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Volumn 271, Issue 5257, 1996, Pages 1858-1860

Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor elF-5A

Author keywords

[No Author keywords available]

Indexed keywords

INITIATION FACTOR; MUTANT PROTEIN; TRANSACTIVATOR PROTEIN;

EID: 0029916299     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5257.1858     Document Type: Article
Times cited : (185)

References (46)
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    • note
    • Oligonucleotide-directed mutagenesis with a bacteriophage M13 mutagenesis system (United States Biochemical, Cleveland, OH) was used to introduce targeted nucleotide substitutions encoding multiple amino acid alterations into the EIF5A gene of pelF-5A (3). All mutations introduced were confirmed by DNA sequence determination (Sequenase 2.0; United States Biochemical). Protein expression of all mutants was confirmed by eIF-5A-specific immunoprecipitation analysis with radiolabeled protein extracts of transiently transfected COS cells.
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    • Wild-type and mutant elF-5A proteins were expressed as COOH-terminal fusions to GST in the Escherichia coli strain BL21. The fusion proteins were purified from crude lysates by affinity chromatography on glutathione-Sepharose 4B as described
    • Wild-type and mutant elF-5A proteins were expressed as COOH-terminal fusions to GST in the Escherichia coli strain BL21. The fusion proteins were purified from crude lysates by affinity chromatography on glutathione-Sepharose 4B as described [M Hammerschmid et al., J. Virol. 68, 7329 (1994)]. Radiolabeled RNA transcripts containing a 252-nucleotide RRE probe were used in the RNA gel retardation assays as described previously (9). For gel retardation assays, samples containing 20,000 cpm of radiolabeled RRE RNA were incubated with 0 076 μM Rev protein (7, 17) for 15 min at ambient temperature to allow the formation of RRE:protain complexes (9) Subsequently, the GST-eIF-5A proteins (3.2 μM of each mutant) were added and incubation continued for an additional 45 min. All reactions also contained 2 μg of MS-2 RNA (Boehringer Mannheim, Mannheim, Germany) and 5 units RNasin (Promega, Madison, WI) in TBE (89 mM tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.4) buffer. The total sample (volume 12 μl) from each reaction was subjected to electrophoresis for 2 hours at 15 mA with native 5% polyacrylamide gels. Finally, the gels were dried and subjected to autoradiography
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    • + human CEM cells were inoculated as described (18) with virus-containing, infectious Am12 supematants and selected for neomycin-resistant CEM cell populations. RNA expression of the transgene was characterized in clonal populations of the transduced CEM cells as described (18). Vectors expressing the elF-5A wild-type and the randomly selected elF-5AM9 gene served as controls in the experiments
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    • 7) were transiently transfected with 3 μg of the Rev reporter pDM 128/CMV (29) or the Tat reporter pBC12/HIV/CAT [J. Berger, J. Hauber, R. Hauber, R. Geiger, B. R. Cullen, Gene 66, 1 (1988)] and 3 μg of the parental vector pBC12/CMV (negative control), pcREV, or pcTAT [
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    • HeLa cells were microinjected in the nucleus with a Zeiss Axiovert inverse microscope and a Zeiss automated injection system AIS (Zeiss, Oberkochen, Germany) in combination with an Eppendorf microinjector 5242 (Eppendorf Geratebau, Hamburg, Germany) The GST-Rev and GST-RevM32 proteins were injected at a concentration of 0.5 mg/ml, together with the respective GST-eIF-5A isoform or BSA (1.25 mg/ml) The injection procedure was carried out as described previously [W. Ansorge, Exp. Cell Res. 140, 31 (1982)]. About 20 min after injection, cells were fixed with 3% paraformaldehyde, 1% BSA in phosphatebuffered saline for 10 min, triton-treated for 10 min, and subsequently stained, as described previously, with a mouse monoclonal antibody to Rev [M. Hammerschmid et al , J Virol. 68, 7329 (1994)]. Immunofluorescence stainings were analyzed with a Zeiss axiophot microscope with a KAF 1400 CCD camera (Photometrics, Tuscon, AZ) and IPLab spectrum and Adobe Photoshop 3.0 software.
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    • note
    • This work is dedicated to the memory of our friend and colleague Peter Dukor. We are indebted to V Magdolen for his initial help and intellectual input and to B. Kappel, L. Hofer, and J. Pertl for their technical assistance. We thank G. Palfi for his advice and help with fluorescence-activated cell sorting analyses and S L. Thomas for her comments on the manuscript Supported in part (M O ) by grant of the Deutsche Forschungsgemeinschaft.


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