Genetic characterization of human immunodeficiency virus type 1 nef gene products translated in vitro and expressed in mammalian cells.
Expression of the human immunodeficiency virus type 1 nef gene was studied by in vitro transcription-translation and by transfection into monkey COS cells. Two Nef-related peptides, of 27 and 25 kDa, were identified by immunoprecipitation with anti-Nef antibodies. The relation between these two proteins was determined by metabolically labeling transfected COS cells and by deleting the initiator methionine of nef. We found that the 25-kDa polypeptide is not a cleavage product of 27-kDa Nef but rather is initiated from an internal ATG 57 bases downstream from the Nef initiation site. Myristoylation of the 27-kDa but not of the 25-kDa Nef was demonstrated by the contranslational modification of Nef in an in vitro reticulocyte translation system. The myristoylation pattern of the two Nef polypeptides further implies that the 25-kDa polypeptide lacks the amino terminus of 27-kDa Nef. Cellular localization of the various forms of Nef was studied in transiently transfected COS cells. Myristoylation was found to be necessary for membrane association of Nef. Myristoylation-deficient 27-kDa Nef mutant and 25-kDa Nef were confined to the soluble cytoplasmic fraction of transfected cells, whereas part of the wild-type 27-kDa Nef was membrane attached.
Kaminchik
J
J
Biotechnology General (Israel) Ltd., Rehovot.
Bashan
N
N
Itach
A
A
Sarver
N
N
Gorecki
M
M
Panet
A
A
D000818:Animals; D002460:Cell Line; D002872:Chromosome Deletion; D003062:Codon / Q000235:genetics; D015870:Gene Expression; D015693:Gene Products, nef / Q000032:analysis / Q000235:genetics*; D015754:Genes, nef; D015497:HIV-1 / Q000235:genetics*; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D014158:Transcription, Genetic; D014162:Transfection; D054311:nef Gene Products, Human Immunodeficiency Virus
D003062:Codon; D015693:Gene Products, nef; D009227:Myristic Acids; D054311:nef Gene Products, Human Immunodeficiency Virus; D019814:Myristic Acid