Biology subjects

Thali, M.

Publications and source records attributed to Thali, M..

3 recordsLinked to original sources

bioRxiv2026

An Aurora Kinase-Dependent Role for Vif in Regulating HIV-1-Induced Cell-Cell Fusion

Viral infectivity factor (Vif) is an HIV-1 accessory protein best known for its counteraction of APOBEC3 enzymes, interferon-inducible host defenses against viral infection, as well as PPP2R5A-E, which are regulatory subunits of the PP2A cellular phosphatase holoenzyme, resulting in striking Vif-dependent phosphoproteome remodeling. One reported consequence of this remodeling is hyperphosphorylation of several Aurora kinase substrates in HIV-1 infected cells, which is reversed when Vif-deficient virus is used. We previously showed that infection of T cells with Vif-deficient HIV-1 results in significantly accelerated formation of syncytia compared to wild-type HIV-1 infection. More recently, others have shown that application of Aurora kinase B inhibitors during HIV-1 infection in T cells also results in a similar hyperfusogenic phenotype. Both effects were specific to Env-driven cell-cell fusion, and did not influence virus infectivity. We thus hypothesized that Vifs influence on the rate of HIV-1-induced cell-cell fusion was mediated by Aurora kinase activity. To start testing this hypothesis, we have evaluated the effects of a small panel of Aurora kinase inhibitors on HIV-1-induced cell-cell fusion in the presence or absence of Vif. Our results replicate the previously documented increase in cell-cell fusion in the absence of Vif, as well as the increase in cell-cell fusion observed upon inhibition of Aurora kinase B in the presence of Vif. Critically, we now present evidence that Vif deletion significantly blunts the impact of Aurora kinase inhibition on cell-cell fusion, supporting our hypothesis that Vif-mediated regulation of cell-cell fusion depends on Aurora kinase signaling dysregulation, likely because of PPP2R5A-E degradation. Further, we document that the cell-cell fusion regulator downstream of Aurora kinase signaling is likely Ezrin, which we have previously shown to prevent excess HIV-1-induced syncytium formation when in its phosphorylated (activated) state. Taken together, these findings establish a Vif, Aurora kinase, and Ezrin-dependent mechanistic framework for the regulation of HIV-1-induced cell-cell fusion in infected T cells which likely helps preserve optimal cell-to-cell virus transmission.

Cell Biology↗

bioRxiv2026

HIV-1-induced small T cell syncytia: susceptibility to direct NK cell killing

Intravital imaging studies of HIV-1-infected humanized mice by three independent groups revealed the presence of small T cell syncytia. Although these multinucleated entities represent only a small fraction of infected T lymphocytes, they arise at the earliest stages of infection, are highly motile, and they make frequent contact with potential target cells. Importantly, while such transient contacts allow for virus transfer, they typically do not result in fusion and these exclusively T cell-based syncytia thus do not grow beyond the three- or four-nuclei stage. Immunofluorescence microscopy analyses, together with initial multiparameter flow cytometric evaluation and proteomic profiling by our lab and collaborators, identified these small T cell syncytia (henceforth referred to merely as syncytia) as a distinct subpopulation of infected cells. Because they appear as soon as any infected cells are detectable and thus likely contribute to early viral spread, they also trigger innate immune responses, including attacks by cytolytic effectors such as natural killer (NK) cells. Here, we first introduce a newly developed surface split GFP system which allows to unequivocally distinguish syncytia from infected mononucleated cells, and which also enables isolating these entities for in-depth functional analyses. Then, using multiparameter flow cytometry with UMAP clustering and transcriptomic profiling, we document that syncytia represent a subpopulation of infected cells that do indeed clearly differ from both uninfected and infected mononucleated T lymphocytes, particularly also regarding expression of immune-regulatory host factors. Finally, we show that the rate of direct killing by NK cells is substantially higher for syncytia than for infected mononucleated cells, both in 2D suspension co-culture and in a 3D collagen coculture system. While the fitness costs imposed on syncytia by such increased susceptibility to NK cytolysis during early infection might be compensated for by yet to be determined syncytia functions that (directly) enhance virus spread, we note that irrespective of what such virus spread-enhancing functions may be, the increased vulnerability of syncytia could possibly be exploited for the development of novel antiviral strategies.

microbiology↗

bioRxiv2018

EWI-2 Inhibits Cell-Cell Fusion at the Virological Synapse

Cell-to-cell transfer of virus particles through the virological synapse (VS) is a highly efficient mode of HIV-1 transmission. Formation of the VS, a transient multiform adhesion structure, is mediated through an interaction between the HIV-1 envelope glycoprotein (Env) and the viral receptor CD4 on the surface of infected cell and target cell, respectively. Given that Env, unlike many other viral fusogens, can mediate the merger of membranes at neutral pH, the close encounter of infected and uninfected cells would seem prone to result in cell-cell fusion and thus the formation of syncytia. However, while it is being recognized now that small, T cell-based syncytia are indeed a defining feature of the natural history of HIV-1, the majority of VSs nevertheless resolve without fusion, thus securing continued virus spread. Gag, the main viral structural component, is partially responsible for restraining Env and preventing it from becoming fusogenic before being incorporated into particles. In addition, a few cellular factors, including tetraspanins and ezrin, have also been shown to inhibit Envs activity while this fusogen is still part of the producer cell. Here, we identify EWI-2, a protein that was previously shown to associate with the tetraspanins CD9 and CD81 and also with ezrin, as a host factor that contributes to the inhibition of Env{-}mediated cell-cell fusion. Using fluorescence microscopy, flow cytometry, and TZM-bl fusion assays, we show that EWI-2, comparable to tetraspanins, while overall being downregulated upon HIV-1 infection, accumulates at the producer cell side of the VS (i.e. the presynapse), where it contributes to the fusion-preventing activities of the other viral and cellular components.

microbiology↗