Abstract
The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.
Highlights
Viral membrane fusion proteins are responsible both for binding to cellular receptors, and the subsequent fusion of viral and cellular membranes
Purified c-irradiated SARSCoV preparations had a fusogenic activity when added to Vero E6 cells at high multiplicity (,500–3000 virions per cell), causing the formation of syncytia in the absence of replication or cytopathic effects (Figure 1A). These syncytia had identical morphology to those observed in cytopathic studies of SARS-CoV in tissues and in tissue cultured cells; syncytia have been observed upon expression of coronavirus S protein, or following addition of cells expressing the S-protein to cells with surface-expressed angiotensin-converting enzyme 2 (ACE2). [22,30,31,32,33,34]
Our cryo-EM results demonstrate that there is a structural transition of the spike that occurs upon receptor binding (Figure 3, Movie S2)
Summary
Viral membrane fusion proteins are responsible both for binding to cellular receptors, and the subsequent fusion of viral and cellular membranes. The paradigm for class I fusion proteins consists of two heptad repeat regions, and a hydrophobic fusion peptide [1]. This motif is present in SARS-CoV [2] and other coronaviruses [3], as well as the hemagglutinin (HA) of influenza [4], gp of human T-cell leukemia virus type 1[5], gp of HIV[6], GP2 of Ebola virus [7,8], and the fusion protein of paramyxovirus [9,10,11,12]. The factors which trigger fusion (endocytosis, pH sensitivity, single receptor vs. primary and co-receptor binding, redox change) differ amongst diverse virus families, all viral fusion proteins are thought to share the same basic fusion mechanism [1,4,17,18,19,20]
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