Structural Molecular Insights into SARS Coronavirus Cellular Attachment, Entry and Morphogenesis
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Coronavirus spikes have the largest mass of any known viral spike molecule. The spike is a type 1 viral fusion protein, a class of trimeric surface glycoprotein proteins from diverse viral families that share many common structural and functional characteristics. Fusion proteins are mainly responsible for host cell receptor recognition and subsequent membrane fusion, and may perform other roles such as virus assembly and release via budding. The conformational changes that occur in the spike of intact SARS coronavirus (SARS-CoV) when it binds to the viral receptor, angiotensin-converting enzyme 2 (ACE2) are described. Clues to the structural/functional relationships of membrane fusion have been made possible by the development of viral purification and inactivation methods, along with cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image processing of many different images containing multiple views of the spikes. These methods have allowed study of the spikes while still attached to virions that are noninfectious, but fusionally competent. The receptor-binding and fusion core domains within the SARS-CoV spike have been precisely localized within the spike. Receptor binding results in structural changes that have been observed in the spike molecule, and these appear to be the initial step in viral membrane fusion. A working model for the stepwise process of receptor binding, and subsequent membrane fusion in SARS-CoV is presented. Uniquely, the large size of the SARS-CoV spike allows structural changes to be observed by cryo-EM in the native state. This provides a useful model for studying the basic process of membrane fusion in general, which forms an essential part of the function of many cellular processes.
KeywordsMembrane Fusion Heptad Repeat Fusion Pore Host Cell Membrane Fusion Mechanism
3.1 Structure of SARS Coronavirus (SARS-CoV)
A striking feature of the SARS-CoV spike is its huge mass (~500 kD per trimer). However, despite the size differences, the SARS-CoV spike performs the same fundamental task in viral entry to the host cell as other smaller type 1 viral fusion proteins, such as the influenza hemagglutinin (HA) (~220 kD per trimer). The SARS-CoV spike can be subdivided into four structural domains (from N to C terminus); two large external domains S1 and S2 are largely responsible for receptor binding and membrane fusion, respectively. In most type 1 viral fusion proteins the analogous peptides are generated by proteolysis of the spike precursor during the maturation process in the host cell, yielding two peptides with the fusion peptide on the N terminus of S2. In SARS-CoV the S1/S2 assignment is given based on sequence homology to other viral fusion proteins, although there appears to be no peptide cleavage. The final two small domains are comprised of a transmembrane domain, and a carboxyterminal cytoplasmic domain. The cell-surface molecule angiotensin-converting enzyme 2 (ACE2) is the receptor for the SARS-CoV S protein (Li et al. 2003) which is a relatively large macromolecule with a diameter of 70 Å. By comparison, the receptor for influenza HA, sialic acid, is much smaller with a 10 Å diameter.
One of the challenges of achieving structural molecular studies with native SARS-CoV is that it is classified as a biological safety level 3 organism, requiring handling in containment. However, once it was shown that specimens could be γ-irradiated with a sufficient dose (2 Mrad) for viral inactivation, while still preserving protein structure, it was possible to carry out cryo-EM of intact virions and to obtain the 3D structure of the native, unfixed virions and the spike, using single particle image processing and averaging from multiple images containing many different viewing angles of the molecule (Booth et al. 2005; Beniac et al. 2006). Immunolabeling showed that these virions were intact antigenically as well as structurally (Fig. 3.1) and still able to bind to the SARS-CoV receptor, ACE2.
3.2 Structure of the Coronavirus Spike
The precise location of ACE2 binding on the distal end of the spike is centered at 70 Å from the central axis of the spike, with a 30 Å gap between the axis of symmetry and ACE2. One ACE2 molecule can bind to each of the three propeller-like blades of the spike, making a structure 220 Å high (Fig. 3.3b). Binding of more than one ACE2 to each spike (on one or both of the other two propeller blades of each trimer) is possible, hence binding of one ACE2 molecule does not stearically hinder binding of additional ACE2 molecules.
The cryo-EM 3D structures of the spike and the spike−ACE2 complex, when combined with the atomic resolution structures of the SARS-CoV spike receptor-binding domain – ACE2 complex (Li et al. 2005a) and the heptad repeat pre- and postfusion cores (Supekar et al. 2004; Hakansson-McReynolds et al. 2006), show that the receptor-binding domain docks to the distal end of the spike with ACE2 filling the extra mass on the spike (shown by the color violet in Fig. 3.3). The empty upper region of the mass appears to be components of the second ACE2 and the Fc component of the chimeric protein, and the location of the C-terminus of the docked ACE2 was consistent with this interpretation.
