A cellular cofactor for the constitutive transport element of type D retrovirus

Science

Volumn 276, Issue 5317, 1997, Pages 1412-1415

  Tang, Hengli ^a   Gaietta, Guido M ^b   Fischer, Wolfgang H ^c   Ellisman, Mark H ^b   Wong Staal, Flossie ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

^c SALK INSTITUTE FOR BIOLOGICAL STUDIES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

5,6 DICHLOROBENZIMIDAZOLE RIBOSIDE; ADENOSINE TRIPHOSPHATE; ANTIBODY; CHLORAMPHENICOL ACETYLTRANSFERASE; CYCLOHEXIMIDE; DACTINOMYCIN; DNA DIRECTED RNA POLYMERASE; DOUBLE STRANDED RNA; HELICASE; MESSENGER RNA; NUCLEAR PROTEIN; PEPTIDE; POLYVINYLIDENE FLUORIDE; PROTEIN; RIBONUCLEOPROTEIN; RNA; RNA BINDING PROTEIN; RNA HELICASE; RNA POLYMERASE; VIRUS RNA;

ARTICLE; CONTROLLED STUDY; GENE EXPRESSION; HUMAN; HUMAN CELL; IMMUNOBLOTTING; IMMUNOHISTOCHEMISTRY; IN SITU HYBRIDIZATION; MUTANT; POLYACRYLAMIDE GEL ELECTROPHORESIS; PRIORITY JOURNAL; PROTEIN LOCALIZATION; PROTEIN RNA BINDING; RETROVIRUS; RNA TRANSCRIPTION; RNA TRANSPORT; SPLICEOSOME;

ADENOSINE TRIPHOSPHATE; BETARETROVIRUS; BIOLOGICAL TRANSPORT; CELL NUCLEUS; HELA CELLS; HUMANS; MUTATION; NUCLEAR PROTEINS; PROTEIN BINDING; RNA HELICASES; RNA NUCLEOTIDYLTRANSFERASES; RNA, VIRAL;

EID: 0030974078     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5317.1412     Document Type: Article

References (25)

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6. Sequences of three microsequenced peptides are as follows: peptide 1, AENN(C)EVGA(C)GYGGVXG (matching amino acids 121 to 136 of helicase A); peptide 2, YTQVGPDHNR (matching amino acids 200 to 209 of helicase A); peptide 3, LAQFEPSQRF (matching amino acids 315 to 323 of helicase A) (22); C. Lee and J. Hurwitz, J. Biol. Chem. 268, 16822 (1993).
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11. H. Tang and F. Wong-Staal, unpublished data.
12. H. Tang, G. M. Gaietta, W. H. Fischer, M. H. Ellisman, F. Wong-Staal, data not shown.
13. The sense and antisense RNA probes were prepared by in vitro transcription, with 2′-deoxyuridine-5′-triphosphate coupled to digoxigenin (digoxigenin-11-dUTP) (Boehringer Mannheim) as a label. HeLa cells were attached to glass cover slips (Fisher) coated with poly-D-lysine (Sigma). Sixteen, 20, and 26 hours after the addition of DNA-calcium precipitate, cells were washed in phosphate-buffered saline (PBS) until no precipitate was visible and then fixed with 4% paraformaldehyde for 30 min. The following steps were performed to facilitate accessibility of CTE and to reduce nonspecific binding of the RNA probe: 2 min in 0.5% Triton X-100 (in PBS), 5 min in 0.5 N HCl, 10 min in acetylation buffer (583 μl of triethanolamine and 125 μl of acetic anhydride in 50 ml of diethyl pyrocarbonate-treated water). Each step was followed by two brief washes in PBS, and then 30 μl of prehybridization solution [containing 50% formamide, 5x standard saline citrate (SSC), 5x Denhardt's reagent, single-stranded DNA (50 μg/ml), tRNA (25 μg/ml)] lacking the probe was applied to each sample. Samples were prehybridized at 42°C for 1 hour in a humid chamber. At the end of the prehybridization step, samples were rinsed in 5x SSC and prepared for the hybridization step: 30 μl of prehybridization solution, containing a 1:100 dilution of the digoxigenin-labeled RNA probes (previously denatured at 65°C for 10 min) was applied to each cover slip, covered with a glass coverslip, and sealed with rubber cement. Hybridization was carried out overnight at 42°C in a humid chamber. The posthybridization washes were performed in SSC wash buffer at high stringency (2x to 0.1x). Samples were treated with ribonuclease to remove RNA probes that did not hybridize to the target RNA. Detection of the hybridized RNA probes was performed with an antibody to digoxigenin F(ab) coupled to alkaline phosphatase (Boehringer Mannheim) followed by a colorimetric reaction with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, or by hybridization with a biotinylated secondary antibody and detection with streptavidin Cy5 conjugate. The protein was detected with a rabbit polyclonal antibody to human helicase A. The polyclonal antibody was then immunodetected with a donkey antibody to rabbit immunoglobulin G coupled to Cy5 (detection of RNA probe with alkaline phosphatase) or to fluorescein isothiocyanate (FITC) (RNA indirectly labeled with Cy5). The samples were viewed with a Bio-Rad MRC 1024 laser-scanning confocal system coupled to a Zeiss Axiovert 35M microscope with a planapochromatic objective (x40, 1.3 numerical aperture, oil immersion).
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17. H. Tang and F. Wong-Staal, unpublished data.
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22. Abbreviations for the amino acid residues are: 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 Y, Tyr.
23. 5 cpm) for 15 min at room temperature. The reaction mixture was then mixed with RNA loading buffer (10% glycerol, 0.01% bromophenol blue, 0.01% xylene cyanol) and run on a 4% polyacrylamide gel (acrylamide:bisacrylamide = 60:1). The gel was subsequently dried and subjected to autoradiography.
24. HeLa cells were grown in slide chambers. Transfections were done by conventional calcium phosphate precipitation and cells were fixed for immunostaining 26 to 28 hours after transfection. Cells were fixed in methanol and acetone (1:1) for 2 min, washed extensively with PBS, and blocked with 3% bovine serum albumin in PBS for 10 min before the first antibodies were applied. The polyclonal antibody against helicase A was provided by Lee and Hurwitz (6). FITC-coupled goat antibody to rabbit immunoglobulin G was used as a second antibody to detect helicase A. Cells grown on coated glass cover slips, fixed with 4% paraformaldehyde, and permeabilized by 0.5% Triton X-100 gave essentially the same results for protein staining.
25. We are indebted to C. Lee and J. Hurwitz for purified helicase A protein and a polyclonal antibody against human helicase A. R. Moy helped with the constructions of pDMCTE and pDCTE. M. Park performed the protein sequence analysis. Supported in part by grants NIH RR 04050 and NS 14718 (M.H.H.) and by the University of California, San Diego, Center for AIDS Research (F.W-S).