Cell Entry of Animal Coronaviruses.

Coronaviruses (CoVs) are a group of enveloped positive-sense RNA viruses and can cause deadly diseases in animals and humans. Cell entry is the first and essential step of successful virus infection and can be divided into two ongoing steps: cell binding and membrane fusion. Over the past two decades, stimulated by the global outbreak of SARS-CoV and pandemic of SARS-CoV-2, numerous efforts have been made in the CoV research. As a result, significant progress has been achieved in our understanding of the cell entry process. Here, we review the current knowledge of this essential process, including the viral and host components involved in cell binding and membrane fusion, molecular mechanisms of their interactions, and the sites of virus entry. We highlight the recent findings of host restriction factors that inhibit CoVs entry. This knowledge not only enhances our understanding of the cell entry process, pathogenesis, tissue tropism, host range, and interspecies-transmission of CoVs but also provides a theoretical basis to design effective preventive and therapeutic strategies to control CoVs infection.

Identifying Distinct Structural Features of the SARS-CoV-2 Spike Protein Fusion Domain Essential for Membrane Interaction.

The SARS-CoV-2 spike protein is the primary antigenic determinant of the virus and has been studied extensively, yet the process of membrane fusion remains poorly understood. The fusion domain (FD) of viral glycoproteins is well established as facilitating the initiation of membrane fusion. An improved understanding of the structural plasticity associated with these highly conserved regions aids in our knowledge of the molecular mechanisms that drive viral fusion. Within the spike protein, the FD of SARS-CoV-2 exists immediately following S2' cleavage at the N-terminus of the S2 domain. Here we have shown that following the introduction of a membrane at pH 7.4, the FD undergoes a transition from a random coil to a more structurally well-defined postfusion state. Furthermore, we have classified the domain into two distinct regions, a fusion peptide (FP, S816-G838) and a fusion loop (FL, D839-F855). The FP forms a helix-turn-helix motif upon association with a membrane, and the favorable entropy gained during this transition from a random coil is likely the driving force behind membrane insertion. Membrane depth experiments then revealed the FP is found inserted within the membrane below the lipid headgroups, while the interaction of the FL with the membrane is shallower in nature. Thus, we propose a structural model relevant to fusion at the plasma membrane in which the FP inserts itself just below the phospholipid headgroups and the FL lays upon the lipid membrane surface.

COVID-19: the CaMKII-like system of S protein drives membrane fusion and induces syncytial multinucleated giant cells.

The SARS-CoV-2 S protein on the membrane of infected cells can promote receptor-dependent syncytia formation, relating to extensive tissue damage and lymphocyte elimination. In this case, it is challenging to obtain neutralizing antibodies and prevent them through antibodies effectively. Considering that, in the current study, structural domain search methods are adopted to analyze the SARS-CoV-2 S protein to find the fusion mechanism. The results show that after the EF-hand domain of S protein bound to calcium ions, S2 protein had CaMKII protein activities. Besides, the CaMKII_AD domain of S2 changed S2 conformation, facilitating the formation of HR1-HR2 six-helix bundles. Apart from that, the Ca2+-ATPase of S2 pumped calcium ions from the virus cytoplasm to help membrane fusion, while motor structures of S drove the CaATP_NAI and CaMKII_AD domains to extend to the outside and combined the viral membrane and the cell membrane, thus forming a calcium bridge. Furthermore, the phospholipid-flipping-ATPase released water, triggering lipid mixing and fusion and generating fusion pores. Then, motor structures promoted fusion pore extension, followed by the cytoplasmic contents of the virus being discharged into the cell cytoplasm. After that, the membrane of the virus slid onto the cell membrane along the flowing membrane on the gap of the three CaATP_NAI. At last, the HR1-HR2 hexamer would fall into the cytoplasm or stay on the cell membrane. Therefore, the CaMKII_like system of S protein facilitated membrane fusion for further inducing syncytial multinucleated giant cells.

Dynamics of SARS-CoV-2 Spike Proteins in Cell Entry: Control Elements in the Amino-Terminal Domains.

