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      Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

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          Significance

          Most cell-cell fusogens have tall ectodomains that drive fusion by undergoing conformational changes that pull two membranes together, akin to the mechanism used by viral fusogens during enveloped virus entry. In contrast, fusion-associated small transmembrane (FAST) proteins from reovirus have short, structurally minimal, membrane-disruptive ectodomains. In this work, we show that evolutionarily distant FAST proteins from aquareovirus and orthoreovirus use different adaptor proteins to hijack host actin assembly and push two membranes together during cell-cell fusion. Despite minimal sequence similarity, the cytoplasmic tails from divergent FAST proteins can be functionally exchanged and even replaced with different actin nucleators while maintaining fusogenicity. This feature suggests a conserved biophysical strategy shared by FAST proteins that could be used by other cell-cell fusogens.

          Abstract

          Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP–mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

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          Most cited references56

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          Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.

          We describe and validate a new membrane protein topology prediction method, TMHMM, based on a hidden Markov model. We present a detailed analysis of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helices. Additionally, TMHMM can discriminate between soluble and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we estimate that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous estimates. We further discovered that proteins with N(in)-C(in) topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for N(out)-C(in) topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/. Copyright 2001 Academic Press.
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            Is Open Access

            Viral membrane fusion.

            Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a "fusion loop" or "fusion peptide") engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics.
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              Myomaker: A membrane activator of myoblast fusion and muscle formation

              Summary Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibers. However, the identity of myogenic proteins that directly govern this fusion process has remained elusive. Here, we discovered a muscle-specific membrane protein, named Myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is down-regulated thereafter. Over-expression of Myomaker in myoblasts dramatically enhances fusion and genetic disruption of Myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibers. Remarkably, forced expression of Myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacologic perturbation of the actin cytoskeleton abolishes the activity of Myomaker, consistent with prior studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein both necessary and sufficient for mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.
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                Author and article information

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                Journal
                Proceedings of the National Academy of Sciences
                Proc. Natl. Acad. Sci. U.S.A.
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                January 05 2021
                December 21 2020
                January 05 2021
                : 118
                : 1
                Affiliations
                [1 ]University of California Berkeley/University of California San Francisco Graduate Group in Bioengineering, Berkeley, CA 94720;
                [2 ]Department of Bioengineering & Biophysics Group, University of California, Berkeley, CA 94720;
                [3 ]Physiology Course, Marine Biological Laboratory, Woods Hole, MA 02543;
                [4 ]Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455;
                [5 ]Department of Chemistry, New York University, New York, NY 10003;
                [6 ]Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
                [7 ]National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, 560065, India;
                [8 ]Science for Life Laboratory, Karolinska Institutet, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Stockholm 17165, Sweden;
                [9 ]Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093;
                [10 ]Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
                [11 ]Chan Zuckerberg Biohub, San Francisco, CA 94158
                Article
                10.1073/pnas.2007526118
                ff615cde-d38a-4c71-a8b3-8da31d5b6ce3
                © 2021

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                Self URI (article page): https://pnas.org/doi/full/10.1073/pnas.2007526118

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