Virological Synapses Research Articles

A-108 Infection through HIV T cell virological synapses

A-108 Infection through HIV T cell virological synapses

Contact-induced mitochondrial polarization supports HIV-1 virological synapse formation.

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.IMPORTANCE HIV-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.

Cell-to-cell transfer of HIV infection: implications for HIV viral persistence.

A major research priority for HIV eradication is the elucidation of the events involved in HIV reservoir establishment and persistence. Cell-to-cell transmission of HIV represents an important area of study as it allows for the infection of cell types which are not easily infected by HIV, leading to the establishment of long-lived viral reservoirs. This phenomenon enables HIV to escape elimination by the immune system. This process may also enable HIV to escape suppressive effects of anti-retroviral drugs. During cell-to-cell transmission of HIV, a dynamic series of events ensues at the virological synapse that promotes viral dissemination. Cell-to-cell transmission involves various types of cells of the immune system and this mode of transmission has been shown to have an important role in sexual and mother-to-child transmission of HIV and spread of HIV within the central nervous system and gut-associated lymphoid tissues. There is also evidence that cell-to-cell transmission of HIV occurs between thymocytes and renal tubular cells. Herein, following a brief review of the processes involved at the virological synapse, evidence supporting the role for cell-to-cell transmission of HIV in the maintenance of the HIV reservoir will be highlighted. Therapeutic considerations and future directions for this area of research will also be discussed.

Cell-to-cell transmission in the pathogenesis of human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis

Virus transmission is an important key factor for understanding the pathomechanism(s) of virus-related neurological diseases. In the past, cell-free transmission was believed to be a major route of virus spread for almost all viruses. However, attention was paid to “cell-to-cell transmission” in the 1960s. Cell-to-cell transmission appears to be more effective, as progeny viruses do not have to encounter antibodies. Although this mode of virus transmission was first highlighted in subacute sclerosing panencephalitis caused by a variant of measles virus, human T-lymphotropic virus type 1 (HTLV-I) also utilizes cell-to-cell transmission. The transmission is undergone through so-called “virological synapses”, a structure first described in HTLV-I. Cell contact induces polarization of the cytoskeleton of the infected cells to the cell–cell junction. HTLV-I core (Gag protein) complexes and the HTLV-I genome accumulate at the cell–cell junction and are transferred to the uninfected cell. The intracellular cyclic adenosine monophosphate regulates the efficiency of intercellular HTLV-I transmission under the control of expression phosphorylated vasodilator- stimulated phosphoprotein.

Galectin-3 translocates to virological synapse and promotes HIV-1 transfer (VIR1P.1000)

Abstract Galectin-3 contains β-galactoside-binding domain and is mainly expressed in immune cells and epithelial cells. Galectin-3 is known to play regulatory roles in the immune system to defend against pathogens. Previous studies showed that Alix is a host factor employed by HIV-1 for its replication. Here, we report that galectin-3 is another such factor. We have reported that galectin-3 is translocated to the immunological synapse and affects T cell cytokine production. We have now studied the role of galectin-3 in the virological synapse (VS). Our data showed that HIV-1 infection induces galectin-3 expression in primary CD4 T cells. Immunofluorescence imaging showed that galectin-3 is translocted to the virological synapse and co-localized with Gag and Env at the cell-to-cell junction of HIV-1-infected cells. We have studied the effects of galectin-3 in the VS by using fluorescent-tagged HIV-1 and incubation of fluorescent-labeled T cells with galectin-3 knockeddown (KD). Our analysis indicates that HIV-1 transmission efficacy is significantly attenuated in galectin-3 KD cells comparing to galectin-3-expressing wild type cells. Our analysis also shows that Alix plays a role in promoting cell-to-cell transfer of HIV-1. Further analysis indicate that effects of galectin-3 in effector cells is likely more important than expression of galectin-3 in target cells. We conclude that endogenous galectin-3 translocates to VS and promotes cell-to-cell viral transfer upon HIV-1 infection.

Ezrin is a component of the HIV-1 virological presynapse and contributes to the inhibition of cell-cell fusion.

