Virological Synapse Formation Research Articles

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse

The availability of cost-effective, highly portable, and easy to use high-resolution live-cell imaging systems could present a significant technological break-through in challenging environments, such as high-level biosafety laboratories or sites where new viral outbreaks are suspected. We describe and demonstrate a cost-effective high-speed fluorescence microscope enabling the live tracking of virus particles across virological synapses that form between infected and uninfected Tcells. The dynamics of HIV-1 proteins studied at the cellular level and the formation of virological synapses in living Tcells reveals mechanisms by which cell-cell interactions facilitate infection between immune cells. Dual-color 3D fluorescence deconvolution microscopy of HIV-1 particles at frames rates of 100 frames per second allows us to follow the transfer of HIV-1 particles across the Tcell virological synapse between living Tcells. We also confirm the successful transfer of virus by imaging Tcell samples fixed at specific time points during cell-cell virus transfer by super-resolution structured illumination microscopy.

Backward bifurcation and stability analysis in a within-host HIV model with both virus-to-cell infection and cell-to-cell transmission, and anti-retroviral therapy

Researches have shown that in addition to direct virus-to-cell infection, viral particles can also be transferred from a productively-infected cell to an uninfected cell through the formation of virological synapses. In order to reduce the viral load in infected individuals, different classes of antiretroviral drugs have been developed, including reverse transcriptase inhibitor (RTI), integrase inhibitor (II), protease inhibitor (PI) and so on. In this paper, we incorporate the mitotic proliferation of target cells which is described by the logistic term, both virus-to-cell infection and cell-to-cell transmission, the intracellular delay and RTI-based therapy into an in-host HIV infection model. Through mathematical analysis, we find that the model undergoes a backward bifurcation when the turn-over rate coefficient of productively-infected cells is smaller than its mitotic proliferation rate coefficient. When the turn-over rate coefficient of productively-infected cells is greater than its mitotic proliferation rate coefficient, the existence of Hopf bifurcation at the chronic-infection equilibrium with and without the intracellular delay is established, respectively. Numerical simulations suggest that the dynamics of the model be sensitive to parameter values and initial conditions, which may be of great significance to control HIV infection. We also show numerical evidence to support the fact that the smaller the therapy efficacy, the higher the viral load in infected individuals.

A Conserved Tryptophan in the Envelope Cytoplasmic Tail Regulates HIV-1 Assembly and Spread.

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.

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

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.

Identification of Host Trafficking Genes Required for HIV-1 Virological Synapse Formation in Dendritic Cells.

Dendritic cells (DCs) are one of the earliest targets of HIV-1 infection acting as a "Trojan horse," concealing the virus from the innate immune system and delivering it to T cells via virological synapses (VS). To explicate how the virus is trafficked through the cell to the VS and evades degradation, a high-throughput small interfering RNA screen targeting membrane trafficking proteins was performed in monocyte-derived DCs. We identified several proteins including BIN-1 and RAB7L1 that share common roles in transport from endosomal compartments. Depletion of target proteins resulted in an accumulation of virus in intracellular compartments and significantly reduced viral trans-infection via the VS. By targeting endocytic trafficking and retromer recycling to the plasma membrane, we were able to reduce the virus's ability to accumulate at budding microdomains and the VS. Thus, we identify key genes involved in a pathway within DCs that is exploited by HIV-1 to traffic to the VS.IMPORTANCE The lentivirus human immunodeficiency virus (HIV) targets and destroys CD4+ T cells, leaving the host vulnerable to life-threatening opportunistic infections associated with AIDS. Dendritic cells (DCs) form a virological synapse (VS) with CD4+ T cells, enabling the efficient transfer of virus between the two cells. We have identified cellular factors that are critical in the induction of the VS. We show that ADP-ribosylation factor 1 (ARF1), bridging integrator 1 (BIN1), and Rab GTPases RAB7L1 and RAB8A are important regulators of HIV-1 trafficking to the VS and therefore the infection of CD4+ T cells. We found these cellular factors were essential for endosomal protein trafficking and formation of the VS and that depletion of target proteins prevented virus trafficking to the plasma membrane by retaining virus in intracellular vesicles. Identification of key regulators in HIV-1 trans-infection between DC and CD4+ T cells has the potential for the development of targeted therapy to reduce trans-infection of HIV-1 in vivo.

Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging

BackgroundHIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell–cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined.ResultsTo study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell–cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS.ConclusionsEnv and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS.

Variable infectivity and conserved engagement in cell-to-cell viral transfer by HIV-1 Env from Clade B transmitted founder clones

HIV-1 transmission is usually initiated by a single viral strain called transmitted/ founder (T/F) virus. In in vitro models, HIV-1 can efficiently spread via cell-free and virological synapse (VS)-mediated cell-to-cell infection. Both modes of infection require the viral glycoprotein Envelope (Env). The efficiency with which T/F Envs initiate VS and mediate cell-to-cell infection has not been well characterized. Here we tested a panel of isogenic HIV-1 molecular clones that carry different Clade B T/F Envs. We found that despite variable infectivity among different Env clones in the two modes of infection, T/F Envs generally mediated efficient VS formation and subsequent cell-to-cell transfer. In contrast, in vitro infectivity of the T/F Env clones was more variable and strongly correlated with intrinsic fusogenicity of various Envs. We speculate that the conservation of cell-to-cell transfer by T/F Env is indicative of a biologically important function of Env.

