Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein

Science

Volumn 285, Issue 5424, 1999, Pages 107-110

  Taylor, Deborah R ^a   Shi, Stephanie T ^a   Romano, Patrick R ^b   Barber, Glen N ^c   Lai, Michael M C ^a  

^a UNIVERSITY OF SOUTHERN CALIFORNIA   (United States)

^b Small Molecule Therapeutics   (United States)

^c UNIVERSITY OF MIAMI MILLER SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

HYBRID PROTEIN; INTERFERON; PROTEIN KINASE;

ARTICLE; AUTOPHOSPHORYLATION; ENZYME ASSAY; ENZYME INHIBITION; GENE SEQUENCE; GENOTYPE; HEPATITIS C; HEPATITIS C VIRUS; PLASMID; PRIORITY JOURNAL; VIRUS INFECTION; VIRUS ISOLATION;

CELL LINE; CHLORAMPHENICOL O-ACETYLTRANSFERASE; DRUG RESISTANCE, MICROBIAL; EIF-2 KINASE; ENDOPLASMIC RETICULUM; ENZYME INDUCTION; EUKARYOTIC INITIATION FACTOR-2; HELA CELLS; HEPACIVIRUS; HUMANS; INTERFERON-ALPHA; PHOSPHORYLATION; PROTEIN BIOSYNTHESIS; RECOMBINANT FUSION PROTEINS; SACCHAROMYCES CEREVISIAE; TRANSFECTION; TRANSFORMATION, GENETIC; VIRAL ENVELOPE PROTEINS;

HEPATITIS C VIRUS;

EID: 0033516497     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5424.107     Document Type: Article

References (30)

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5. M. J. Clemens, in (2), pp. 575-605 . Four autophosphorylation sites (serine 83 and threonines 88, 89, and 90) have been identified in the RNA-binding domain of PKR (D. R. Taylor and M. B. Mathews, in preparation). These sequences are not conserved between human and rodent species. These sites and 10 others have been observed by mass spectroscopy by X. Zhang et al. [Anal. Chem. 70, 2050 (1998)].
6. M. J. Clemens, in (2), pp. 575-605 . Four autophosphorylation sites (serine 83 and threonines 88, 89, and 90) have been identified in the RNA-binding domain of PKR (D. R. Taylor and M. B. Mathews, in preparation). These sequences are not conserved between human and rodent species. These sites and 10 others have been observed by mass spectroscopy by X. Zhang et al. [Anal. Chem. 70, 2050 (1998)].
7. M. J. Clemens, in (2), pp. 575-605 . Four autophosphorylation sites (serine 83 and threonines 88, 89, and 90) have been identified in the RNA-binding domain of PKR (D. R. Taylor and M. B. Mathews, in preparation). These sequences are not conserved between human and rodent species. These sites and 10 others have been observed by mass spectroscopy by X. Zhang et al. [Anal. Chem. 70, 2050 (1998)].
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11. 32P-labeled proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to autoradiography.
12. BL21(DE3) bacterial cells were transformed with plasmids expressing His-tagged wild-type and mutant (K296R) PKR (pET-14b; Novagen) or green fluorescent protein (pRSET-GFP; Invitrogen), and were induced for 3 hours in the presence of 0.5 M isopropyl-β-D-thiogalactopyranoside. Proteins were extracted by sonication, and equal amounts of His-tagged proteins were incubated with nickel nitrilotriacetic acid agarose (Qiagen) for 18 hours at 4°C. The beads were washed with phosphate-buffered saline (PBS) containing 0.02 M imidazole (Amresco) and 0.3% NP-40 and were then incubated for 2 hours at 4°C in binding buffer [0.04 M Hepes-K (pH 7.5), 0.1 M KCl, 0.1% NP-40, 0.02 M β-mercaptoethanol]. The beads were washed seven times with binding buffer plus 0.02 M imidazole and 0.85% NP-40. Bound radiolabeled proteins were eluted with SDS sample buffer and boiled before SDS-PAGE analysis.
13. Cell lysates were assayed for CAT enzyme activity 2 days after transfection. Transfections were performed in triplicate and the results represent three separate experiments. CAT activity is expressed in relative units, and error bars were calculated on the basis of standard deviation of the mean. Cells that were infected with adenovirus dl331 strain (multiplicity of infection of 10) were also treated with IFN-α (+; 1000 U/ml). Immunoblots were performed with antibody to actin (Santa Cruz Biotechnology) and polydonal antibody to PKR (23) and were developed by chemiluminescence (Amersham; ECL). For immunofluorescent staining of E2 and PKR, HEK-293 cells plated in eight-well chamber slides were transfected with 1 μg of COOH-terminal Flag-tagged E2L and treated with IFN-α 18 hours before fixation. Three days after transfection, the cells were fixed in 4% formaldehyde and permeabilized in acetone. The cells were then double-stained with a rabbit polyclonal antibody to Flag (Zymed) (1:300) and a monoclonal antibody to PKR (1:5) (Ribogene). Confocal microscopy was performed on a Nikon PCM2000 confocal microscope.
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27. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and V, Tyr.
28. RY1-1 carries two chromosomal copies of PKR at the Leu2 locus (18) and has wild-type elF2α.
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30. We thank M. B. Mathews for polyclonal antiserum to PKR; B. Thimmapaya for dl331 adenovirus; T.-Y. Hsieh for hnRNPK-pcDNA3; and P. Koetters, J.-W. Oh, and members of the Lai laboratory for helpful discussions. Confocal microscopy was performed at the cell biology core laboratory of University of Southern California Liver Center. Supported by a NIH grant (AI 40038) and by a postdoctoral fellowship to D.R.T. from the NIH.