HIV-1 Entry in SupT1-R5, CEM-ss, and Primary CD4+ T Cells Occurs at the Plasma Membrane and Does Not Require Endocytosis

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

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Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4+ T cells

Virology Journal ◽

10.1186/1743-422x-7-3 ◽

2010 ◽

Vol 7 (1) ◽

pp. 3 ◽

Cited By ~ 6

Author(s):

Ajit Patil ◽

Archana Gautam ◽

Jayanta Bhattacharya

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Cd4 T Cells ◽

Detergent Resistant Membranes ◽

Envelope Incorporation ◽

Hiv 1

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Specific Lipid Recruitment by the Retroviral Gag Protein upon HIV-1 Assembly: From Model Membranes to Infected Cells

Proceedings ◽

10.3390/proceedings2020050102 ◽

2020 ◽

Vol 50 (1) ◽

pp. 102

Author(s):

Cyril Favard ◽

Jakub Chojnacki ◽

Naresh Yandrapalli ◽

Johnson Mak ◽

Christian Eggeling ◽

...

Keyword(s):

T Cells ◽

Plasma Membrane ◽

In Silico ◽

Cd4 T Cells ◽

Virus Assembly ◽

Gag Protein ◽

Infected Cells ◽

Specific Lipid ◽

Hiv 1

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. Here, we addressed the question whether HIV-1 Gag was able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in silico, in vitro on reconstituted membranes and in cellulo at the plasma membrane of the host CD4+ T cells. In silico, we could observe the first PI(4,5)P2 preferential recruitment by HIV-1 MA or Gag while protein docked on artificial membranes. In vitro, using biophysical techniques, we observed the specific trapping of PI(4,5)P2, and, to a lesser extent, cholesterol and the exclusion of sphingomyelin, during HIV-1 myr(-)Gag self-assembly on LUVs and SLBs. Finally, in infected living CD4+ T cells, we measured lipid dynamics within and away from HIV-1 assembly sites using super-resolution stimulated emission depletion (STED) microscopy coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). The analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol but not phosphatidylethanolamine (PE) or sphingomyelin (SM) at the cellular membrane. Furthermore, analyzing CD4+ T cells expressing only HIV-1 Gag protein showed that Gag is the main driving force restricting the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. Our data provide the first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly by selectively recruiting lipids to generate PI(4,5)P2/cholesterol-enriched nanodomains favoring virus assembly, and that HIV-1 does not assemble on pre-existing lipid domains.

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Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

The Journal of Cell Biology ◽

10.1083/jcb.201409082 ◽

2015 ◽

Vol 209 (3) ◽

pp. 435-452 ◽

Cited By ~ 33

Author(s):

Pehuén Pereyra Gerber ◽

Mercedes Cabrini ◽

Carolina Jancic ◽

Luciana Paoletti ◽

Claudia Banchio ◽

...

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Cd4 T Cells ◽

Membrane Association ◽

Late Endosomes ◽

Phosphatidylinositol 4 ◽

Viral Polyprotein ◽

Late Stages ◽

Hiv 1

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

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