3.3 Viral Membrane Fusion in SARS-CoV
Viral membrane fusion proteins are responsible both for binding to cellular receptors, and the subsequent fusion of viral and cellular membranes. The paradigm for type 1 fusion proteins consists of two heptad repeat regions, and a hydrophobic fusion peptide (Dutch et al. 2000). This motif is present in SARS-CoV (Hakansson-McReynolds et al. 2006) and other coronaviruses (Xu et al. 2004), as well as the hemagglutinin (HA) of influenza (Skehel and Wiley 2000), gp21 of human T-cell leukemia virus type 1 (Kobe et al. 1999), gp41 of HIV(Weissenhorn et al. 1997), GP2 of Ebola (Weissenhorn et al. 1998; Malashkevich et al. 1999), and the fusion protein of paramyxovirus (Zhao et al. 2000; Chen et al. 2001). Type 1 viral fusion proteins can also be divided into two subtypes: those whose fusion mechanism is low pH-dependent such as influenza HA, and those that are pH-independent like the retroviral fusion proteins. In retroviruses, receptor binding itself can trigger fusion, with temperature and redox conditions also influencing the fusion mechanism (Hernandez et al. 1997; Damico et al. 1998). The SARS-CoV spike appears to be insensitive to redox conditions (Fenouillet et al. 2007). Although the factors which trigger fusion (endocytosis, pH sensitivity, single receptor vs. primary and coreceptor binding, redox change) differ amongst diverse virus families, all viral fusion proteins are thought to share the same basic fusion mechanism (Baker et al. 1999; Skehel and Wiley 2000; Dutch et al. 2000; Colman and Lawrence 2003; Dimitrov 2004; Hofmann and Pohlmann 2004).
The precise mechanisms by which type 1 viral fusion proteins gain access to the host cell remain unknown. The hypothetical entry process includes several steps that take place in sequence: receptor binding, fusion core rearrangement, fusion peptide insertion in host cell membrane, refolding of heptad repeats, membrane fusion, and finally viral nucleocapsid transfer (Earp et al. 2005).
3.4 Cellular Attachment and Entry of SARS-CoV
In most proposed models of membrane fusion it is postulated that the S1 domain or analogous receptor-binding domains dissociate from the spike during the membrane fusion process. This dynamic process was demonstrated for influenza HA by Kemble et al. (1992) in their investigation where they engineered intermonomer disulfide bonds between the HA S1 subunits. The result of this was that fusion activity was impaired; however it could be restored under reducing conditions. It is probable that the SARS-CoV spike shares a similar mechanism, with the structural changes detected by cryo-EM representing the initial step in this process.
The next step in the membrane fusion process involves the refolding of the fusion core back upon itself to adopt the postfusion configuration (Supekar et al. 2004) (Fig. 3.5; models M4–M6). In this configuration three heptad 1 repeats (HR1) form a parallel coiled-coil trimer, and the three HR2 pack in an antiparallel fashion in the hydrophobic grooves of the HR1 trimer. The result of this is that both the FP and transmembrane regions of the spike are brought in close proximity to each other, resulting in the host and viral membranes being in close contact. Each SARS-CoV virion has an average of 65 spikes (Beniac et al. 2006). At this density, several spikes would be close enough together to act in concert to disrupt the plasma membrane and induce pore formation between viral and cellular membranes. Opening of these pores would allow the SARS-CoV nucleocapsid to enter the host cell cytoplasm, as shown in our model in Fig. 3.6. Multiple SARS-CoV spike trimers may be involved in formation of the fusion pore, similar to the situation in influenza, where it has been suggested that between three and six HA trimers may be involved in the production of each fusion pore (Skehel and Wiley 2000). In Figs. 3.5 and 3.6 we have presented the ACE2–SARS:S1 domains as they were solved in this cryo-EM investigation, for the model of membrane fusion that we present. For other fusion proteins like influenza HA1 and HIV GP120 it has been modeled that the rearrangements upon membrane fusion are dramatic involving a shedding of the above-mentioned domains. The cryo-EM investigations have detected structural movement of S1 upon ACE2 binding, which could represent the initial phase of this dramatic process that is postulated to occur in the course of membrane fusion.
An analysis of the structure of the spike–receptor complex demonstrates how SARS-CoV can adapt to utilize receptors from different species and how they may evolve to gain specificity for new receptor types, in that there is redundancy and a great deal of protein mass that can accommodate evolutionary changes. RNA viruses have a high rate of mutation and recombination (Moya et al. 2004). In SARS-CoV the spike is able to retain specific binding affinity for the ACE2 of more than one host species, and rapid evolution to gain specificity for novel ACE2 species has been demonstrated (Li et al. 2005b, 2006). The large size of the spike of coronaviruses may be related to the use of large host cell-surface molecules such as ACE2 as specific receptors. Amongst the coronavirus family, specific cell-surface receptors for the S protein are all in the range of 60–110 kD (Wentworth and Holmes 2001). These large host receptor molecules are of course functionally constrained and, in turn, relatively well conserved across species barriers. In utilizing binding to a large receptor molecule, the spike S1 domain also acts as a “spacer arm” holding the receptor far enough away from the threefold axis of symmetry of the spike S2 domain to permit fusion core rearrangement and subsequent membrane fusion. Such a property necessitates having a large spike molecule. Moreover, multiple receptor binding can have functional significance, enhancing the binding and entry of viruses. Cross-linking of adjacent host receptor molecules could increase the affinity of the virus for its target cell, as well as improving the kinetics of fusion. The SARS-CoV spike is a useful model system for the investigation of type 1 viral fusion protein dynamics. Utilizing this system for further research may lead to the possibility of developing broad-spectrum antivirals that target conserved cell fusion mechanisms shared by diverse virus families.
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