Immune adaptor ADAP in T cells regulates HIV-1 transcription and cell-cell viral spread via different co-receptors

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.

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Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

Journal of Virology ◽

10.1128/jvi.02651-09 ◽

2010 ◽

Vol 84 (7) ◽

pp. 3516-3527 ◽

Cited By ~ 139

Author(s):

Nicola Martin ◽

Sonja Welsch ◽

Clare Jolly ◽

John A. G. Briggs ◽

David Vaux ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Virological Synapse ◽

Viral Spread ◽

Immunodeficiency Virus ◽

Virological Synapses ◽

Hiv 1 ◽

Entry Inhibition ◽

Cell Cell

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4+ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.

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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.

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Limited expression of R5-tropic HIV-1 in CCR5-positive type 1–polarized T cells explained by their ability to produce RANTES, MIP-1α, and MIP-1β

Blood ◽

10.1182/blood.v95.4.1167.004k11_1167_1174 ◽

2000 ◽

Vol 95 (4) ◽

pp. 1167-1174 ◽

Cited By ~ 37

Author(s):

Francesco Annunziato ◽

Grazia Galli ◽

Filomena Nappi ◽

Lorenzo Cosmi ◽

Roberto Manetti ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Memory T Cells ◽

Receptor Expression ◽

Th2 Cells ◽

Th1 Cells ◽

Naive T Cells ◽

Naïve T Cells ◽

Viral Spread ◽

Hiv 1

Human T helper (Th) cells (Th1- or Th2-oriented memory T cells as well as Th1- or Th2-polarized naive T cells) were infected in vitro with an R5-tropic HIV-1 strain (BaL) and assessed for their profile of cytokine production, CCR5 receptor expression, and HIV-1 p24 antigen (p24 Ag) production. Higher p24 Ag production was found in CCR5-negative Th2-like memory T cells than in CCR5-positive Th1-like memory T cells. By contrast, p24 Ag production was higher in Th1-polarized activated naive T cells in the first 4 days after infection. However, p24 Ag production in Th1-polarized T cells became comparable or even lower than the production in Th2-polarized populations later in infection or when the cells were infected with HIV-1BaL after secondary stimulation. The higher levels of p24 Ag production by Th1-polarized naive T cells soon after infection reflected a higher virus entry, as assessed by the single round infection assay using the HIV–chloramphenicol acetyl transferase (HIV-CAT) R5-tropic virus that contains the envelope protein of HIV-1 YU2 strain. The limitation of viral spread in the Th1-polarized populations, despite the initial higher level of T-cell entry of R5-tropic strains, was due to the ability of Th1 cells to produce greater amounts of β-chemokines than Th2 cells. In fact, an inverse correlation was observed between Th1-polarized naive T cells and Th1-like memory-activated T cells in regards to p24 Ag production and the release of the following CCR5-binding chemokines: regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein–1 (MIP-1), and MIP-1β. Moreover, infection with the HIV-1BaL strain of Th1-polarized T cells in the presence of a mixture of anti-RANTES, anti–MIP-1, and anti–MIP-1β neutralizing antibodies resulted in a significant increase of HIV-1 expression. These findings suggest that Th1-type responses may favor CD4+ T-cell infection by R5-tropic HIV-1 strains, but HIV-1 spread in Th1 cells is limited by their ability to produce CCR5-binding chemokines.

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The envelope cytoplasmic tail regulates HIV-1 assembly and spread

10.1101/2021.02.08.430194 ◽

2021 ◽

Author(s):

Xenia Snetkov ◽

Tafhima Haider ◽

Dejan Mesner ◽

Nicholas Groves ◽

Schuyler van Engelenburg ◽

...

Keyword(s):

T Cells ◽

Structural Integrity ◽

Alpha Helix ◽

Cytoplasmic Tail ◽

Virological Synapse ◽

Cell Contact ◽

Viral Fusion ◽

And Function ◽

Hiv 1 ◽

Cell Cell

AbstractThe 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 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. Strikingly we find that mutating W757 had wide-ranging consequences including altering Env mobility in the plasma membrane, preventing Env and Gag recruitment to sites of cell-cell contact for virological synapse (VS) formation and cell-cell spread, and impeding viral fusion. Notably, W757 was also required for efficient virus budding, revealing a previously unappreciated role for the EnvCT in regulating HIV-1 assembly and egress. We conclude that W757 is a key residue that stabilises the structural integrity and function of Env, consistent with the recent model that this region of the EnvCT acts as a critical supporting baseplate for Env.

