key: cord-0776912-tj0szotn
authors: Pattnaik, Gourab Prasad; Bhattacharjya, Surajit; Chakraborty, Hirak
title: Enhanced Cholesterol-dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to its N-terminal Counterpart
date: 2021-01-15
journal: bioRxiv
DOI: 10.1101/2021.01.14.426613
sha: 12ea75beca06f9ab06b8fa4f20eb369b161e48cd
doc_id: 776912
cord_uid: tj0szotn

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 viruses contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion, and is termed as ‘fusion peptide’. However, Severe Acute Respiratory Syndrome Coronavirus (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV 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 pore.

Membrane fusion is a crucial step for successful entry and infection of the enveloped viruses, leading to the transfer of viral genetic materials into the host cell. [1] [2] [3] [4] [5] The fusion event is triggered by the viral fusion protein that comes into action after the receptor binding domain interacts with the cell surface receptor proteins. 6 Generally, for class I viruses, a 20-25 amino acid stretch present in the Nterminus of the fusion protein is known as fusion peptide, which is instrumental in binding with the host cell and initiating the fusion process. [7] [8] Severe acute respiratory syndrome (SARS) is an emerging form of pneumonia caused by SARS-CoVs, and the entire world is now going through a crisis due to the attack of SARS-CoV-2. The fusion domain of SARS-CoV spike protein (S2) contains three putative fusion peptides recognized as N-terminal fusion peptide (FP), internal fusion peptide 1 (IFP1), and internal fusion peptide 2 (IFP2). [9] [10] [11] [12] [13] The S2 protein contains heptad repeats, HR1 and HR2, and a transmembrane region at the C-terminus in addition to these fusion peptides. Interestingly, the FP and IFP1 are highly homologous between SARS-CoV-1 and SARS-CoV-2 (Table 1) . Therefore, proper understanding of the role of FP and IFP1 in inducing membrane fusion would provide valuable mechanistic insights of the entry of both SARS-CoV-1 and SARS-CoV-2. The atomic resolution structure of the complex formed by two heptad regions revealed the formation of a six-helix bundle; considered to facilitate close apposition of two fusing membranes. [14] [15] Membrane composition plays a significant role in the fusion process as it alters the fusion protein or peptide conformation as well as the membrane organization and dynamics. 16 The role of cholesterol in membrane fusion is firmly established from the results obtained from viral and model membrane fusion. [17] [18] Cho-lesterol is also known to promote oligomerization of the SARS-CoV FP. 19 The lipid stalk hypothesis assumes that the sequential evolution of the intermediates toward the opening of fusion pore. Initially, two bilayers come close and the outer leaflets of both bilayers mix to form the stalk intermediate. Subsequently, the inner leaflets of the apposed membranes come in contact with each other to form transmembrane contact, which finally undergoes mixing of inner leaflets to open fusion pore. The stalk and transmembrane contact structures are collectively called hemifusion intermediates. A schematic representation of the fusion process is shown in scheme 1. Scheme 1. Schematic representation of different intermediates during the course of membrane fusion.

In this work, we have studied the effectiveness of FP and IFP1-induced fusion of small unilamellar vesicles (SUVs), and evaluated the effect of membrane cholesterol on the fusion process. Our results demonstrate that the IFP1 promotes lipid mixing in a cholesterol-dependent fashion.

Both the rate and extent of lipid mixing increase significantly in presence of cholesterol. On the contrary, the FP is not that efficient to induce lipid mixing, however, there is a slight increase in rate and extent of lipid mixing in presence of membrane cholesterol. Interestingly, both FP and IFP1 fail to demonstrate substantial content mixing highlighting the role of other domains of S2 protein for the pore formation. The extent of content leakage remains about 10%, which confirms the overall integrity of fusing membranes.

