Multimerization of Human Immunodeficiency Virus Type 1 Gag Promotes Its Localization to Barges, Raft-Like Membrane Microdomains

Abstract

The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed "barges." Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two-to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process.

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

“…This method uses Optiprep (Iodixanol) (Axis-Shield, Norton, MA) gradients to monitor raft associated and soluble proteins derived from non-ionic detergent treated cell lysates [48]. Decreased palmitoylation has been shown to lead to decreased lipid raft association.…”

Do lipid rafts mediate virus assembly and pseudotyping?

“…The Triton X-100-extractability assay, while widespread, is not used with sufficient uniformity that comparison of results from different laboratories is straightforward. In the case of HIV there is evidence that the coarseness of commonly used density gradients disguises the fact that Gag-membrane complexes float at a slightly higher density than conventional rafts (Lindwasser & Resh, 2001). Other combinations of detergent and temperature can be used to isolate raft-like membrane domains, which may have different compositions to those preserved in Triton X-100 at 4˚C (Roper et al, 2000).…”

“…Rousso and colleagues observed that insolubility in cold Triton X-100 was dependent on the palmitoylation of at least one of the two cytoplasmic palmitoylation sites. Lindwasser & Resh (2001) examined the flotation of Gag on multiple-step Optiprep gradients and found that while 6-8 % of Gag floats in the raft fraction (marked by GM1 and caveolin-1), about 30 % of Gag was found at a slightly higher density. A reduction in Gag multimerization relocates some of this Gag to the raft fraction.…”

Role of Membrane Rafts in Viral Infection

“…Interaction of Gag proteins with the plasma membrane and membrane rafts may be enhanced or stabilized by lower-ordered Gag multimerization, but not required for higher-order Gag multimerization. The role of membrane rafts in enhancement of Gag-Gag interaction is not clear [165]. The viral surface glycoprotein complex (gp120 and gp41) is incorporated into the HIV-1 envelope together with membrane rafts by interactions of Gag protein with the cytoplasmic tail of gp41, which stabilizes the gp120-gp41 interaction.…”

“…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) …”