Mapping and characterization of the N-terminal I domain of human immunodeficiency virus type 1 Pr55(Gag).
Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55(Gag) molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55(Gag). The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.
Sandefur
S
S
Departments of Pediatrics and Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Smith
R M
RM
Varthakavi
V
V
Spearman
P
P
D000595:Amino Acid Sequence; D003902:Detergents / Q000494:pharmacology; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
D003902:Detergents; D015683:Gene Products, gag; D008164:Luminescent Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D049452:Green Fluorescent Proteins