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      The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner


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          Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2′ cleavage site is the FP (amino acids 798–818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816–835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca 2 +-dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion “platform.”

          Graphical Abstract


          • ESR and infectivity assays were used to study the SARS-CoV fusion peptide.

          • Membrane-ordering ability was used as a criterion for identifying bona fide FP.

          • FP1 (downstream of S2′ site, aa 798–818) and FP2 (aa 816–835) were identified.

          • FP1 and FP2 act in a cooperative manner to induce greater membrane ordering.

          • FP1- and FP2-mediated membrane fusion is calcium dependent.

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          Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein

          Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes—A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.
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            Dissecting virus entry via endocytosis.

            Numerous virus families utilize endocytosis to infect host cells, mediating virus internalization as well as trafficking to the site of replication. Recent research has demonstrated that viruses employ the full endocytic capabilities of the cell. The endocytic pathways utilized include clathrin-mediated endocytosis, caveolae, macropinocytosis and novel non-clathrin, non-caveolae pathways. The tools to study endocytosis and, consequently, virus entry are becoming more effective and specific as the amount of information on endocytic component structure and function increases. The use of inhibitory drugs, although still quite common, often leads to non-specific disruptions in the cell. Molecular inhibitors in the form of dominant-negative proteins have surpassed the use of chemical inhibitors in terms of specificity to individual pathways. Dominant-negative molecules are derived from both structural proteins of endocytosis, such as dynamin and caveolin, and regulatory proteins, primarily small GTPases and kinases. This review focuses on the experimental approaches taken to examine virus entry and provides both classic examples and recent research on a variety of virus families.
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              Nonlinear-Least-Squares Analysis of Slow-Motion EPR Spectra in One and Two Dimensions Using a Modified Levenberg–Marquardt Algorithm


                Author and article information

                J Mol Biol
                J. Mol. Biol
                Journal of Molecular Biology
                19 October 2017
                8 December 2017
                19 October 2017
                : 429
                : 24
                : 3875-3892
                [1 ]Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, United States
                [2 ]Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853, United States
                [3 ]School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, United States
                Author notes
                © 2017 Elsevier Ltd. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 21 June 2017
                : 2 October 2017
                : 12 October 2017

                Molecular biology
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