Mapping and Characterization of the N-Terminal I Domain of Human Immunodeficiency Virus Type 1 Pr55 ^Gag

Abstract

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 Pr55Gag 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.

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: Structural and mechanistic implications of p17 myristoylation

“…It is plausible that the myristoyl switch in p17 at the membrane surface, although not caused by any structural change associated with proteolysis, may be triggered energetically. Because regions of the HIV-1 Gag polyprotein outside the p17 domain contribute to membrane binding as well as to protein oligomerization (18,21,(61)(62)(63)(64), the departure of downstream Gag subdomains upon proteolytic cleavage predictably weakens oligomeric structures of p17 at the membrane surface. Such destabilization may lead to the dissociation of ''clustered'' myristoyl moieties or p17 molecules from each other and, ultimately, the detachment of some p17 molecules from the membrane.…”

Basic Residues in the Nucleocapsid Domain of Gag Are Required for Interaction of HIV-1 Gag with ABCE1 (HP68), a Cellular Protein Important for HIV-1 Capsid Assembly

“…23). The nonspecific RNA interaction is important for promoting Gag multimerization and capsid formation and appears to be governed by basic charge in NC (24,25,(42)(43)(44)(45). In contrast, packaging of the genome of HIV-1 and many other retroviruses requires intact zinc fingers within NC (46 -51).…”

Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly

“…For example, mutations that inhibit Gag assembly also disrupt membrane binding (55)(56)(57), and C-terminal truncations lead to progressive decreases in both Gag multimerization and membrane affinity (58). In addition, the binding of Gag to nucleic acid templates, which promotes Gag self association (59)(60)(61)(62), also enhances Gag interactions with membranes (63)(64)(65). The relative influence of Gag self association and PI(4,5)P 2 binding for triggering myristate exposure in vivo is not clear.…”