Selective pressures drive adaptive changes in the coronavirus spike proteins directing virus-cell entry. These changes are concentrated in the amino-terminal domains (NTDs) and the receptor-binding domains (RBDs) of complex modular spike protein trimers. The impact of this hypervariability on virus entry is often unclear, particularly with respect to sarbecovirus NTD variations. Therefore, we constructed indels and substitutions within hypervariable NTD regions and used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus-like particles and quantitative virus-cell entry assays to elucidate spike structures controlling this initial infection stage. We identified NTD variations that increased SARS-CoV-2 spike protein-mediated membrane fusion and cell entry. Increased cell entry correlated with greater presentation of RBDs to ACE2 receptors. This revealed a significant allosteric effect, in that changes within the NTDs can orient RBDs for effective virus-cell binding. Yet, those NTD changes elevating receptor binding and membrane fusion also reduced interdomain associations, leaving spikes on virus-like particles susceptible to irreversible inactivation. These findings parallel those obtained decades ago, in which comparisons of murine coronavirus spike protein variants established inverse relationships between membrane fusion potential and virus stability. Considerable hypervariability in the SARS-CoV-2 spike protein NTDs also appear to be driven by counterbalancing pressures for effective virus-cell entry and durable extracellular virus infectivity. These forces may selectively amplify SARS-CoV-2 variants of concern. IMPORTANCE Adaptive changes that increase SARS-CoV-2 transmissibility may expand and prolong the coronavirus disease 2019 (COVID-19) pandemic. Transmission requires metastable and dynamic spike proteins that bind viruses to cells and catalyze virus-cell membrane fusion. Using newly developed assays reflecting these two essential steps in virus-cell entry, we focused on adaptive changes in SARS-CoV-2 spike proteins and found that deletions in amino-terminal domains reset spike protein metastability, rendering viruses less stable yet more poised to respond to cellular factors that prompt entry and subsequent infection. The results identify adjustable control features that balance extracellular virus stability with facile virus dynamics during cell entry. These equilibrating elements warrant attention when monitoring the evolution of pandemic coronaviruses.

Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry.

SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.

Enhanced Cholesterol-Dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to Its N-Terminal Counterpart.

Membrane fusion is an important step for the entry of the lipid-sheathed viruses into the host cells. The fusion process is being carried out by fusion proteins present in the viral envelope. The class I virus contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion and is called as a "fusion peptide". However, severe acute respiratory syndrome (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV-2 is far more efficient than its N-terminal counterpart (FP) to induce hemifusion between small unilamellar vesicles. Moreover, the ability of IFP1 to induce hemifusion formation increases dramatically with growing cholesterol content in the membrane. Interestingly, IFP1 is capable of inducing hemifusion but fails to open the pore.

Dynamics of transmissible gastroenteritis virus internalization unraveled by single-virus tracking in live cells.

Transmissible gastroenteritis virus (TGEV) is a swine enteropathogenic coronavirus that causes significant economic losses in swine industry. Current studies on TGEV internalization mainly focus on viral receptors, but the internalization mechanism is still unclear. In this study, we used single-virus tracking to obtain the detailed insights into the dynamic events of the TGEV internalization and depict the whole sequential process. We observed that TGEVs could be internalized through clathrin- and caveolae-mediated endocytosis, and the internalization of TGEVs was almost completed within ~2 minutes after TGEVs attached to the cell membrane. Furthermore, the interactions of TGEVs with actin and dynamin 2 in real time during the TGEV internalization were visualized. To our knowledge, this is the first report that single-virus tracking technique is used to visualize the entire dynamic process of the TGEV internalization: before the TGEV internalization, with the assistance of actin, clathrin, and caveolin 1 would gather around the virus to form the vesicle containing the TGEV, and after ~60 seconds, dynamin 2 would be recruited to promote membrane fission. These results demonstrate that TGEVs enter ST cells via clathrin- and caveolae-mediated endocytic, actin-dependent, and dynamin 2-dependent pathways.

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors1-4, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein5-7. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage8-10. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide11,12. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp61413-15 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.

Nicotinic acetylcholine receptors regulate clustering, fusion and acidification of the rat brain synaptic vesicles.

The brain nicotinic acetylcholine receptors (nAChRs) expressed in pre-synaptic nerve terminals regulate neurotransmitter release. However, there is no evidence for the expression of nAChRs in synaptic vesicles, which deliver neurotransmitter to synaptic cleft. The aim of this paper was to investigate the presence of nAChRs in synaptic vesicles purified from the rat brain and to study their possible involvement in vesicles life cycle. According to dynamic light scattering analysis, the antibody against extracellular domain (1-208) of α7 nAChR subunit inhibited synaptic vesicles clustering. Sandwich ELISA with nAChR subunit-specific antibodies demonstrated the presence of α4ß2, α7 and α7ß2nAChR subtypes in synaptic vesicles and showed that α7 and ß2 nAChR subunits are co-localized with synaptic vesicle glycoprotein 2A (SV2A). Pre-incubation with either α7-selective agonist PNU282987 or nicotine did not affect synaptic vesicles clustering but delayed their Ca2+-dependent fusion with the plasma membranes. In contrast, nicotine but not PNU282987 stimulated acidification of isolated synaptic vesicles, indicating that α4ß2 but not α7-containing nAChRs are involved in regulation of proton influx and neurotransmitter refilling. Treatment of rats with levetiracetam, a specific modulator of SV2A, increased the content of α7 nAChRs in synaptic vesicles accompanied by increased clustering but decreased Ca2+-dependent fusion. These data for the first time demonstrate the presence of nAChRs in synaptic vesicles and suggest an active involvement of cholinergic regulation in neurotransmitter release. Synaptic vesicles may be an additional target of nicotine inhaled upon smoking and of α7-specific drugs widely discussed as anti-inflammatory and pro-cognitive tools.