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

2016 ◽  
Vol 91 (2) ◽  
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.

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

2014 ◽  
Vol 89 (1) ◽  
pp. 14-24 ◽  
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.

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

Viruses ◽  
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.

scholarly journals A Replication-Competent HIV Clone Carrying GFP-Env Reveals Rapid Env Recycling at the HIV-1 T Cell Virological Synapse

Viruses ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 38
Author(s):  
Lili Wang ◽  
Alice Sandmeyer ◽  
Wolfgang Hübner ◽  
Hongru Li ◽  
Thomas Huser ◽  
...  
Keyword(s):  
Virological Synapse ◽  
Target Cells ◽  
Structural Protein ◽  
Combined Use ◽  
Infected Cells ◽  
Virological Synapses ◽  
Dynamic Exchange ◽  
Endocytic Compartments ◽  
Viral Transfer ◽  
Hiv 1

HIV-1 infection is enhanced by cell–cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS.

Efficient HIV-1 transmission from macrophages to T cells across transient virological synapses

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4660-4663 ◽  
Author(s):  
Fedde Groot ◽  
Sonja Welsch ◽  
Quentin J. Sattentau
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Human Monocyte ◽  
Productive Infection ◽  
Virological Synapse ◽  
Primary Target ◽  
Virological Synapses ◽  
Adhesive Contacts ◽  
Hiv 1

Abstract Macrophages are reservoirs of HIV-1 infection, proposed to transmit virus to CD4+ T cells, the primary target of the virus. Here we report that human monocyte-derived macrophages (MDMs) rapidly spread HIV-1 to autologous CD4+ T cells resulting in productive infection. Transmission takes place across transient adhesive contacts between T cells and MDMs, which have the features of a virological synapse including copolarization of CD4 on the T cell with HIV-1 Gag and Env on the macrophage. We propose that an infected MDM can infect at least one T cell every 6 hours. Since HIV-1–infected macrophages can survive for many weeks, these results highlight the central role played by macrophages in HIV-1 infection and pathogenesis.

scholarly journals Recruitment of Env to the HIV-1 T cell virological synapse by targeted and sustained Env recycling

2020 ◽  
Author(s):  
Lili Wang ◽  
Alice Sandmeyer ◽  
Wolfgang Hübner ◽  
Hongru Li ◽  
Thomas Huser ◽  
...  
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Virological Synapse ◽  
Target Cells ◽  
Structural Protein ◽  
Rapid Exchange ◽  
Infected Cells ◽  
Virological Synapses ◽  
Dynamic Exchange ◽  
Viral Transfer ◽  
Hiv 1

ABSTRACTHIV-1 infection is enhanced by cell-cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Using surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, we observed targeted accumulation and sustained recycling of Env between the endocytic recycling compartment (ERC) and the VS. We observed dynamic exchange of Env at the VS while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse indicate that retention of Env is not likely to be maintained by entrapment into an immobile Gag lattice or through immobilizing interactions with CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus.

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

2016 ◽  
Vol 90 (21) ◽  
pp. 9841-9854 ◽  
Author(s):  
Shimona Starling ◽  
Clare Jolly
Keyword(s):  
T Cell ◽  
Cell Polarization ◽  
Cell Contact ◽  
Cell Remodeling ◽  
T Cell Polarization ◽  
Infected Cells ◽  
Virological Synapses ◽  
Cell Spread ◽  
Hiv 1 ◽  
Cell Cell

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.

scholarly journals Expansion of Monocytic Myeloid-Derived Suppressor Cells Dampens T Cell Function in HIV-1-Seropositive Individuals

2012 ◽  
Vol 87 (3) ◽  
pp. 1477-1490 ◽  
Author(s):  
Aiping Qin ◽  
Weiping Cai ◽  
Ting Pan ◽  
Kang Wu ◽  
Qiong Yang ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Suppressor Cells ◽  
Cell Contact ◽  
Myeloid Derived Suppressor Cells ◽  
Cell Dysfunction ◽  
Arginase 1 ◽  
T Cell Function ◽  
T Cell Dysfunction ◽  
Hiv 1

ABSTRACTT lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1 infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLA-DR−/lowCD11b+CD33+/highCD14+CD15−cells) in the peripheral blood of HIV-1-seropositive subjects (n= 61) compared with healthy controls (n= 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1+subjects correlated with prognostic HIV-1 disease markers, including the HIV-1 load (r= 0.5957;P< 0.0001), CD4+T cell loss (r= −0.5312;P< 0.0001), and activated T cells (r= 0.4421;P= 0.0004). Functional studies showed that M-MDSCs from HIV-1+subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manners; this effect was dependent on the induction of arginase 1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generationin vitro, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating that a positive-feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechanism of T cell dysfunction in HIV-1-infected individuals and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.

Cost-Effective High-Speed, Three-Dimensional Live-Cell Imaging of HIV-1 Transfer at the T Cell Virological Synapse

2021 ◽  
Author(s):  
Alice Sandmeyer ◽  
Lili Wang ◽  
Wolfgang Hübner ◽  
Marcel Müller ◽  
Benjamin Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Cost Effective ◽  
Live Cell ◽  
Virological Synapse ◽  
Hiv 1
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