Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain

Abstract

Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation. We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain. Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known. Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast Saccharomyces cerevisiae. However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2. Another synthetic peptide, corresponding to the central region of PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently. Neither of the PKR peptides was able to disrupt the HCV E2-PKR interaction. Taken together, these results show that PKR is autophosphorylated on serine 83 and threonines 88, 89, and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of and competition with these autophosphorylation sites.

FIG. 1

CNBr digestion of PKR. (A) Sites of cleavage resulting from CNBr digestion of PKR are predicted, based on the specificity of CNBr, to cleave peptide bonds at the C-terminal side of methionine residues. (B) Radiolabeled phosphopeptides derived by CNBr cleavage of PKR were separated by reverse-phase HPLC. Fractions were collected and assayed for radioactivity. (C) Radiolabeled peptides resulting from CNBr cleavage of PKR were resolved in Tris-Tricine gels. Lane 1, digest of ³²P-PKR; lane 2: pool C. (D) Radioactive sequencing was performed after secondary digestion with the proteases listed. Radioactive peaks were obtained at the cycle numbers shown. The cycle numbers containing the largest amounts of radioactivity are underlined and in boldface. (E) Interpretation of the sequencing results. The amino acid sequence of a PKR peptide (residues 69 to 90) is shown. Arrows, protease recognition sites within the peptide for proteases Lys-C, trypsin, chymotrypsin, and V8. Numbers were obtained from analysis of Edman degradation reactions shown in panel D. ∗, autophosphorylated amino acids.

FIG. 1

CNBr digestion of PKR. (A) Sites of cleavage resulting from CNBr digestion of PKR are predicted, based on the specificity of CNBr, to cleave peptide bonds at the C-terminal side of methionine residues. (B) Radiolabeled phosphopeptides derived by CNBr cleavage of PKR were separated by reverse-phase HPLC. Fractions were collected and assayed for radioactivity. (C) Radiolabeled peptides resulting from CNBr cleavage of PKR were resolved in Tris-Tricine gels. Lane 1, digest of ³²P-PKR; lane 2: pool C. (D) Radioactive sequencing was performed after secondary digestion with the proteases listed. Radioactive peaks were obtained at the cycle numbers shown. The cycle numbers containing the largest amounts of radioactivity are underlined and in boldface. (E) Interpretation of the sequencing results. The amino acid sequence of a PKR peptide (residues 69 to 90) is shown. Arrows, protease recognition sites within the peptide for proteases Lys-C, trypsin, chymotrypsin, and V8. Numbers were obtained from analysis of Edman degradation reactions shown in panel D. ∗, autophosphorylated amino acids.

FIG. 1

CNBr digestion of PKR. (A) Sites of cleavage resulting from CNBr digestion of PKR are predicted, based on the specificity of CNBr, to cleave peptide bonds at the C-terminal side of methionine residues. (B) Radiolabeled phosphopeptides derived by CNBr cleavage of PKR were separated by reverse-phase HPLC. Fractions were collected and assayed for radioactivity. (C) Radiolabeled peptides resulting from CNBr cleavage of PKR were resolved in Tris-Tricine gels. Lane 1, digest of ³²P-PKR; lane 2: pool C. (D) Radioactive sequencing was performed after secondary digestion with the proteases listed. Radioactive peaks were obtained at the cycle numbers shown. The cycle numbers containing the largest amounts of radioactivity are underlined and in boldface. (E) Interpretation of the sequencing results. The amino acid sequence of a PKR peptide (residues 69 to 90) is shown. Arrows, protease recognition sites within the peptide for proteases Lys-C, trypsin, chymotrypsin, and V8. Numbers were obtained from analysis of Edman degradation reactions shown in panel D. ∗, autophosphorylated amino acids.

FIG. 2

Phosphorylation of peptides by PKR. (A) Kinetics of peptide phosphorylation. Reactions were stopped at different time points (a, 0 min; b, 4 min; c, 8 min; d, 16 min; e, 28 min; f, 44 min; g, 66 min; h, 108 min; I, 133 min; j, 190 min). (B) Quantitation of PKR autophosphorylation and peptide phosphorylation. Data from panel A are presented as percentages of maximal ³²P labeling. □, P1 phosphorylation; ■, PKR phosphorylation in the presence of P1; ▵, P2 phosphorylation; ▴, PKR phosphorylation in the presence of P2. (C) Phosphorylated peptides were subjected to phosphoamino acid analysis by hydrolysis, two-dimensional separation, and autoradiography. Positions of markers, detected by ninhydrin staining, are circled.

FIG. 3

PKR mutations and yeast growth. (A) Schematic of PKR autophosphorylation site mutations. Serine and threonine residues were changed to alanine to generate Triple and RBD mutants and the combination mutant; Tri-RBD, which has all seven sites changed to alanine. (B) Results of yeast growth assays. Transformed cells (H1816) were grown on SD medium and were replica plated to SGAL and SD+3AT medium for growth analysis (43). +, strong growth; ±, slow growth; −, no growth.

FIG. 4

Properties of PKR mutants. (A) PKR phosphorylation state. Total cellular protein extracts from transformed H1817 cells were treated with lambda phosphatase (+) or phosphatase buffer alone (−), resolved in SDS–10% polyacrylamide gels, and analyzed by Western blotting with a monoclonal anti-PKR antibody (30) and chemiluminescence detection. wt, wild type. (B) Kinase activity. Autophosphorylation of wild-type and mutant PKR and phosphorylation of eIF2α were conducted in vitro. Kinase assay mixtures contained PKR, isolated on antibody-coated beads, in the presence (+) or absence (−) of eIF2. Detection was by gel electrophoresis and autoradiography.

FIG. 5

Expression of PKR mutants in mammalian cells. COS1 cells were transfected in duplicate with plasmids encoding wild-type (WT) or mutant PKR together with pEGFP-C1 (Clontech) encoding green fluorescent protein. Transfection efficiency was monitored by observing the ratio of green fluorescent cells to the total number of cells. Protein expression was monitored by immunoblotting with a PKR monoclonal antibody (30) and an antiactin antibody.

FIG. 6

HCV E2 competition with PKR autophosphorylation site peptides. (A) Kinase assay mixtures contained PKR immunoprecipitated from yeast extracts. PKR was incubated with GST or GST-E2 proteins and peptide P1 or P2 before adding [³²P]ATP. Incorporation of ³²P into PKR and peptides was monitored by gel electrophoresis and autoradiography. (B) E2 binding assays. Nickel nitrilotriacetic acid-agarose-bound histidine-tagged PKR was incubated with peptide P1 (1, 5, 15, and 60 mg/ml [lanes 2 to 5]) or peptide P2 (0.6 and 2.4 mg/ml [lanes 6 and 7]) or without added peptide (lane 1) and then with ³⁵S-labeled E2 protein. Binding was monitored by gel electrophoresis and autoradiography.