Coronavirus : Triggers For Membrane Fusion.

Triggers for Membrane Fusion by Coronavirus Spike Proteins:

The triggers for coronavirus spikes to undergo conformational transitions demonstrate a more complex pattern than many other class I membrane fusion proteins, probably a reflection of their unique structural features discussed above. For example, although influenza virus HA is primed by proteolysis during virus packaging, many coronavirus spikes are not. Instead, coronavirus spikes are often subjected to proteolysis later in the cell entry process, sometimes after receptor binding. Thus, proteolysis of coronavirus spikes can lead directly to membrane fusion and thereby serves as an essential trigger for membrane fusion. The host proteases that cleave coronavirus spikes mainly come from four different stages of the virus infection cycle: (a) proprotein convertases (e.g., furin) during virus packaging in virus-producing cells, (b) extracellular proteases (e.g., elastase) after virus release into extracellular space, (c) cell surface proteases [e.g., type II transmembrane serine protease (TMPRSS2)] after virus attachment to virus-targeting cells, and (d) lysosomal proteases (e.g., cathepsin L and cathepsin B) after virus endocytosis in virus-targeting cells. In addition to proteolysis, traditional triggers such as receptor binding and low pH may also play a role in membrane fusion. Here, I discuss detailed triggers for membrane fusion by the spikes from three representative coronaviruses.

As the prototypic coronavirus, MHV has been extensively examined for its cell entry mechanism. The findings are complicated and in some cases contradictory. First, MHV spike is cleaved by proprotein convertases during virus packaging in the virus-producing cells . This proteolysis is critical for MHV entry into virus-targeting cells. Second, the binding of CEACAM1 triggers the conformational transition of MHV spike and hence membrane fusion. This is supported by the observations that incubation of MHV spike with recombinant soluble CEACAM1 led to enhanced hydrophobicity of MHV S2 and the appearance of a protease-resistant S2 fragment. These observations indicated the exposure of fusion peptides and formation of the six-helix bundle, respectively, in postfusion S2. Third, there have been contradictory reports about whether MHV enters target cells at the cell membrane or through endocytosis and whether MHV spike undergoes conformational transitions at low, neutral, or even elevated pH. This may depend on the MHV strains examined or experimental approaches used in the studies. The roles of pH and endocytosis in MHV entry still need to be further clarified. Interestingly, the neurotropic strain MHV-JHM can mediate virus entry into host cells that do not express CEACAM1. This receptor-independent entry by MHV-JHM is unique among viruses. Biochemical characterization of MHV-JHM spike suggested that it is more labile than the spikes from other MHV strains, meaning that it undergoes conformational transitions spontaneously in the absence of the receptor. It is believed that MHV-JHM is able to infect neural cells where CEACAM1 expression level is very low, at least in part because its spike can mediate receptor-independent entry. Taken together, the membrane fusion mechanism of MHV spike depends on both proteolysis and receptor binding, and it may or may not depend on the low pH of endosomes; in addition, receptor-independent membrane fusion by MHV-JHM spike contributes to the neutral tropism of MHV-JHM.

Research on the cell entry mechanism of SARS-CoV has led to novel findings. First, SARS-CoV spike is not cleaved by proprotein convertases during virus packaging and hence remains intact on mature virions. Instead, SARS-CoV enters host cells through endocytosis, and its spike is processed by lysosomal proteases (e.g., cathepsin L and cathepsin B). This is supported by the observation that inhibitors against either endosomal acidification or lysosomal cysteine proteases block SARS-CoV entry. However, low pH itself is not a trigger for SARS-CoV entry. This is supported by the observation that when expressed on the cell surface and cleaved by exogenous proteases, SARS-CoV spike can mediate cell-cell fusion with ACE2-expressing cells at neutral pH. Thus, the role of low pH in SARS-CoV entry is to activate lysosomal proteases, which further activate SARS-CoV spike for membrane fusion. This is different from influenza virus HA, which is activated through binding protons in the low-pH environment of endosomes. Second, in addition to lysosomal proteases, both extracellular proteases (e.g., elastases in the respiratory tract) and cell surface proteases (e.g., TMPRSS2 on the surface of lung cells) also activate SARS-CoV spike for membrane fusion. Due to their cell and tissue specificities, these proteases likely contribute to the respiratory tract and lung tropism of SARS-CoV. Third, in addition to the cleavage site at the S1/S2 boundary, a second site, S2′, has been identified at the N terminus of the internal fusion peptide within S2 . Whereas the cleavage at the S1/S2 boundary removes the structural constraint of S1 on S2, the cleavage at the S2′ site releases the internal fusion peptide for insertion into target membranes. Fourth, it is not clear whether the binding of receptor ACE2 is a trigger for SARS-CoV spike to fuse membranes. Two electron microscopy studies observed no or moderate conformational changes of SARS-CoV spike associated with ACE2 binding . However, other studies suggested that ACE2 binding triggers a conformational change in SARS-CoV spike, which exposes previously cryptic protease sites for cleavage. The role of ACE2 binding in triggering membrane fusion waits to be further investigated. Nevertheless, SARS-CoV entry does not depend on low pH, but it requires at least two protease cleavages in the spike by lysosomal proteases, extracellular proteases, or cell surface proteases.

The overall cell entry mechanism of MERS-CoV is similar to that of SARS-CoV. Like SARS-CoV spike, MERS-CoV spike must be cleaved at both the S1/S2 boundary and the S2′ site for membrane fusion to occur . MERS-CoV also enters host cells through endocytosis and is activated by lysosomal cysteine proteases for membrane fusion . Moreover, extracellular proteases and cell surface proteases help activate MERS-CoV entry. Unlike SARS-CoV, MERS-CoV spike is cleaved by host proprotein convertases during virus packaging. Interestingly, despite recognizing the same receptor DPP4, MERS-CoV and HKU4 spikes differ in their activities to mediate virus entry: HKU4 spike mediates virus entry into bat cells but not human cells, whereas MERS-CoV spike mediates virus entry into both bat and human cells. Two residue differences have been identified between MERS-CoV and HKU4 spikes that account for this functional difference ; they allow MERS-CoV spike, but not HKU4 spike, to be activated by human proprotein convertases and lysosomal cysteine proteases. Thus, the corresponding two mutations played a critical role in the transmission of MERS-CoV from its likely natural reservoir, bats, to humans, either directly or through intermediate host camels. On the other hand, HKU4 spike can be activated by bat lysosomal proteases but not human lysosomal proteases, suggesting that human and bat lysosomal proteases process viral spikes differently. These studies on MERS-CoV entry reveal that different activities of spike-processing proteases from different hosts can pose a barrier for cross-species transmissions of viruses.

In sum, proteolysis has been established as an essential trigger for coronavirus spikes to fuse membranes, as cleavages at the S1/S2 boundary and S2′ site can remove the structural constraint of S1 on S2 and release the internal fusion peptide, respectively. Among the host proteases, lysosomal proteases provide the most reliable source for spike processing because they are ubiquitous and abundant in many cell types. The availability of some other proteases (e.g., proprotein convertases, extracellular proteases, and cell surface proteases) depends on the types of cells and tissues, regulating tissue tropisms of coronaviruses. Moreover, protease activities from different host species may vary, regulating host ranges of coronaviruses. Some other triggers for coronavirus entry (e.g., receptor binding and low pH) may depend on specific coronaviruses or different strains of the same coronavirus. The overall goal of these triggers is to overcome the energy barrier for the conformational transition of coronavirus spikes.

Acknowledgement:

This work is supported by Nation Institutes of Health.