Live Imaging of HIV-1 Transfer across T Cell Virological Synapse to Epithelial Cells that Promotes Stromal Macrophage Infection

During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed invitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4+ Tcells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa.

Dissemination of HIV-1 from latently infected cells to resting CD4+ T cells requires CD4 and actin remodeling.

Abstract The major barrier to HIV-1 eradication is the replication-competent proviruses in long-lived latently infected CD4+ T cells. Current efforts to purge latently HIV-1-infected cells have focused on developing “shock and kill” approaches to force reactivation of the proviruses in these cells to be killed after reactivation of virus gene expression by viral cytopathic effects, host immune responses, or both. Cell-free virus infection and cell-to-cell transmission via the virological synapses (VS) are the two modes of HIV-1 dissemination between CD4+ T cells. Several studies have demonstrated that cell-to-cell spread, in which HIV-1 disseminates through direct contact of infected cells with surrounding uninfected cells, is a far more efficient process than that of cell-free virus infection. In this work, we studied the mechanism of cell-to-cell transmission between primary CD4+ T cells and the HIV-1 latently infected cell line ACH-2. When primary resting CD4+ T cells were co-cultured with ACH-2 cells under latency reversing agent Romidepsin stimulation, about 20.7% cells had been infected without becoming activated. Additionally, blocking with anti-CD4 or anti-gp120 neutralizing antibodies (VRC01) decreased de novo infection, and neither anti-CXCR4 antibodies nor chemokine inhibitors demonstrated the ability to inhibit cell-to-cell transmission. We also found that co-cultured resting CD4+ T cells carried higher levels of active cofilin, the actin-depolymerizing factor, suggesting actin plays a role in VS formation and viral pathogenesis. These results suggest HIV-1 Env gp120 binds with the CD4 receptor, not chemokine coreceptor to form VS during cell-to-cell transmission.

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

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.IMPORTANCE HIV-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.

Analysis of an age structured HIV infection model with virus-to-cell infection and cell-to-cell transmission

Recent studies reveal that cell-to-cell transmission via formation of virological synapses can contribute significantly to virus spread, and hence, may play a more important role than virus-to-cell infection in some situations. Age-structured models can be employed to study the variations w.r.t. infection age in modeling the death rate and virus production rate of infected cells. Considering the above characteristics for within-host dynamics of HIV, in this paper, we formulate an age-structured hybrid model to explore the effects of the two infection modes in viral production and spread. We offer a rigorous analysis for the model, including addressing the relative compactness and persistence of the solution semiflow, and existence of a global attractor. By subtle construction and estimates of Lyapunov functions, we show that the global attractor actually consists of an singleton, being either the infection free steady state if the basic reproduction number is less than one, or the infection steady state if the basic reproduction number is larger than one.

HIV-1 Gag, Envelope, and Extracellular Determinants Cooperate To Regulate the Stability and Turnover of Virological Synapses.

Retroviruses spread more efficiently when infected and uninfected cells form tight, physical interfaces known as virological synapses (VSs). VS formation is initiated by adhesive interactions between viral Envelope (Env) glycoproteins on the infected cell and CD4 receptor molecules on the uninfected cell. How high-avidity Env-CD4 linkages are resolved over time is unknown. We describe here a tractable two-color, long-term (>24 h) live cell imaging strategy to study VS turnover in the context of a large cell population, quantitatively. We show that Env's conserved cytoplasmic tail (CT) can potently signal the recruitment of Gag capsid proteins to the VS, a process also dependent on residues within Gag's N-terminal matrix (MA) domain. Additionally, we demonstrate that Env's CT and Gag's MA domain both regulate the duration of interactions between viral donor and target cells, as well as the stability of this interaction over time (i.e., its capacity to resolve or form a syncytium). Finally, we report the unexpected finding that modulating extracellular fluid viscosity markedly impacts target T cell trafficking and thus affects the duration, stability, and turnover of virus-induced cell-cell contacts. Combined, these results suggest a stepwise model for viral cell-to-cell transmission wherein (i) Env-receptor interactions anchor target cells to infected cells, (ii) Env signals Gag's recruitment to the cell-cell contact dependent on an intact Env CT and Gag MA, and (iii) Env CT and Gag MA, in conjunction with extracellular forces, combine to regulate VS stability and infectious outcomes. HIV-1 spreads efficiently at physical, cell-cell interfaces known as virological synapses (VSs). The VS provides for spatiotemporal coupling of virus assembly and entry into new host cells and may transmit signals relevant to pathogenesis. Disrupting this mode of transmission may be critical to the goal of abolishing viral persistence in infected individuals. We describe here a long-term live cell imaging strategy for studying virus-induced effects on cell behavior in the context of a large cell population. We demonstrate cooperative roles for viral Gag capsid proteins and Envelope glycoproteins in regulating VS formation and turnover. We also show that modulating fluid viscosity markedly affects T cell trafficking and VS stability. Thus, extracellular factors also play an important role in modulating the nature of infectious cell-cell interactions. In sum, our study provides new tools and insights relevant to exposing vulnerabilities in how HIV-1 and other viruses spread infection among cells, tissues, and people.

Mitochondria polarization in the contact regions of virus-infected effector cells during cell-to-cell transmission of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) has developed many unique strategies to evade the immune system, one of which is via cell-to-cell transfer through a virological synapse (VS). Cell-to-cell transmission results in more efficient virus transmission than infection with cell-free virions. Cell-to-cell transmission occurs at the VS, where a tight cleft forms between HIV-1-infected cells (effectors) and receptor-expressing (target) cells. Upon VS formation, viral budding polarizes toward target cells; the budding virus, viral RNA, and virus-related proteins are likely transmitted to target cells via this cleft. Furthermore, the movement of HIV-1 across an adhesive junction might help to protect the virus from attack by humoral factors, such as antibodies or complements. Therefore, cell-to-cell transfer is considered to be the predominant mechanism of viral spread during infection.1 Recent reports have shown that cell-to-cell spread of HIV-1 between T cells is more efficient than the equivalent cell-free infection.1,2 Normally, T cells are not inherently polarized and do not display front–rear polarity in the absence of stimulation. However, T cells can adapt and show front–rear polarity during stimulation, including cell–cell contact with antigen-presenting cells at the immunological synapse (IS), T cell migration, and chemokine or cytokine stimulation. IS formation triggers cytoplasmic and membrane remodeling within the T cell and recruits organelles, such as the secretory apparatus, signaling machinery, and mitochondria, to the contact region. Mitochondria has been reported to play an important role in the formation of IS by promoting sustained calcium influx to support synaptic signaling.3 VS formation shares many similarities with the IS.2 Mitochondria have long been known to be essential organelles that provide metabolic energy. They are involved in many physical functions and regulations including cellular signaling pathways, apoptosis, autophagy, and innate immunity.3 There is limited information regarding the role of mitochondria in VS. To address this question, we used transmission electron microscopy (TEM) to observe the location of mitochondria during HIV-1 VS formation using HIV-1-infected Jurkat T cells cocultured with uninfected Jurkat T cells. Our results illustrated in Fig. 1 showed that mitochondria were polarized and were engaged primarily in the contact regions of the HIV-1-infected T cells (effectors) during cell-to-cell contact. Recently, Groppelli et al. also indicated that polarization of mitochondria supports HIV-1 VS formation.2 Lee-Huang and Huang used live-cell real-time imaging to observe mitochondria in HIV-1-infected cells.4 FIG. 1. Cell-to-cell contact between HIV-1-infected (the lower cell) and HIV-uninfected (the upper cell) Jurkat T cells under transmission electron microscopy (TEM). We therefore suggest that mitochondria are important for VS formation and supply energy for cell-to-cell transmission of HIV-1. At present, the detailed mechanism of VS formation and cell-to-cell transmission is still not fully understood. These findings provide further insight into intercellular interactions and organelle localization during HIV-1 VS formation.

Macrophage infection via selective capture of HIV-1-infected CD4+ T cells.

SummaryMacrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4+ T cells leading to efficient macrophage infection. Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.

DC-T cell virological synapses and the skin: novel perspectives in dermatology.

Virological synapses (VS) increase cell-to-cell viral transmission and facilitate propagation of human immunodeficiency virus type 1 (HIV-1) and human T-cell leukaemia virus type 1 (HTLV-1). VS formation also plays a more general role in viral replication and dissemination. VS have been observed in vitro and ex vivo between uninfected T cells and T cells infected with HIV-1 or HTLV-1. In addition, dendritic cells (DC) infected with HIV-1 also play an important role in viral transmission to uninfected CD4+ T cells via VS formation. Recent studies revealed that several DC subsets are also infected with HTLV-1. These findings may help explain the rapid dissemination of both viruses within secondary lymphoid tissues in vivo. VS also explain, at least in part, why HIV-1 can propagate in the mucosal sites during sexual transmission. Furthermore, in the case of HTLV-1, VS can potentially explain some of the features of HTLV-1-associated dermatitis as infected T cells in the skin contribute to the pathogenesis of this condition.

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.

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.

Mathematical models of HIV replication and pathogenesis.

This review outlines how mathematical models have been helpful, and continue to be so, for obtaining insights into the in vivo dynamics of HIV infection. The review starts with a discussion of a basic mathematical model that has been frequently used to study HIV dynamics. Some crucial results are described, including the estimation of key parameters that characterize the infection, and the generation of influential theories which argued that in vivo virus evolution is a key player in HIV pathogenesis. Subsequently, more recent concepts are reviewed that have relevance for disease progression, including the multiple infection of cells and the direct cell-to-cell transmission of the virus through the formation of virological synapses. These are important mechanisms that can influence the rate at which HIV spreads through its target cell population, which is tightly linked to the rate at which the disease progresses towards AIDS.

Antiviral treatments over cell-to-cell infection

Antiviral treatments over cell-to-cell infection