During cell-to-cell transmission of HIV-1, viral and cellular proteins transiently accumulate at the contact zone between infected (producer) and uninfected (target) cells, forming the virological synapse. Rearrangements of the cytoskeleton in producer and target cells are required for proper targeting of viral and cellular components during synapse formation, yet little is known about how these processes are regulated, particularly within the producer cell. Since ezrin-radixin-moesin (ERM) proteins connect F-actin with integral and peripheral membrane proteins, are incorporated into virions, and interact with cellular components of the virological presynapse, we hypothesized that they play roles during the late stage of HIV-1 replication. Here we document that phosphorylated (i.e., active) ezrin specifically accumulates at the HIV-1 presynapse in T cell lines and primary CD4(+) lymphocytes. To investigate whether ezrin supports virus transmission, we sought to ablate ezrin expression in producer cells. While cells did not tolerate a complete knockdown of ezrin, even a modest reduction of ezrin expression (~50%) in HIV-1-producing cells led to the release of particles with impaired infectivity. Further, when cocultured with uninfected target cells, ezrin-knockdown producer cells displayed reduced accumulation of the tetraspanin CD81 at the synapse and fused more readily with target cells, thus forming syncytia. Such an outcome likely is not optimal for virus dissemination, as evidenced by the fact that, in vivo, only relatively few infected cells form syncytia. Thus, ezrin likely helps secure efficient virus spread not only by enhancing virion infectivity but also by preventing excessive membrane fusion at the virological synapse. While viruses, in principal, can propagate through successions of syncytia, HIV-1-infected cells in the majority of cases do not fuse with potential target cells during viral transmission. This mode of spread is coresponsible for key features of HIV-1 pathogenesis, including killing of bystander cells and establishment of latently infected T lymphocytes. Here we identify the ERM protein family member ezrin as a cellular factor that contributes to the inhibition of cell-cell fusion and thus to suppressing excessive syncytium formation. Our analyses further suggest that ezrin, which connects integral membrane proteins with actin, functions in concert with CD81, a member of the tetraspanin family of proteins. Additional evidence, documented here and elsewhere, suggests that ezrin and CD81 cooperate to prevent cytoskeleton rearrangements that need to take place during the fusion of cellular membranes.

104 Cell-to-cell transmission of HIV

HIV-1 infects CD4+ T-lymphocyte by cell free viral particles released from the surface of effector cells or after the formation of intercellular contact between infected cell and healthy lymphocyte. The last way of infection was designated as cell-to-cell viral transmission. The current studies suggest that cell-to-cell transmission of HIV occurs in peripheral lymph nodes at the early stage of infection and is more efficient pathway of virus dissemination than cell-free infection. One of the most accepted models of HIV transmission is the formation of virological synapse (VS); tight intercellular adhesion junction sharing structural similarity with immunological synapse. VS forms after HIV surface protein gp120 expressed on the plasma membrane of infected cells in prefusogenic conformation recognizes CD4 receptor on target cell. Viruses transmitted across VS undergo endocytosis by target cells, so that viral entry occurs via endocytic compartments and not via cell surface. This feature of cell-to-cell transmission accounts for the resistance of HIV to the neutralizing antibodies. Although the different stages of HIV cell-to-cell transmission have been extensively studied, the levels of correlation between virus transmission and targets infection were not accurately quantified. To fill up the gap in our understanding of cell-mediated viral infection, we have developed a new methods and improved vectors to quantify the levels of VS formation, cell-to-cell infection, and macromolecular transport across the cell-cell contact. New T cell lines stably producing recombinant virus like particles and reporter RNA, as well as modified target cells were generated.

Evidence Showing that Tetraspanins Inhibit HIV-1-Induced Cell-Cell Fusion at a Post-Hemifusion Stage

Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening.

Electron Tomography of HIV-1 Infection in Gut-Associated Lymphoid Tissue

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1–infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1–infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue.

Comparative infection of autologous primary T cells and monocytes derived DCs using cell-free virus preparation, viral biofilm, pseudotyped-virus or viral synapse

HTLV-1 infected T cells and blood dentritic cells (DCs) can be found in HTLV-1 carriers. It is currently believed that T cell infection requires cell-cell contact, while DCs could also be infected with cell-free virus. However, a comparative study using different modes of viral preparation for infecting human primary autologous T and DC cells has not been performed. Supernatant from HTLV-1 chronically infected T cells, cell conjugates between HTLV-1 infected cells and target cells, or purified viral biofilm were used side-by-side to infect autologous human primary lymphocytes and monocytes-derived dendritic cells. To monitor the early steps of viral infection, we also used pseudotyped viruses carrying either the HTLV-1 or the VSV envelopes. Infection was then followed by flow cytometry, ELISA, immunofluorescence, real-time PCR and alu-PCR. Our first data show that, depending on the source of viruses, lymphocytes have distinct susceptibility to HTLV-1 infection. The impact of the viral source in the spread of HTLV-1 infection will be discussed.

High-multiplicity HIV-1 infection and neutralizing antibody evasion mediated by the macrophage-T cell virological synapse.

Macrophage infection is considered to play an important role in HIV-1 pathogenesis and persistence. Using a primary cell-based coculture model, we show that monocyte-derived macrophages (MDM) efficiently transmit a high-multiplicity HIV-1 infection to autologous CD4(+) T cells through a viral envelope glycoprotein (Env) receptor- and actin-dependent virological synapse (VS), facilitated by interactions between ICAM-1 and LFA-1. Virological synapse (VS)-mediated transmission by MDM results in high levels of T cell HIV-1 integration and is 1 to 2 orders of magnitude more efficient than cell-free infection. This mode of cell-to-cell transmission is broadly susceptible to the activity of CD4 binding site (CD4bs) and glycan or glycopeptide epitope-specific broadly neutralizing monoclonal antibodies (bNMAbs) but shows resistance to bNMAbs targeting the Env gp41 subunit membrane-proximal external region (MPER). These data define for the first time the structure and function of the macrophage-to-T cell VS and have important implications for bNMAb activity in HIV-1 prophylaxis and therapy. IMPORTANCE The ability of HIV-1 to move directly between contacting immune cells allows efficient viral dissemination with the potential to evade antibody attack. Here, we show that HIV-1 spreads from infected macrophages to T cells via a structure called a virological synapse that maintains extended contact between the two cell types, allowing transfer of multiple infectious events to the T cell. This process allows the virus to avoid neutralization by a class of antibody targeting the gp41 subunit of the envelope glycoproteins. These results have implications for viral spread in vivo and the specificities of neutralizing antibody elicited by antibody-based vaccines.

Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission

The neutralizing activity of anti-HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4(+) lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies.

Antiviral treatments over cell-to-cell infection

Antiviral treatments over cell-to-cell infection

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

BackgroundImmune cell adaptor protein ADAP (adhesion and degranulation-promoting adaptor protein) mediates aspects of T-cell adhesion and proliferation. Despite this, a connection between ADAP and infection by the HIV-1 (human immunodeficiency virus-1) has not been explored.ResultsIn this paper, we show for the first time that ADAP and its binding to SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) regulate HIV-1 infection via two distinct mechanisms and co-receptors. siRNA down-regulation of ADAP, or expression of a mutant that is defective in associating to its binding partner SLP-76 (termed M12), inhibited the propagation of HIV-1 in T-cell lines and primary human T-cells. In one step, ADAP and its binding to SLP-76 were needed for the activation of NF-κB and its transcription of the HIV-1 long terminal repeat (LTR) in cooperation with ligation of co-receptor CD28, but not LFA-1. In a second step, the ADAP-SLP-76 module cooperated with LFA-1 to regulate conjugate formation between T-cells and dendritic cells or other T-cells as well as the development of the virological synapse (VS) and viral spread between immune cells.ConclusionsThese findings indicate that ADAP regulates two steps of HIV-1 infection cooperatively with two distinct receptors, and as such, serves as a new potential target in the blockade of HIV-1 infection.

Contact-induced polarization shapes HIV-1 infected T cells for efficient HIV-1 dissemination at the virological synapse

Background Transmission of HIV-1 between CD4+ T cells is by cellfree infection or direct cell-to-cell spread at intercellular junctions called virological synapses (VS). Transmission at VS has been shown to be orders of magnitude more efficient than cell-free infection. Increasing evidence suggests a key role for cell-cell spread in HIV-1 dissemination, replication and pathogenesis. Our previous work showed that HIV-1 VS formation is associated with striking organelle polarization to intercellular junctions and that a large volume of the synaptic cytoplasm in the infected cell is occupied by mitochondria. However, whether this occurs in response to contact and the functional consequences imparted by this are unclear. Therefore, we set out to investigate the potential role of mitochondria in the formation of the HIV-1 VS and in HIV-1 cell-cell spread.

