Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells

The HIV-1 envelope (Env) is an essential determinant of viral infectivity, tropism and spread between T cells. Lentiviral Env contain an unusually long 150 amino acid cytoplasmic tail (EnvCT), but the function of the EnvCT and many conserved domains within it remain largely uncharacterised. Here, we identified a highly conserved tryptophan motif at position 757 (W757) in the LLP-2 alpha helix of the EnvCT as a key determinant for HIV-1 replication and spread between T cells. Alanine substitution at this position potently inhibited HIV-1 cell–cell spread (the dominant mode of HIV-1 dissemination) by preventing recruitment of Env and Gag to sites of cell–cell contact, inhibiting virological synapse (VS) formation and spreading infection. Single-molecule tracking and super-resolution imaging showed that mutation of W757 dysregulates Env diffusion in the plasma membrane and increases Env mobility. Further analysis of Env function revealed that W757 is also required for Env fusion and infectivity, which together with reduced VS formation, result in a potent defect in viral spread. Notably, W757 lies within a region of the EnvCT recently shown to act as a supporting baseplate for Env. Our data support a model in which W757 plays a key role in regulating Env biology, modulating its temporal and spatial recruitment to virus assembly sites and regulating the inherent fusogenicity of the Env ectodomain, thereby supporting efficient HIV-1 replication and spread.

Download Full-text

Identification and Characterization of Virus Assembly Intermediate Complexes in HIV-1-Infected CD4+T Cells

Virology ◽

10.1006/viro.1998.9064 ◽

1998 ◽

Vol 243 (1) ◽

pp. 78-93 ◽

Cited By ~ 61

Author(s):

Young-Min Lee ◽

Xiao-Fang Yu

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Virus Assembly ◽

Assembly Intermediate ◽

Identification And Characterization ◽

Hiv 1

Download Full-text

Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

Viruses ◽

10.3390/v13010142 ◽

2021 ◽

Vol 13 (1) ◽

pp. 142

Author(s):

Yue Yuan ◽

Caron A. Jacobs ◽

Isabel Llorente Garcia ◽

Pedro M. Pereira ◽

Scott P. Lawrence ◽

...

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Single Molecule ◽

Cd4 T Cells ◽

Super Resolution ◽

Fold Increase ◽

Membrane Receptors ◽

Virus Binding ◽

Resolution Imaging ◽

Hiv 1

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.

Download Full-text

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.

Download Full-text

Engineered Bacteriophage T4 Nanoparticle as a Potential Targeted Activator of HIV-1 Latency in CD4+ Human T cells

10.1101/2021.07.20.453091 ◽

2021 ◽

Author(s):

Himanshu Batra ◽

Jingen Zhu ◽

Swati Jain ◽

Neeti Ananthaswamy ◽

Marthandan Mahalingam ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Virus Assembly ◽

Bacteriophage T4 ◽

Latent Reservoir ◽

Major Barrier ◽

Hiv 1

The latent HIV-1 reservoir containing stably integrated and transcriptionally silent proviruses in CD4+ T cells is a major barrier for virus eradication. Targeted reactivation of the latent reservoir remains a major challenge in establishing a path for an HIV-1 cure. Here, we investigated the possibility of reactivating the HIV-1 reservoir by targeting engineered bacteriophage T4 capsid nanoparticles to reservoir cells. The surface lattice of the 120 x 86 nm phage capsid was arrayed with CD4 binding ligands such as recombinant CD4DARPin or the HIV-1 gp140 envelope protein. When exposed to either PBMCs or the resting CD4+ T cells in vitro, these nanoparticles caused T cells activation without inducing global T cell activation. Furthermore, the nanoparticles reactivated HIV-1 proviral transcription that led to virus assembly and release in the J-Lat cells, a cell line model of HIV-1 latency. Intriguingly, the observed T cell activation and HIV-1 latency reversal did not occur through the classic PKC or NFAT pathways suggesting the involvement of a yet unknown pathway. These studies demonstrate that engineered non-infectious bacteriophages could be potentially exploited for HIV-1 cure and other targeted T cell therapies.

Download Full-text