Inefficient human immunodeficiency virus replication in mobile lymphocytes

Cell-to-cell viral transfer facilitates the spread of lymphotropic retroviruses such as human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), likely through the formation of "virological synapses" between donor and target cells. Regarding HIV replication, the importance of cell contacts has been demonstrated, but this phenomenon remains only partly characterized. In order to alter cell-to-cell HIV transmission, we have maintained cultures under continuous gentle shaking and followed viral replication in this experimental system. In lymphoid cell lines, as well as in primary lymphocytes, viral replication was dramatically reduced in shaken cultures. To document this phenomenon, we have developed an assay to assess the relative contributions of free and cell-associated virions in HIV propagation. Acutely infected donor cells were mixed with carboxyfluorescein diacetate succinimidyl ester-labeled lymphocytes as targets, and viral production was followed by measuring HIV Gag expression at different time points by flow cytometry. We report that cellular contacts drastically enhance productive viral transfer compared to what is seen with infection with free virus. Productive cell-to-cell viral transmission required fusogenic viral envelope glycoproteins on donor cells and adequate receptors on targets. Only a few syncytia were observed in this coculture system. Virus release from donor cells was unaffected when cultures were gently shaken, whereas virus transfer to recipient cells was severely impaired. Altogether, these results indicate that cell-to-cell transfer is the predominant mode of HIV spread and help to explain why this virus replicates so efficiently in lymphoid organs.     

Modulation of HIV pathogenesis and T-cell signaling by HIV-1 Nef

HIV-1 Nef protein is an approximately 27-kDa myristoylated protein that is a virulence factor essential for efficient viral replication and infection in CD4(+) T cells. The functions of CD4(+) T cells are directly impeded after HIV infection. HIV-1 Nef plays a crucial role in manipulating host cellular machinery and in HIV pathogenesis by reducing the ability of infected lymphocytes to form immunological synapses by promoting virological synapses with APCs, and by affecting T-cell stimulation. This article reviews the current status of the efficient Nef-mediated spread of virus in the unreceptive environment of the immune system by altering CD4(+) T-lymphocyte signaling, intracellular trafficking, cell migration and apoptotic pathways.     

Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function.

ZAP-70 is a protein tyrosine kinase (PTK) required for T-cell development and T-cell antigen receptor (TCR) function. ZAP-70 is associated with the phosphorylated antigen receptor and undergoes tyrosine phosphorylation following receptor activation. We demonstrate here that tyrosine phosphorylation of ZAP-70 results in an increase in its catalytic activity and that this activation is mediated by the phosphorylation of tyrosine residue 493 by the src family of PTKs. The activity of baculoviral expressed ZAP-70 was up-regulated 10-fold when ZAP-70 was co-infected and phosphorylated by the src family PTK, lck. Mutation of Y493 alone abrogated the ability of ZAP-70 to be activated by lck. Moreover, we demonstrate that phosphorylation of Y493 and activation of ZAP-70 is required for antigen receptor-mediated induction of interleukin-2 (IL-2) secretion in lymphocytes.     

Masters of manipulation: Viral modulation of the immunological synapse

In order to thrive, viruses have evolved to manipulate host cell machinery for their own benefit. One major obstacle faced by pathogens is the immunological synapse. To enable efficient replication and latency in immune cells, viruses have developed a range of strategies to manipulate cellular processes involved in immunological synapse formation to evade immune detection and control T‐cell activation. In vitro, viruses such as human immunodeficiency virus 1 and human T‐lymphotropic virus type 1 utilise structures known as virological synapses to aid transmission of viral particles from cell to cell in a process termed trans‐infection. The formation of the virological synapse provides a gateway for virus to be transferred between cells avoiding the extracellular space, preventing antibody neutralisation or recognition by complement. This review looks at how viruses are able to subvert intracellular signalling to modulate immune function to their advantage and explores the role synapse formation has in viral persistence and cell‐to‐cell transmission.     

HIV-1 at the immunological and T-lymphocytic virological synapse

Cell-cell transmission of human immunodeficiency virus type 1 (HIV-1) is considered the most effective mode of viral spread in T-lymphocyte cultures. Evidence has accumulated that HIV-1 assembles polarized synaptic-like structures, referred to as virological synapses, as specialized sites of viral transfer. Interestingly, it was recently also discovered that HIV-1 impairs the formation of the structurally similar immunological synapse, thereby modulating exogenous T-lymphocyte stimulation to yield an optimal activation state for productive HIV-1 infection. The careful dissection of these opposing effects will contribute to our understanding of retroviral spread and cellular signal transduction machineries.     

Dynamic movement of the calcium sensor STIM1 and the calcium channel Orai1 in activated T-cells: puncta and distal caps

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca(2+) influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca(2+) channel components to existing and newly forming immunological synapses.     

The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM.     

Effect of Synaptic Transmission on Viral Fitness in HIV Infection

HIV can spread through its target cell population either via cell-free transmission, or by cell-to-cell transmission, presumably through virological synapses. Synaptic transmission entails the transfer of tens to hundreds of viruses per synapse, a fraction of which successfully integrate into the target cell genome. It is currently not understood how synaptic transmission affects viral fitness. Using a mathematical model, we investigate how different synaptic transmission strategies, defined by the number of viruses passed per synapse, influence the basic reproductive ratio of the virus, R₀, and virus load. In the most basic scenario, the model suggests that R₀ is maximized if a single virus particle is transferred per synapse. R₀ decreases and the infection eventually cannot be maintained for larger numbers of transferred viruses, because multiple infection of the same cell wastes viruses that could otherwise enter uninfected cells. To explain the relatively large number of HIV copies transferred per synapse, we consider additional biological assumptions under which an intermediate number of viruses transferred per synapse could maximize R₀. These include an increased burst size in multiply infected cells, the saturation of anti-viral factors upon infection of cells, and rate limiting steps during the process of synapse formation.     

HIV-1 Nef employs two distinct mechanisms to modulate Lck subcellular localization and TCR induced actin remodeling

The Nef protein acts as critical factor during HIV pathogenesis by increasing HIV replication in vivo via the modulation of host cell vesicle transport and signal transduction processes. Recent studies suggested that Nef alters formation and function of immunological synapses (IS), thereby modulating exogenous T-cell receptor (TCR) stimulation to balance between partial T cell activation required for HIV-1 spread and prevention of activation induced cell death. Alterations of IS function by Nef include interference with cell spreading and actin polymerization upon TCR engagement, a pronounced intracellular accumulation of the Src kinase Lck and its reduced IS recruitment. Here we use a combination of Nef mutagenesis and pharmacological inhibition to analyze the relative contribution of these effects to Nef mediated alterations of IS organization and function on TCR stimulatory surfaces. Inhibition of actin polymerization and IS recruitment of Lck were governed by identical Nef determinants and correlated well with Nef's association with Pak2 kinase activity. In contrast, Nef mediated Lck endosomal accumulation was separable from these effects, occurred independently of Pak2, required integrity of the microtubule rather than the actin filament system and thus represents a distinct Nef activity. Finally, reduction of TCR signal transmission by Nef was linked to altered actin remodeling and Lck IS recruitment but did not require endosomal Lck rerouting. Thus, Nef affects IS function via multiple independent mechanisms to optimize virus replication in the infected host.     

Tracking and quantitation of fluorescent HIV during cell-to-cell transmission

The green fluorescent protein (GFP) is a powerful genetic marking tool that has enabled virologists to monitor and track viral proteins during HIV infection. Expression-optimized Gag-GFP constructs have been used to study virus-like particle (VLP) assembly and localization in cell types that are easily transfected. The development of HIV-1 variants carrying GFP within the context of the viral genome has facilitated the study of infection and has been particularly useful in monitoring the transfer of virus between cells following virological synapse formation. HIV Gag-iGFP, a viral clone that contains GFP inserted between the matrix (MA) and capsid (CA) domains of Gag, is the first replication competent molecular clone that generates fluorescent infectious particles. Here, we discuss some methods that exploit HIV Gag-iGFP to quantify cell-to-cell transmission of virus by flow cytometry and to track the proteins during assembly and transmission using live-cell imaging.     

Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.     

A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse

Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCgamma1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or "antisynapse".     

Mechanism of recruitment of WASP to the immunological synapse and of its activation following TCR ligation

F-actin polymerization following engagement of the T cell receptor (TCR) is dependent on WASP and is critical for T cell activation. The link between TCR and WASP is not fully understood. In resting cells, WASP exists in a complex with WIP, which inhibits its activation by Cdc42. We show that the adaptor protein CrkL binds directly to WIP. Further, TCR ligation results in the formation of a ZAP-70-CrkL-WIP-WASP complex, which is recruited to lipid rafts and the immunological synapse. TCR engagement also causes PKCtheta-dependent phosphorylation of WIP, causing the disengagement of WASP from the WIP-WASP complex, thereby releasing it from WIP inhibition. These results suggest that the ZAP-70-CrkL-WIP pathway and PKCtheta link TCR to WASP activation.     

T cell activation stimulates the association of enzymatically active tyrosine-phosphorylated ZAP-70 with the Crk adapter proteins.

Engagement of the T cell antigen receptor initiates signal transduction involving tyrosine phosphorylation of multiple effector molecules and the formation of multimolecular complexes at the receptor site. Adapter proteins that possess SH2 and SH3 protein-protein interaction domains are implicated in the assembly of cell activation-induced signaling complexes. We found that Crk adapter proteins undergo activation-induced interaction with the zeta-chain associated protein (ZAP-70) tyrosine kinase in the human T cell line, Jurkat. Incubation of various glutathione S-transferase fusion proteins with a lysate of activated Jurkat cells resulted in selective association of ZAP-70 with Crk, but not Grb2 or Nck, adapter proteins. In addition, tyrosine-phosphorylated ZAP-70 co-immunoprecipitated with Crk from a lysate of activated Jurkat cells, and ZAP-70 association with GST-Crk was observed in a lysate of activated human peripheral blood T cells. Association between the two molecules was mediated by direct physical interaction and involved the Crk-SH2 domain and phosphotyrosyl-containing sequences on ZAP-70. The association required intact Lck, considered to be an upstream regulator of ZAP-70, because it could not take place in activated JCaM1 cells, which express normal levels of ZAP-70 but are devoid of Lck. Finally, glutathione S-transferase-Crk fusion proteins were found to interact predominantly with membrane-residing tyrosine-phosphorylated ZAP-70 that exhibited autophosphorylation activity as well as phosphorylation of an exogenous substrate, CFB3. These findings suggest that Crk adapter proteins play a role in the early activation events of T lymphocytes, apparently, by direct interaction with, and regulation of, the membrane-residing ZAP-70 protein tyrosine kinase.     

Information transfer at the immunological synapse

Antigen-specific activation of T lymphocytes requires the interaction of their clonally distributed T-cell receptors with plasma membrane ligands composed of foreign peptide antigens bound to major histocompatibility complex molecules. For proliferation and differentiation to ensue, a variety of other adhesive and accessory proteins must also interact with their counter-receptors on the antigen-presenting cell to facilitate and complement the T-cell receptor-antigen recognition event. Recent studies have revealed that these various proteins show an unexpected degree of spatial organization in the zone of cell-cell contact. This region of membrane approximation is now referred to as the "immunological synapse" because of its functional analogy to the site of intercellular information transfer between neurons. Here, we review the evidence for signaling-dependent control of the dynamic changes in protein distribution that gives rise to the synapse and try to relate the emerging spatio-temporal information on synapse formation to T-cell biology.     

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.     

Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.     

RhoH regulates subcellular localization of ZAP-70 and Lck in T cell receptor signaling

RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3ζ was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3ζ phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development, Rhoh(-/-) bone marrow cells were transduced with a chimeric myristoylation-tagged ZAP-70. Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3ζ in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway.