Mobility of Human Immunodeficiency Virus Type 1 Pr55 Gag in Living Cells

101

Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-␤-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.

Section: Discussionmentioning

confidence: 99%

Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane

Finzi

Orthwein

Mercier

et al. 2007

101

“…Immunofluorescence microscopy studies have revealed that Gag colocalizes/copatches with lipid raft markers in cells (59,98,105) and cofractionates with lipid raft markers in detergent-resistant membranes (DRMs) (9,31,32,53,59,84,85,98,104,107) although qualitative differences between canonical DRMs and Gag-containing DRMs have been noted (31,59,84). Depletion of cellular cholesterol, which disrupts lipid rafts, reduces virus particle production and disrupts the behavior of Gag in cells as measured by a variety of assays (43,104,106,113). Importantly, one study loaded cells with an unsaturated myristate analogue which blocked Gag fractionation into DRMs and reduced virus production, suggesting that N-terminal saturated acylation is a necessary molecular determinant of lipid raft association and assembly of Gag (85).…”

mentioning

confidence: 99%

Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane

Hogue

Grover

Soheilian

et al. 2011

108

116

The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.

“…The spatial and temporal aspects of HIV-1 Gag assembly at the plasma membrane have been investigated using live-cell microscopy techniques, such as FRAP (fluorescence recovery after photobleaching) and TIRF (total internal reflection fluorescence) microscopy (37)(38)(39). More recently, such studies have been extended to analyze the dynamics of viral RNA-Gag interactions and the recruitment of individual ESCRT components to the sites of assembly (40)(41)(42)(43).…”

mentioning

confidence: 99%

Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

Roy

Chan

Lambelé

et al. 2013

HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells.