key: cord-253366-03cg831z
authors: Chakraborty, Hirak; Bhattacharjya, Surajit
title: Mechanistic insights of host cell fusion of SARS-CoV-1 and SARS-CoV-2 from atomic resolution structure and membrane dynamics
date: 2020-07-22
journal: Biophys Chem
DOI: 10.1016/j.bpc.2020.106438
sha: 
doc_id: 253366
cord_uid: 03cg831z

The emerging and re-emerging viral diseases are continuous threats to the wellbeing of human life. Previous outbreaks of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS had evidenced potential threats of coronaviruses in human health. The recent pandemic due to SARS-CoV-2 is overwhelming and has been going beyond control. Vaccines and antiviral drugs are ungently required to mitigate the pandemic. Therefore, it is important to comprehend the mechanistic details of viral infection process. The fusion between host cell and virus being the first step of infection, understanding the fusion mechanism could provide crucial information to intervene the infection process. Interestingly, all enveloped viruses contain fusion protein on their envelope that acts as fusion machine. For coronaviruses, the spike or S glycoprotein mediates successful infection through receptor binding and cell fusion. The cell fusion process requires merging of virus and host cell membranes, and that is essentially performed by the S2 domain of the S glycoprotein. In this review, we have discussed cell fusion mechanism of SARS-CoV-1 from available atomic resolution structures and membrane binding of fusion peptides. We have further discussed about the cell fusion of SARS-CoV-2 in the context of present pandemic situation.

Membrane fusion, one of the most fundamental processes for the survival of eukaryotes, occurs when two closely apposed lipid bilayers merge into a continuous single bilayer and the inner contents are mixed with each other. Several cellular events such as endocytosis, exocytosis, cellular trafficking, compartmentalization, import of nutrients and export of waste, vesiculation, inter cellular communication, fertilization and many others involve membrane fusion, though they vary vastly in space and time. The fusion of synaptic vesicles is almost 10,000-fold faster than the fusion of vacuoles. Despite many diversities all fusion processes follow a common route that includes membrane contact (docking), membrane merger (stalk and transmembrane contact formation), and opening of pore to transfer the intracellular material (pore formation).

There are several models that describe the mechanism of membrane fusion, out of which the stalk model is the most accepted one [1] . The continuum model of membrane fusion or stalk model proposes that the fusion starts from a point-like membrane protrusion that transform into an hourglass like connection between two apposed monolayers [2] . This early hemifusion connection is called as the fusion stalk, which further expanded to form the hemifusion diaphragm [3] . The further expansion of the hemifusion diaphragm opens the pore either on the diaphragm or its perimeter [4, 5] . Fusion pore can also be formed directly from stalk by avoiding the hemifusion state [6, 7] . Though membrane fusion is an integral event for the survival of eukaryotes, enveloped viruses utilize this process to enter the host cell [8, 9] . The emerging and reemerging viral pandemics prompted us to understand the mechanism of fusion for better intervention against viral diseases. The recent worldwide outburst of coronavirus-2 (CoV-2) causing severe acute respiratory syndrome (SARS), COVID-19, has created havoc in terms of morbidity and mortality. Middle East severe acute respiratory syndrome (MERS) coronavirus and SARS coronavirus-1 (SARS-CoV-1), which belong to the same family, coronaviradae, were confined in a certain part of the world [10] . SARS-CoV-2 has shown much higher infectivity, and has been spread all over the world.

There is no vaccine for MERS and SARS, and now the whole world is eagerly waiting for COVID-19 vaccine. The fusion between the host cell and virus being the first step of infection process, it is important to understand the fusion mechanism. This review discusses the mechanistic insights of the fusion of SARS-CoV-1 and SARS-CoV-2 from their atomistic structure, and membrane interaction.

Enveloped viruses exploit membrane fusion for their entry to the host cell. The enveloped viruses cover their genetic material with the host cell derived lipid bilayer, which houses the fusion protein that is instrumental in the fusion between virus and host cell [11] .

The receptor binding domain of the fusion protein finds its interaction partner on the cell surface and the interaction between these two allows the virus to dock on the host cell surface. Several viruses such as human immunodeficiency virus and coronavirus fuse at the cell surface to transfer the genetic material in the host cell, whereas viruses like influenza enters the host cell through endocytosis and then fuses with the endosome upon pH trigger [12] . Therefore, the viral fusion with the host cell is considered as the first step of viral infection.

The fusion protein presents on the viral envelope is called fusion machine as it orchestrates the fusion process between the virus and host cell. Interestingly, there is no sequence homology among the fusion proteins of different viruses however, all the fusion proteins share several common features [13] . Now we have a decent understanding on the fusion mechanisms aided by three dimensional structures of fusion proteins and peptides of several viruses with the help of NMR spectroscopy, cryo-electron microscopy and x-ray crystallography.

In recent years, the fusion proteins are considered as the target for antiviral intervention due to our growing understanding on the proteins and protein complexes that mediate fusion between virus and host cell. The amino acid sequences of fusion proteins are remarkably different among the viruses, but all initiates membrane fusion through trimer formation, and a common pathway of membrane dynamics [13] . The trimeric glycoproteins of class I virus generally contain a signal peptide, a receptor binding domain, a fusion domain, and a cytoplasmic tail (Figure 2) . The signal peptide is being cleaved by the signal peptidase but remains in contact with the lipid and subunit of peptidase complex before it is released in the cytosol. The signal peptide of HIV fusion protein, gp160 is known to interact with a calcium binding cytosolic protein, calmodulin [14] . However, the direct role of signal peptide in membrane fusion is not yet clear. The receptor binding domain (RBD) is extremely critical as it provides host tropism and zoonotic transmission of the virus [15] . There are specific receptors on the host cell surfaces, which are being recognized by the receptor binding proteins to dock on the host cell surface. The CD4 receptor binds to the RBD of HIV (gp120) [16] , whereas the RBD of influenza hemagglutinin (HA1) binds to the cell surface sialic acid to anchor on the host cell [17] . The spike protein (S1) of coronaviruses utilizes host angiotensin converting enzyme (ACE) to find the host cell for infection [18] . Any small alteration in the RBD leads to inefficient binding resulting in reduced infectivity of the virus [19] . Interestingly, binding of virus glycoprotein with cell surface proteins other than the specific receptor does not promote entry of the virus to the cell [20] .

The interaction between RBD and the cell surface receptor induces dramatic conformational changes in the fusion protein leading to exposure of the fusion domain [21] . [17] . Mutations in this stretch of amino acids have been shown to block fusion mediated viral infection for many viruses [22] [23] [24] . Interestingly, fusion peptide itself is capable to induce fusion between lipid vesicles, and several putative mechanisms have been proposed on how fusion peptide promotes membrane fusion [19, [25] [26] [27] [28] . The α-helical trimeric heptad region plays a crucial role in inducing fusion by the formation of six helix bundle. The six helix bundle formation, a trademark of class I viral fusion protein, brings two apposing membranes close to each other [18] . The heptad region is further divided into two regions, portions close to the N-terminal (or near the fusion peptide) and the C-terminal (or near transmembrane domain) are termed as fusion peptide proximal region (FPPR) and membrane-proximal external region (MPER), respectively [29] . The interaction between FPPR and MPER promotes the formation of six helix bundle. The transmembrane domain is a stretch of 20-25 hydrophobic amino acids that remains anchored to the viral envelope. It is hypothesized that the fusion peptide (partitioned in the host membrane) interacts with the transmembrane domain (anchored in the viral envelope) to facilitate pore formation [30] . residue influenza virus HA peptide into SUV at pH 5.0 [38] . In addition, it had been proposed that binding of 8-18 FPs of HA would yield required free energy change, 60-120 kcal/mol, for the stabilization of curved membrane lipid membrane which is initially essential for fusion process [38] . Insertion of FPs into lipid bilayer is known to support stable structures with propensity for stable oligomerizations [41] . The FPs of influenza virus HA peptide, HIV-1 gp41 belonging to the type I fusion protein have been serving as an archetypal example for mechanistic studies with model membranes. Both HA and HIV-1 gp41 FPs assume monomeric -helical structure, determined by solution NMR methods, in detergent micelle (Figure 4 ) [44, 45, 55] . A large part (residues Ile4-Ala15) of helical structure of the FP of HIV-1 gp41 in SDS micelle was deduced to be deeply inserted into the micelle core akin to the orientation of transmembrane helices [55] .

Initial solution NMR and EPR studies determined an inverted "V" shaped or boomerang like helical structure of the 20-residue long HA FP in DPC micelle [44] . The non-polar face of the amphipathic helical structure probably be embedded into the monolayer of the lipid bilayer.

Interestingly, a tightly packed helical hairpin structure of HA FP was determined in DPC micelle by solution NMR. This 23-residue construct of HA FP contains additional three conserved residues Trp 21 -Tyr 22 -Gly 23 at the C-terminus [45] . The helical hairpin structure of HA FP might undergo a structural change to a continuous long helix during fusion pore formation. It is conceivable that virus fusion would require higher order oligomerization or association of the fusion proteins. These oligomeric states are likely to confine fusion- Solid state NMR methods showed transition of -helical to -sheet conformations of FPs of HIV-1 gp41 and HA in membrane bilayers [50, 58] . Structures of FPs could significantly be influenced depending on membrane mimetic environments. As stated above, the FP of HIV-1 gp41 assumed helical structure whereas aggregated -sheet conformations were deduced in cholesterol containing lipid bilayers.

