Effect of myristoylation on p27 nef subcellular distribution and suppression of HIV-LTR transcription.
The effect of myristoylation on p27nef subcellular distribution and suppression of HIV-1 transcription was examined by transfecting COS-7 cells with plasmids expressing either myristoylated (pSVnef) or nonmyristolyated p27nef (pSVnefala2). Similar levels of myristoylated and nonmyristoylated p27nef were expressed with only the product of the pSVnef plasmid being myristoylated. Immuno-histochemical microscopy and radioimmunoprecipitation revealed myristolyated p27nef only in the membrane fraction while nonmyristolyated p27nef was found distributed between the nucleus and the cytosol fractions. The effect of myristoylation on p27nef suppression of HIV LTR controlled transcription was examined in transient transfected COS cells and in CEM human T-cell clones consituitively expressing either myristolyated or nonmyristolyated p27nef by cotransfecting with a chloramphenicol acetyltransferase (CAT) plasmid under control of the HIV-1 LTR. In both systems, myristoylated p27nef exhibited a 13- to 18-fold inhibition of basal CAT activity while the nonmyristolyated mutant and the same plasmid carrying the nef gene in a reverse orientation inhibited CAT activity one- to two-fold. These results confirm the cytoplasmic membrane localization of p27nef and establish that its subcellular targeting is dependent on covalently attached myristate. The data also provide further evidence that p27nef acts as a transcriptional suppressor and establishes for the first time that myristolyation is required for the full manifestation of this effect.
Yu
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Laboratory of Biological Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Felsted
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RL
D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D015693:Gene Products, nef / Q000235:genetics* / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D009227:Myristic Acids; D054311:nef Gene Products, Human Immunodeficiency Virus; D019814:Myristic Acid; D015500:Chloramphenicol O-Acetyltransferase