Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.
Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.
Spearman
P
P
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Wang
J J
JJ
Vander Heyden
N
N
Ratner
L
L
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D015685:Fusion Proteins, gag-pol / Q000235:genetics / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000254:growth & development* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014616:Vaccinia virus / Q000235:genetics; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism
D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D009227:Myristic Acids; D011498:Protein Precursors; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D019814:Myristic Acid