These observations appear to indicate that multiple conformations of FPs may be playing important roles in membrane fusion. Although, determination of atomic resolution structures is challenging in lipid bilayers, therefore, structural works utilizing systems of lipid nanodisks or bicelles could be employed for better understating on the effect of membranes [59] [60] [61] [62] . It is worthy to mentioned that host protein is known to be important to aid in membrane 

Coronaviruses (CoVs) are zoonotic pathogens subdivided, based on genomes and serology, into four different categories alpha, beta, gamma and delta [68, 69] . CoVs are enveloped viruses with a positive sense single stranded RNA as genetic element [68] . CoVs The trimeric S or spike glycoprotein at the envelop of the CoVs appears to be determining in host specificity [20, 75] . The S protein of CoVs including SARS-CoV-1 and MERS helps the virus to bind to the receptor of the host cells and host-virus fusion. The S protein can be divided into two domains S1 and S2 which can be proteolytically cleaved in some CoVs [76, 77] . The S1 domain of S is largely involve in receptor binding whereas the S2 domain is responsible for host-virus membrane fusion [78] [79] [80] [81] [82] . The full-length S of SARS-CoV-1 is 1255 amino acid long with several functional domains and potential multiple proteolytic cleavage sites at the boundary of S1 and S2, and also S2' site (Figure 7) . Cryo-EM structures of S proteins from mouse coronavirus (MHV), human coronavirus KHU1, SARS-CoV-1 and MERS-CoV are reported in the prefusion state [83] [84] [85] . These structures revealed trimeric architecture of the S protein with extensive packing J o u r n a l P r e -p r o o f between S1 and S2 domains. Further, the fusogenic components of the S2 domain including HR1, fusion peptides are found to be buried within the core of the trimeric structure, whereas, the receptor binding domain of S1 can be amenable for interactions with the host cell. The S protein is expected to undergo a large conformational change from the latent prefusion state to fusion active state upon receptor binding and priming by host proteases. The x-ray structures of the S2 domain of S protein largely on HR1, HR2 and HR1/HR2 complex revealed canonical trimeric coiled-coil helical structures akin to type I fusion system [79, 80] .

The HR1/HR2 complex forms a bundle of trimeric six helix whereby three short helices of HR2 can be found to be tightly packed along the long three helices of HR1. The quaternary association of HR1 and HR2 would bring the fusion peptides and viral TM helix at proximity, presumably facilitate membrane fusion process (Figure 3 ).

Although membrane fusion of SARS-CoV-1 belongs to the type I fusion system, the large S protein of SARS-CoV-1 encodes number of regions in the S2 domain with membrane binding and/or membrane fusion activity [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] . The existence of multiple potential FPs in the S2 domain of S protein in SARS-CoV-1 is unique in comparison to type I fusion proteins of HIV and influenza viruses. Notably, the single FP, located at the N-terminus, has been known to be responsible for membrane fusion of HIV-1 gp41 and HA influenza viruses [24] [25] [26] . Intuitively, membrane fusion mechanism of SRAS-CoV-1 and other CoVs may be complex involving several distinct stages. The upstream part of the HR1 (residues 892-972) of S2 domain of S protein of SARS-CoV-1 appears to be involved in membrane fusion (Figure 7) . Several research groups have identified membrane binding and fusogenic peptides or potential FPs from the upstream region (residues 758-890) of HR1 ( Table 1) . (Table 1) , also termed as internal fusion peptide or IFP2, demonstrating high binding affinity and leakage from model membranes of various compositions [88] . The IFP2 appeared to be binding with negatively charged phospholipids with higher affinity compared to zwitterionic lipids [88] . It may be noteworthy that the IFP1 is located at the C-terminus of a proteolytic cleavage at residues 797/798 at S2' site. This would suggest that after proteolytic priming of S protein at S1/S2 and S2' sites, the IFP1 would remain a part of the fusion protein whereas FP, residues 770-788, will not be covalently bonded to the rest of the S2 domain (Figure 7) . In a later study, Whittaker and co-workers has defined a yet another segment of residues 798-815 in proximity to the S2'cleavage site as a fusion peptide or IFP1 [93] . Mutagenesis studies on the full-length S protein identified residues L803, L804 and F805 that are critically involved in fusion. Biochemical analyses with synthetic peptide of IFP1 demonstrated ability of this peptide in membrane fusion. In addition to these fusogenic peptides, a C-terminal segment adjacent to TM or PTM (residues 1185-1202) is known to confer strong membrane partitioning (Figure 7) . Further, ~170 amino acids long polypeptide between HR1 and HR2 also appeared to be containing peptide motifs with membrane interacting properties and virus-cell fusion inhibiting activity [94] . 873 GAALQIPFAMQMAYRF 888 (IFP2) 88 858 ALISGTATAGWTFGAGAALQIPFAMQMAYR 886 79 J o u r n a l P r e -p r o o f

We have determined 3-D structures of FPs and PTM of SARS-CoV-1 in solution of DPC detergent micelle by NMR methods [96, 97] . The atomic resolution structure of the FP ( 770 MYKTPTLKYFGGFNFSQIL 788 ) revealed a bend helical structure presumably resulting from two Gly residues G780 and G781 at the center of the sequence (Figure 8) . 1185 LGKYEQYIKWPWYVWLGF 1202 ) of S protein has also been determined in DPC micelle.