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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.

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Multinucleated HIV-1-infected T cells exhibit unique migration patterns and frequent virus-dependent cell-cell interactions

10.22443/rms.lsfm2020.2 ◽

2020 ◽

Author(s):

Menelaos Symeonides

Keyword(s):

T Cells ◽

Cell Interactions ◽

Migration Patterns ◽

Dependent Cell ◽

Hiv 1 ◽

Cell Cell

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HIV Infection Stabilizes Macrophage-T Cell Interactions To Promote Cell-Cell HIV Spread

Journal of Virology ◽

10.1128/jvi.00805-19 ◽

2019 ◽

Vol 93 (18) ◽

Cited By ~ 2

Author(s):

Paul Lopez ◽

Wan Hon Koh ◽

Ryan Hnatiuk ◽

Thomas T. Murooka

Keyword(s):

T Cells ◽

T Cell ◽

Live Cell Imaging ◽

Cell Contact ◽

Cell Infection ◽

Cell Contacts ◽

3 Dimensional ◽

Viral Spread ◽

Large Numbers ◽

Cell Cell

ABSTRACTMacrophages are susceptible to HIV infection and play an important role in viral dissemination through cell-cell contacts with T cells. However, our current understanding of macrophage-to-T cell HIV transmission is derived from studies that do not consider the robust migration and cell-cell interaction dynamics between these cells. Here, we performed live-cell imaging studies in 3-dimensional (3D) collagen that allowed CD4+T cells to migrate and to locate and engage HIV-infected macrophages, modeling the dynamic aspects of thein situenvironment in which these contacts frequently occur. We show that HIV+macrophages form stable contacts with CD4+T cells that are facilitated by both gp120-CD4 and LFA-1–ICAM-1 interactions and that prolonged contacts are a prerequisite for efficient viral spread. LFA-1–ICAM-1 adhesive contacts function to restrain highly motile T cells, since their blockade substantially destabilized macrophage-T cell contacts, resulting in abnormal tethering events that reduced cell-cell viral spread. HIV-infected macrophages displayed strikingly elongated podosomal extensions that were dependent on Nef expression but were dispensable for stable cell-cell contact formation. Finally, we observed persistent T cell infection in dynamic monocyte-derived macrophage (MDM)-T cell cocultures in the presence of single high antiretroviral drug concentrations but achieved complete inhibition with combination therapy. Together, our data implicate macrophages as drivers of T cell infection by altering physiological MDM-T cell contact dynamics to access and restrain large numbers of susceptible, motile T cells within lymphoid tissues.IMPORTANCEOnce HIV enters the lymphoid organs, exponential viral replication in T cells ensues. Given the densely packed nature of these tissues, where infected and uninfected cells are in nearly constant contact with one another, efficient HIV spread is thought to occur through cell-cell contactsin vivo. However, this has not been formally demonstrated. In this study, we performed live-cell imaging studies within a 3-dimensional space to recapitulate the dynamic aspects of the lymphoid microenvironment and asked whether HIV can alter the morphology, migration capacity, and cell-cell contact behaviors between macrophages and T cells. We show that HIV-infected macrophages can engage T cells in stable contacts through binding of virus- and host-derived adhesive molecules and that stable macrophage-T cell contacts were required for high viral spread. Thus, HIV alters physiological macrophage-T cell interactions in order to access and restrain large numbers of susceptible, motile T cells, thereby playing an important role in HIV progression.

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Human Immunodeficiency Virus Type 1 Assembly, Budding, and Cell-Cell Spread in T Cells Take Place in Tetraspanin-Enriched Plasma Membrane Domains

Journal of Virology ◽

10.1128/jvi.01845-06 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7873-7884 ◽

Cited By ~ 126

Author(s):

Clare Jolly ◽

Quentin J. Sattentau

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Membrane Domains ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Cell Cell

ABSTRACT Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4+ T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4+ T cells and primary CD4+ T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs.

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