LFA-1 Engagement Triggers T Cell Polarization at the HIV-1 Virological Synapse

ABSTRACTHIV-1 efficiently disseminates by cell-cell spread at intercellular contacts called virological synapses (VS), where the virus preferentially assembles and buds. Cell-cell contact triggers active polarization of organelles and viral proteins within infected cells to the contact site to support efficient VS formation and HIV-1 spread; critically, however, which cell surface protein triggers contact-induced polarization at the VS remains unclear. Additionally, the mechanism by which the HIV-1 envelope glycoprotein (Env) is recruited to the VS remains ill defined. Here, we use a reductionist bead-coupled antibody assay as a model of the VS and show that cross-linking the integrin LFA-1 alone is sufficient to induce active T cell polarization and recruitment of the microtubule organizing center (MTOC) in HIV-1-infected cells. Mutant cell lines coupled with inhibitors demonstrated that LFA-1-induced polarization was dependent on the T cell kinase ZAP70. Notably, immunofluorescent staining of viral proteins revealed an accumulation of surface Env at sites of LFA-1 engagement, with intracellular Env localized to a Golgi compartment proximal to the polarized MTOC. Furthermore, blocking LFA-1-induced MTOC polarization through ZAP70 inhibition prevented intracellular Env polarization. Taken together, these data reveal that LFA-1 is a key determinant in inducing dynamic T cell remodeling to the VS and suggest a model in which LFA-1 engagement triggers active polarization of the MTOC and the associated Env-containing secretory apparatus to sites of cell-cell contact to support polarized viral assembly and egress for efficient cell-cell spread.IMPORTANCEHIV-1 causes AIDS by spreading within immune cells and depletion of CD4 T lymphocytes. Rapid spread between these cells occurs by highly efficient cell-cell transmission that takes place at virological synapses (VS). VS are characterized by striking T cell remodeling that is spatially associated with polarized virus assembly and budding at sites of cell contact. Here, we show that the integrin LFA-1 triggers organelle polarization and viral protein recruitment, facilitating formation of the VS, and that this requires the T cell kinase ZAP70. Taken together, these data suggest a mechanism by which HIV-1-infected T cells sense and respond to cell contact to polarize viral egress and promote cell-cell spread. Understanding how cell-cell spread is regulated may help reveal therapeutic targets to specifically block this mode of HIV-1 dissemination.

Download Full-text

Contact-Induced Mitochondrial Polarization Supports HIV-1 Virological Synapse Formation

Journal of Virology ◽

10.1128/jvi.02425-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 14-24 ◽

Cited By ~ 16

Author(s):

Elisabetta Groppelli ◽

Shimona Starling ◽

Clare Jolly

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Virological Synapse ◽

Cell Contact ◽

Target Cells ◽

Cell Contacts ◽

Cell Spread ◽

Hiv 1 ◽

Cell Cell

ABSTRACTRapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles, including mitochondria, to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remain unclear. Here, we report that contact between an HIV-1-infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live-cell imaging, we show that mitochondrial and Gag polarization in HIV-1-infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts, and is an active, calcium-dependent process. We also find that perturbation of mitochondrial polarization impairs cell-cell spread of HIV-1 at the VS. Taken together, these data suggest that HIV-1-infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes.IMPORTANCEHIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the virological synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles, including mitochondria. Here, we show that cell-cell contact induces rapid recruitment of mitochondria to the contact site and that this supports efficient VS formation and consequently cell-cell spread. Additionally, we observed that cell-cell contact induces a mitochondrion-dependent increase in intracellular calcium, indicative of cellular signaling. Taken together, our data suggest that VS formation is a regulated process and thus a potential target to block HIV-1 cell-cell spread.

Download Full-text

Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.01469-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5547-5560 ◽

Cited By ~ 134

Author(s):

Clare Jolly ◽

Ivonne Mitar ◽

Quentin J. Sattentau

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Plasma Membrane ◽

T Cell ◽

Immunodeficiency Virus ◽

Fixed Cell ◽

Cell Spread ◽

Hiv 1 ◽

Cell Cell

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

Download Full-text

Endocytic Motif on a Biotin-Tagged HIV-1 Env Modulates the Co-Transfer of Env and Gag during Cell-to-Cell Transmission

Viruses ◽

10.3390/v13091729 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1729

Author(s):

María Inés Barría ◽

Raymond A. Alvarez ◽

Kenneth Law ◽

Deanna L. Wolfson ◽

Thomas Huser ◽

...