The above observation indicates that the IFP1 (and partially FP) induces hemifusion, however incapable of opening the pore between two fusing membranes. Our results support the requirement of interaction between FP and transmembrane domain of fusion protein for pore opening as proposed earlier in HIV. 20 Table 1 : Sequences of FP and IFP1 for SARS-CoV-1, SARS-CoV-2 and peptides used in the study In order to evaluate the effect of FP and IFP in membrane fusion, we have measured lipid mixing, content mixing, and content leakage kinetics using fluorescence-based methodologies described in method section (Supplementary material). IFP1 induced about 51% of lipid mixing in DOPC/DPOE/DOPG (60/30/10 mol%) SUVs in a lipid to peptide 100:1. The rate and extent of lipid mixing increases with increasing cholesterol concentration, and extents are about 71% and 84% in DOPC/DOPE/DOPG/CH (50/30/10/10 mol%) and DOPC/DOPE/DOPG/CH (40/30/10/20 mol%) SUVs, respectively ( Figure 1A , Table-2). This result suggests that the efficiency of IFP1 in promoting lipid mixing is extremely dependent on the concentration of membrane cholesterol. Though it promotes significant amount of lipid mixing, does not induce content mixing, and brings about 10% content leakage in the membrane containing 20 mol% of cholesterol ( Figure  1B&C Generally, for the entry of class I viruses the N-terminal FP is considered to be crucial. Though SARS-coronaviruses belong to class I category, our results demonstrated that IFP1 is more fusogenic than its N-terminal counterpart. The higher fusogenicity of IFP1 could be correlated to its higher hydrophobicity compared to the N-terminal FP (Figure 3 ). Ten out of sixteen (62.5%) amino acids are hydrophobic in IFP1, whereas seven out of nineteen (36.8%) amino acids are hydrophobic in FP as per the Kyte-Doolittle hydrophobicity scale. 21 Our results further demonstrated the important role of cholesterol in the enhancement of IFP1 and FP-induced hemifusion; an important link between the membrane cholesterol and higher risk of viral infection. The stringency of cholesterol in class I viral infection has been shown earlier, and our results indicate that the higher fusogenicity could be due to the higher effectiveness of fusion peptides in inducing hemifusion intermediate in presence of cholesterol. Cholesterol might promote membrane fusion either by modulating the peptide conformation 22,23 and depth of penetration 18 or changing membrane physical properties such as intrinsic negative curvature and stiffness. 24 Cholesterol has an inverted cone like structure that generates intrinsic negative curvature to the membrane, which promotes the formation of nonlamellar fusion intermediates. In addition, cholesterol enhances overall membrane stiffness which provides mechanical stability to the highly curved intermediate structures Taken together, our work provides three important information regarding the fusion peptide-induced membrane fusion for SARS-coronaviruses. Firstly, it is clearly demonstrated that the IFP1 is more fusogenic than the FP and it could be due to higher hydrophobicity of IFP1. Secondly, the importance of cholesterol in the peptide-induced membrane fusion, and finally the requirement of interaction between fusion peptide and transmembrane domain for pore opening. 

SARS-CoV-1 MYKTPTLKDFGGFNFSQIL SARS-CoV-2 IYKTPTLKDFGGFNFSQIL Internal Fusion Peptide 1 SARS-CoV-1 GAALQIPFAMQMAYRF SARS-CoV-2 GAALQIPFAMQMAYRF

Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin

Enveloped viruses: a common mode of membrane fusion?

Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41

Initial stages of HIV-1 envelope glycoprotein-mediated cell fusion monitored by a new assay based on redistribution of fluorescent dyes

The many mechanisms of viral membrane fusion proteins

Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme

Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus

H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region

Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndromeassociated coronavirus spike protein

A second SARS-CoV S2 glycoprotein internal membrane-active peptide. Biophysical characterization and membrane interaction

Mechanistic insights of host cell fusion of SARS-CoV-1 and SARS-CoV-2 from atomic resolution structure and membrane dynamics

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Acid stability of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity

Membrane Composition Modulates Fusion by Altering Membrane Properties and Fusion Peptide Structure

Target Membrane Cholesterol Modulates Single Influenza Virus Membrane Fusion Efficiency but Not Rate

Cholesterol alters the inhibitory efficiency of peptide-based membrane fusion inhibitor

Membrane Cholesterol Modulates Oligomeric Status and Peptide-Membrane Interaction of Severe Acute Respiratory Syndrome Coronavirus Fusion Peptide

HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion

A simple method for displaying the hydropathic character of a protein

Major antiparallel and minor parallel beta sheet populations detected in the membraneassociated human immunodeficiency virus fusion peptide

Solid-state NMR spectroscopy of human immunodeficiency virus fusion peptides associated with host-cell-like membranes: 2D correlation spectra and distance measurements support a fully extended conformation and models for specific antiparallel strand registries

Membrane curvature at a glance