Inhibition of human immunodeficiency virus type 1 particle formation by alterations of defined amino acids within the C terminus of the capsid protein.

Crystal structures of human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) reveal that the last 11 C-terminal amino acids are disordered. This disordered region contains a glycine-rich sequence 353-GVGGP-357 (numbering refers to the initiation methionine of Gag) that is highly conserved within the Gag proteins of HIV-1, HIV-2, and simian immunodeficiency virus, which suggests the importance of this sequence in virus replication. In the present study, we demonstrate that changing any individual residue within this short region in the context of the full-length HIV-1 genome virtually abolishes production of extracellular virus particles, in either the presence or absence of viral protease activity. This severe defect in virus particle production results from impaired Gag multimerization, as well as from decreased Gag association with the cellular membranes, as demonstrated by the results of gradient sedimentation and membrane flotation centrifugation assays. These findings are further supported by the diffuse distribution pattern of the mutant Gag within the cytoplasm, as opposed to the punctate distribution of the wild-type Gag on the plasma membrane. On the basis of these results, we propose that the disordered feature of amino acid stretch 353-GVGGP-357 in the CA crystal forms may have allowed Gag to adopt multiple conformations and that such structural flexibility is needed by Gag in order to construct geometrically complex particles.

mentioning

confidence: 99%

“…In addition to these three functional domains, the C-terminal domain (CTD) of CA (CA amino acids 150 to 231) is also indispensable for virus particle formation (11,31,52,61,63,66). Within this domain exists a major homology region (MHR) that is highly conserved among retroviral Gag proteins.…”

mentioning

confidence: 99%

A Structurally Disordered Region at the C Terminus of Capsid Plays Essential Roles in Multimerization and Membrane Binding of the Gag Protein of Human Immunodeficiency Virus Type 1

Liang

Whitney

et al. 2003

“…Structural studies and protein-protein interaction analyses have demonstrated that CA and SP1 likely contribute to Gag multimerization by facilitating direct interactions between Gag molecules (24, 29, 31, 33, 59-61, 67, 96). Mutations and deletions in the C-terminal domain of CA (12,21,42,50,56,58,76,82,87,88,90) and SP1 (1,45,49,93) cause defects in virus particle production, indicating that the Gag-Gag interaction mediated by this region is physiologically important for virus assembly. Unlike CA and SP1, NC is thought to promote Gag multimerization primarily through interaction with RNA.…”

mentioning

confidence: 99%

Association of Human Immunodeficiency Virus Type 1 Gag with Membrane Does Not Require Highly Basic Sequences in the Nucleocapsid: Use of a Novel Gag Multimerization Assay

Ono

Waheed

Joshi

et al. 2005

111

Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterolenriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.

“…Bacterially expressed CA has been shown to assemble in vitro (10,20,51), and the C-terminal domain of CA dimerizes in the crystal structure in the absence of other domains (16). Mutations affecting the C-terminal domain of HIV-1 CA have also been shown to disrupt virus assembly (8,26,45,50,54,59). In contrast, mutations in the N-terminal domain of CA generally do not disrupt virus assembly but rather perturb proper virion maturation (45,52).…”

mentioning

confidence: 99%

Relationship between Human Immunodeficiency Virus Type 1 Gag Multimerization and Membrane Binding

Ono

Demirov

Freed

2000

104

106

The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55Gag , is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between membrane binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the membrane-binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55Gag was truncated at the C terminus of matrix (MAstop), between the N-and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using membrane flotation centrifugation, we observe that MA shows significantly reduced membrane binding relative to full-length Gag but that CA146 displays steady-state membrane-binding properties comparable to those of Pr55Gag . The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state membrane binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient binding of HIV-1 Gag to membrane.