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

Abstract

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.

Properties and functions of the nucleocapsid protein in virus assembly

“…46,85 In the absence of NC, MLV Gag is still targeted and associated to the plasma membrane but no Gag-RNA refs. 19,44,[74][75][76][77][78][79][80][81][82][83][84][85][86]. Mutations in the polybasic region of NC or the zinc fingers impair and even abrogate genomic RNA packaging with the notion that the ZnF and hydrophobic plateau are driving the specificity of gRNA encapsidation, while the basic residues are more involved in Gag-Gag multimerization and assembly.…”

“…It binds the HIV-1 genomic RNA and Gag via its NC domain complexes accumulate and no viral buds are visible by microscopy. 76 As for HIV-1, a multimerization-defective NC mutant still binds to the plasma membrane raft microdomains, 86 suggesting that NC is dispensable for Gag membrane association. In contrast, basic residues in MA are required for Gag membrane binding via their interactions with the phosphinositide PI(4,5)P2.…”

The Functional Roles of Lipid Rafts in T Cell Activation, Immune Diseases and HIV Infection and Prevention

“…There are a few characterized processed proteins for viral assembly the protein Env-gp41 is located in lipid raft fraction when HIV-infected cells were extracted with cold Triton X-100. Polyprotein precursor Pr55 gag is also associated with membrane rafts, and can be easily colocalized with GM1 (26). In the presence of normal amount of cholesterol, Nef significantly enhances HIV-1 infectivity, and this effect is abolished when virus is produced from cholesterol-depleted cells.…”

Role of Membrane Rafts in Viral Infection

“…The palmitoylated cytoplasmic tails of gp41 are required for association with rafts, whereas interactions between gp41 and Gag protein are not involved in the association with rafts. Moreover, membrane raft association with gp41 or Gag protein is not essential for HIV-1 assembly, infectious progeny virus production, and surface trafficking [164,168,169]. However, quantal association of Gag protein with cholesterol-enriched rafts facilitates HIV-1 particle production by enhancement of both Gag-membrane interaction and Gag multimerization [172,173].…”

“…The role of membrane rafts in the assembly and budding of enveloped viruses has been investigated for influenza virus [59,63,[150][151][152][153][154][155][156][157][158][159][160], HIV-1 [63,[161][162][163][164][165][166][167][168][169][170][171][172][173], HTLV-1 [174], measles virus (Paramyxoviridae) [63,175,176], Sendai virus [Paramyxoviridae] [177,178], Newcastle disease virus (NDV; Paramyxoviridae) [179,180], RSV [147,148,[181][182][183][184], HSV [185,186], murine cytomegalovirus (MCMV; Herpesviridae) [187], Ebola virus [70,188], Marburg virus [70], and vesicular stomatitis virus (VSV; Rhabdoviridae) …”