Murine leukemia virus targets innate-like B1 B cells to establish infection in mice

Background Retroviruses are believed to efficiently spread via sites of cell-cell contact designated virological synapses. This model was developed based on in vitro evidence in which infected cells establish cell-cell contact with uninfected cells. Applying intravital microscopy, we were recently able to provide in vivo support for the existence of virological synapses within the lymph node of living mice. Visualizing cells infected with fluorescently labeled murine leukemia virus (MLV) we identified B cells that were able to form long-lived virological synapses with uninfected lymphocytes [1]. In vivo virological synapses were, like their in vitro counterpart, dependent on the expression of the viral envelope glycoprotein (Env) and characterized by a prolonged polarization of viral capsid to the cell-cell interface. B cells were among the first cells to become infected by incoming MLV. However, the specific subtype of B cells that is susceptible to MLV had remained unknown.

Investigating contact-induced T cell polarisation at virological synapses

Background Human Immunodeficiency Virus Type-1 (HIV-1), the main causative agent of Acquired Immunodeficiency Syndrome (AIDS) is a major global health problem. Cell-cell spread of HIV-1 between CD4 T cells confers many advantages including more rapid infection kinetics, evasion of neutralising antibodies and cellular restriction factors, and may pose a barrier to eradicating HIV-1 from the host. HIV-1 cell-cell spread occurs at intercellular contacts called virological synapses (VS) at which HIV-1 preferentially assembles and buds. We have previously shown that the microtubule organising centre (MTOC) and associated organelles are polarised within the HIV-1 infected cell at the VS; however the causes and consequences of this are unknown.

Synaptic transmission and the susceptibility of HIV infection to anti-viral drugs

Cell-to-cell viral transmission via virological synapses has been argued to reduce susceptibility of the virus population to anti-viral drugs through multiple infection of cells, contributing to low-level viral persistence during therapy. Using a mathematical framework, we examine the role of synaptic transmission in treatment susceptibility. A key factor is the relative probability of individual virions to infect a cell during free-virus and synaptic transmission, a currently unknown quantity. If this infection probability is higher for free-virus transmission, then treatment susceptibility is lowest if one virus is transferred per synapse, and multiple infection of cells increases susceptibility. In the opposite case, treatment susceptibility is minimized for an intermediate number of virions transferred per synapse. Hence, multiple infection via synapses does not simply lower treatment susceptibility. Without further experimental investigations, one cannot conclude that synaptic transmission provides an additional mechanism for the virus to persist at low levels during anti-viral therapy.

Multiple proviral integration events after virological synapse-mediated HIV-1 spread

HIV-1 can move directly between T cells via virological synapses (VS). Although aspects of the molecular and cellular mechanisms underlying this mode of spread have been elucidated, the outcomes for infection of the target cell remain incompletely understood. We set out to determine whether HIV-1 transfer via VS results in productive, high-multiplicity HIV-1 infection. We found that HIV-1 cell-to-cell spread resulted in nuclear import of multiple proviruses into target cells as seen by fluorescence in-situ hybridization. Proviral integration into the target cell genome was significantly higher than that seen in a cell-free infection system, and consequent de novo viral DNA and RNA production in the target cell detected by quantitative PCR increased over time. Our data show efficient proviral integration across VS, implying the probability of multiple integration events in target cells that drive productive T cell infection.

Basic Residues in the Matrix Domain and Multimerization Target Murine Leukemia Virus Gag to the Virological Synapse

Murine leukemia virus (MLV) can efficiently spread in tissue cultures by polarizing assembly to virological synapses. The viral envelope glycoprotein (Env) establishes cell-cell contacts and subsequently recruits Gag by a process that depends on its cytoplasmic tail. MLV Gag is recruited to virological synapses through the matrix domain (MA) (J. Jin, F. Li, and W. Mothes, J. Virol. 85:7672-7682, 2011). However, how MA targets Gag to sites of cell-cell contact remains unknown. Here we report that basic residues within MA are critical for directing MLV Gag to virological synapses. Alternative membrane targeting domains (MTDs) containing multiple basic residues can efficiently substitute MA to direct polarized assembly. Similarly, mutations in the polybasic cluster of MA that disrupt Gag polarization can be rescued by N-terminal addition of MTDs containing basic residues. MTDs containing basic residues alone fail to be targeted to the virological synapse. Systematic deletion experiments reveal that domains within Gag known to mediate Gag multimerization are also required. Thus, our data predict the existence of a specific "acidic" interface at virological synapses that mediates the recruitment of MLV Gag via the basic cluster of MA and Gag multimerization.