The PTM is a conserved sequence among SARS-CoVs, and also observed in other viruses like HIV-1 and Eob. It has been postulated that the PTM segment could be involved in viral membrane fusion process [87] . Micelle-bound PTM assumes a fold of helix (residues K1187-Y1191)-loop (I1192-K1193)-helix (W1194-F1202) structure whereby the two helices were found to be independently oriented (Figure 8) . Observations of multiple adjacent fusogenic peptides in the S2 domain prompted us to examine structure and membrane localization of a 64-residue long, residues R758-E821, or LFP (long fusion peptide) in detergent micelle solution [97] . The primary structure of LFP contains FP (residues 770-788) and IFP1 (residues 798-815) with additional residues at the N and C-termini. LFP was over-expressed in E. coli as a fusion protein containing an 81-resiude long prodomain of human furin followed by an Asp-Pro sequence for formic acid digestion [89, 90] . The construct also contains an additional six residue His-tag at the N-terminus for affinity purification resulting a fusion protein His 6 -Prodomain-D-P-LFP. Atomic resolution structure, 15 N relaxation and micelle localization were investigated by heteronuclear NMR methods in DPC micelle solution (Figures 9, 10) . 3-D structure of LFP demonstrated existence of discretely folded helices connected by several loops (Figure 9A ). The Cterminal region of LPF defines a long -helix including residues T795-Y819 with a kink at residue D812. It may be noted that IFP1, residues 798-815, is included within the C-terminal J o u r n a l P r e -p r o o f helix. The FP segment (residues 770-788) in LFP assumed a helix-loop-helix structure, although, the isolated FP delineated a bend helical structure (Figure 8) . Interestingly, the Nterminal residues R761-Q769 of LFP assumed an amphipathic helical conformation ( Figure   9A ). The helical structures of LPF were found to be motionally rigid experiencing fast motion in ns-ps time scale. 

The fusogenic property of the membrane generally depends on the its composition and curvature [100] . protein undergoes oligomerization and may be important for infection process [108, 109] .

Pandemic due to coronavirus was predicted after SARS and MERS outbreaks.

However, no vaccines or effective drugs were developed for the mitigation of the threats. A new strain of coronavirus called SARS-CoV-2 or COVID-19 is demonstrating a rapid spread J o u r n a l P r e -p r o o f all over the world which was initially found in Wuhan, China. SARS-CoV-2 pandemic, so far, has caused nearly 500,000 deaths globally with an infection of over 11 million people.

The high level of infectivity of SARS-CoV-2 compared to SARS-CoV-1 could be related to an efficient cell entry of the virus. Although, the molecular mechanism of the cell entry process remains unclear, binding of the virus to the host cell receptor is an important step in successful infection. Studies have shown that the isolated RBD of SARS-CoV-2 binds to ACE2 with tighter affinity compared to SARS-CoV-1, indicating potential for higher infectivity [107, [110] [111] [112] [113] . However, paradoxically, experiments with the full-length S protein have evidenced either similar binding affinity or even lower affinity of RBD to human ACE2 in comparison to SARS-CoV-1 [111] [112] [113] . The cryo-EM structure of SARS-CoV-2 showed that the RBD domain could be hidden within the S protein structure for immune evasion [111] . Therefore, other factors might be responsible for high infectivity of SARS-CoV-2. Protease cleavage of S protein has been postulated to be one of the factors responsible for high infectivity of SARS-CoV-2 [112] [113] [114] . An efficient membrane fusion mechanism between SARS-CoV-2 and host cell could also be responsible for the high level of infection. Sequence comparison of S proteins domain between SARS-CoV-1 and SARS-CoV-2 indicated high level of conservation both for the S1 and S2 domains [115] .

Nevertheless, variations can be observed for the fusogenic regions of the S2 domain between two viruses. Although, effect of these variations for an efficient membrane fusion remains to be examined.

Emerging and re-emerging viral infections are continuous threats to human kind. 

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Mechanistic Insights of Host Cell Fusion of SARS-CoV-1 and SARS-CoV-2 from Atomic Resolution Structure and Membrane Dynamics Hirak Chakraborty 1,2 and Surajit Bhattacharjya 3

Odisha 768 019, India 2 Centre of Excellence in Natural Products and Therapeutics