Clare Jolly

Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission.

Transmission of HIV-1 via intercellular connections has been estimated as 100–1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virological synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.

HIV-1 Cell to Cell Transfer across an Env-induced, Actin-dependent Synapse

Direct cell–cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1–infected effector T cells to primary CD4+/CXCR4+ target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function–associated antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was observed by 1 h after conjugate formation, was independent of cell–cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

Retroviral Spread by Induction of Virological Synapses

Cells of the immune system communicate via the formation of receptor‐containing adhesive junctions termed immunological synapses. Recently, retroviruses have been shown to subvert this process in order to pass directly from infected to uninfected immune cells. Such cell–cell viral dissemination appears to function by triggering existing cellular pathways involved in antigen presentation and T‐cell communication. This mode of viral spread has important consequences for both the virus and the host cells in terms of viral pathogenesis and viral resistance to immune and therapeutic intervention. This review summarises the current knowledge concerning virological synapses induced by retroviruses.

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

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.

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

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.

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

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.

Cell-Cell Spread of Human Immunodeficiency Virus Type 1 Overcomes Tetherin/BST-2-Mediated Restriction in T cells

ABSTRACT Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferon-induced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4+ T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.

Adhesion Molecule Interactions Facilitate Human Immunodeficiency Virus Type 1-Induced Virological Synapse Formation between T Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can spread between CD4+ T cells by using a virological synapse (VS). The VS assembly is a cytoskeleton-driven process dependent on HIV-1 envelope glycoprotein (Env)-receptor engagement and is hypothesized to require adhesion molecule interactions. Here we demonstrate that leukocyte function-associated antigen 1 (LFA-1), intercellular adhesion molecule 1 (ICAM-1), and ICAM-3 are enriched at the VS and that inhibition of these interactions influences conjugate formation and reduces VS assembly. Moreover, CD4+ T cells deficient in LFA-1 or with modified LFA-1 function were less able to support VS assembly and cell-cell transfer of HIV-1. Thus, cognate adhesion molecule interactions at the VS are important for HIV-1 spread between T cells.

Human Immunodeficiency Virus Type 1 Virological Synapse Formation in T Cells Requires Lipid Raft Integrity

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can spread directly between T cells by forming a supramolecular structure termed a virological synapse (VS). HIV-1 envelope glycoproteins (Env) are required for VS assembly, but their mode of recruitment is unclear. We investigated the distribution of GM1-rich lipid rafts in HIV-1-infected (effector) T cells and observed Env colocalization with polarized raft markers GM1 and CD59 but not with the transferrin receptor that is excluded from lipid rafts. In conjugates of effector T cells and target CD4+ T cells, GM1, Env, and Gag relocated to the cell-cell interface. The depletion of cholesterol in the infected cell dispersed Env and GM1 within the plasma membrane, eliminated Gag clustering at the site of cell-cell contact, and abolished assembly of the VS. Raft integrity is therefore critical for Env and Gag coclustering and VS assembly in T-cell conjugates.

Transportin 3 Promotes a Nuclear Maturation Step Required for Efficient HIV-1 Integration

The HIV/AIDS pandemic is a major global health threat and understanding the detailed molecular mechanisms of HIV replication is critical for the development of novel therapeutics. To replicate, HIV-1 must access the nucleus of infected cells and integrate into host chromosomes, however little is known about the events occurring post-nuclear entry but before integration. Here we show that the karyopherin Transportin 3 (Tnp3) promotes HIV-1 integration in different cell types. Furthermore Tnp3 binds the viral capsid proteins and tRNAs incorporated into viral particles. Interaction between Tnp3, capsid and tRNAs is stronger in the presence of RanGTP, consistent with the possibility that Tnp3 is an export factor for these substrates. In agreement with this interpretation, we found that Tnp3 exports from the nuclei viral tRNAs in a RanGTP-dependent way. Tnp3 also binds and exports from the nuclei some species of cellular tRNAs with a defective 3′CCA end. Depletion of Tnp3 results in a re-distribution of HIV-1 capsid proteins between nucleus and cytoplasm however HIV-1 bearing the N74D mutation in capsid, which is insensitive to Tnp3 depletion, does not show nucleocytoplasmic redistribution of capsid proteins. We propose that Tnp3 promotes HIV-1 infection by displacing any capsid and tRNA that remain bound to the pre-integration complex after nuclear entry to facilitate integration. The results also provide evidence for a novel tRNA nucleocytoplasmic trafficking pathway in human cells.