Keyword(s):

Fluorescent Imaging ◽

Productive Infection ◽

Live Cells ◽

Cell Contact ◽

Target Cells ◽

Structural Protein ◽

Cell Infection ◽

Virological Synapses ◽

Hiv 1 ◽

Cell Cell

During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell–cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell–cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.

Download Full-text

Cytoskeletal Polarization of T Cells Is Regulated by an Immunoreceptor Tyrosine-based Activation Motif–dependent Mechanism

The Journal of Cell Biology ◽

10.1083/jcb.140.4.861 ◽

1998 ◽

Vol 140 (4) ◽

pp. 861-871 ◽

Cited By ~ 185

Author(s):

Bente Lowin-Kropf ◽

Virginia Smith Shapiro ◽

Arthur Weiss

Keyword(s):

T Cells ◽

T Cell ◽

Actin Polymerization ◽

Gene Activation ◽

Attachment Site ◽

Cell Polarization ◽

Cell Contact ◽

Microtubule Organizing Center ◽

T Cell Polarization ◽

Cytoskeletal Rearrangements

Abstract. Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell–cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex–dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-ζ and CD3ε, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-ζ and once in CD3ε, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.

Download Full-text

Visualization of HIV T Cell Virological Synapses and Virus-Containing Compartments by Three-Dimensional Correlative Light and Electron Microscopy

Journal of Virology ◽

10.1128/jvi.01605-16 ◽

2016 ◽

Vol 91 (2) ◽

Cited By ~ 13

Author(s):

Lili Wang ◽

Edward T. Eng ◽

Kenneth Law ◽

Ronald E. Gordon ◽

William J. Rice ◽

...

Keyword(s):

Electron Microscopy ◽

T Cell ◽

Ion Beam ◽

Light And Electron Microscopy ◽

Viral Proteins ◽

Virological Synapse ◽

Cell Contact ◽

Target Cells ◽

Virological Synapses ◽

Hiv 1

ABSTRACT Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells. IMPORTANCE This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bona fide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses.

Download Full-text

Engagement of specific T-cell surface molecules regulates cytoskeletal polarization in HTLV-1–infected lymphocytes

Blood ◽

10.1182/blood-2004-07-2850 ◽

2005 ◽

Vol 106 (3) ◽

pp. 988-995 ◽

Cited By ~ 74

Author(s):

Amanda L. Barnard ◽

Tadahiko Igakura ◽

Yuetsu Tanaka ◽

Graham P. Taylor ◽

Charles R.M. Bangham

Keyword(s):

T Cell ◽

Cell Surface ◽

Contact Region ◽

Intercellular Adhesion Molecule ◽

Cell Junction ◽

Cell Contact ◽

Intercellular Adhesion Molecule 1 ◽

Microtubule Organizing Center ◽

Infected Cells ◽

Cell Cell

Abstract Cell-cell contact is required for efficient transmission of human T-lymphotropic virus type 1 (HTLV-1). An HTLV-1–infected cell polarizes its microtubule-organizing center (MTOC) toward the cell-cell junction; HTLV-1 core (Gag) complexes and the HTLV-1 genome accumulate at the point of contact and are then transferred to the uninfected cell. However, the mechanisms involved in this cytoskeletal polarization and transport of HTLV-1 complexes are unknown. Here, we tested the hypothesis that engagement of a specific T-cell surface ligand is synergistic with HTLV-1 infection in causing polarization of the MTOC to the cell contact region. We show that antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54) caused MTOC polarization at a higher frequency in HTLV-1–infected cells. ICAM-1 is upregulated on HTLV-1–infected cells, and, in turn, ICAM-1 on the cell surface upregulates HTLV-1 gene expression. We propose that a positive feedback loop involving ICAM-1 and HTLV-1 Tax protein facilitates the formation of the virologic synapse and contributes to the T-cell tropism of HTLV-1. In contrast, MTOC polarization induced in T cells by antibodies to CD3 or CD28 was significantly inhibited by HTLV-1 infection.

Download Full-text