitored by Western blotting. Plates were transfected in duplicate, and the immunoblot was probed with antibodies against human PKR (top) and actin (bottom). As observed previously (42, 54) PKR mutant K296R was expressed at a much higher level than wild-type PKR, indicating that this mutant is unable to down-regulate its expression because it is inactive. Both the Triple and RBD mutants were expressed at very low levels, indicating that these mutants are active. The Tri-RBD mutant, however, was expressed at an intermediate level, lower than that for K296R but higher than those for the other mutants, indicating that this mutant is less active than either the Triple or RBD mutant but not as inactive as K296R. Thus, the RBD sites contribute to, but are not essential for, the activity of the kinase.

Peptide competition and HCV E2 inhibition.

We previously showed that HCV E2 binds to PKR and inhibits its kinase activity, as measured by its ability both to become autophosphorylated and to phosphorylate a histone H2a substrate (52). To determine whether HCV E2 can inhibit phosphorylation of peptides corresponding to its autophosphorylation sites, activated PKR was incubated with E2 in a kinase assay containing P1 or P2. Figure 6A shows that GST-E2 efficiently blocked the phosphorylation of peptide P1 (lane 6). The phosphorylation of P2 was also inhibited but to a lesser extent (lane 7), but GST protein did not significantly inhibit P1 or P2 phosphorylation (lanes 3 and 4). As discussed previously (52), GST-E2 did not affect PKR autophosphorylation in this system due to its preactivation state.

Deletion of or mutations in the PePHD in HCV E2 abrogate its functional interaction with PKR (52). We therefore tested the ability of the two PKR peptides to interfere with the binding of E2 to PKR. As shown in Fig. 6B, the interaction between PKR and HCV E2 was not blocked by either peptide P1 or P2. Concentrations of P1 as high as 60 mg/ml failed to prevent the binding of E2 to PKR. Thus E2 is a strong ligand for PKR and can compete with substrates that are trans-autophosphorylation sites, but it is not displaced from PKR by high concentrations of peptide substrates corresponding to these sites.