Characterization of human immunodeficiency virus type 1 Pr55gag membrane association in a cell-free system: requirement for a C-terminal domain.
Association of the human immunodeficiency virus type 1 (HIV-1) gag polyprotein precursor with cellular membranes is necessary for assembly of virions. We used in vitro synthesized HIV-1 gag to study its association with isolated cellular membranes. Rabbit reticulocyte lysates programmed with HIV-1 gag mRNA incorporated [35S]methionine and [3H]myristate into two predominant species of 55 kDa and 40 kDa. Radioimmunoprecipitation with HIV-1-specific antibodies suggested that the 55-kDa protein represented the polyprotein precursor (Pr55gag), while the 40-kDa protein was a mixture of N- or C-terminal truncations of the gag precursor. The Pr55gag protein bound to cellular membranes, while the 40-kDa mixed protein species did not. Membrane binding studies with C terminus-truncated and point mutants revealed that the seven-amino acid sequence located between the two Cys-His arrays in the nucleocapsid region was necessary for stable association to occur. Therefore, we propose that signals in addition to myristate are required for the membrane association of HIV-1 gag proteins and that these signals include a domain in the nucleocapsid protein.
Platt
E J
EJ
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121.
Haffar
O K
OK
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D015683:Gene Products, gag / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D014158:Transcription, Genetic
D003062:Codon; D017931:DNA Primers; D015683:Gene Products, gag; D011498:Protein Precursors; C059767:p55 gag precursor protein, Human immunodeficiency virus 1