The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner

Abstract

Coronaviruses are a major infectious disease threat, and include the pathogenic human pathogens of zoonotic origin: SARS-CoV and MERS-CoV. Entry of coronaviruses into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the coronavirus fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2′ cleavage site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous ESR spectroscopic studies, the membrane ordering effect of influenza virus, HIV and Dengue virus FPs have been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium-dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca2+-dependency membrane ordering was not observed with influenza FP, indicating that the coronavirus FP exhibits a mechanistically different behavior. Membrane ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion “platform”.

An exploration of conditions proposed to trigger the Ebola virus glycoprotein for fusion

“…Because of these conflicting conclusions we re-examined the sufficiency of NPC1 to trigger the fusion activity of EBOV GP cl. In the process, we also assessed the potential enhancing role of additional factors shown, as outlined in the Results section, to augment fusion of other endocytosed enveloped viruses [23,[33][34][35]38,39,45,50,53]. Our findings are consistent with and extend the result stated by Miller et al [10].…”

“…Based on findings stated by Miller et al [10] and presented in Figs 1, 2 and 5, we postulated that a factor(s) in addition to low pH and NPC1 is needed to elicit the fusion activity of EBOV GP cl . We therefore tested factors required for fusion or uncoating of other endocytosed enveloped viruses (Ca ++ , K + , BMP, proteases) [38,39,41,45,53], proposed for EBOV in particular (Ca ++ , additional cathepsin action, reducing agent) [6,25,55], and even the non-physiological condition of elevated temperature, which can substitute for biological viral fusion triggers [33][34][35]. However, none of these factors or treatments elicited detectable CCF between effector cells bearing EBOV GP cl and target cells bearing NPC1 or its C-Loop (at any pH tested), while robust CCF was consistently detected with effector cells bearing LCMV GP at low pH.…”

“…The third additional factor we tested was cations, specifically Ca ++ and K + . EBOV entry requires TPC2, an endosomal calcium channel [25] and the glycoproteins of Rubella [38], Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) [39,40], have been reported to require Ca ++ for optimal functioning. K + is needed for productive entry of influenza and bunyaviruses [41,42].…”

A Tale of Two Viruses: The Distinct Spike Glycoproteins of Feline Coronaviruses

“…Mutation at this amino acid was first described by Chang et al in 2012, were they described that this mutation allowed to distinguish FECV from FIP in most of their analyzed cases [11]. The region of S containing M1058 was originally attributed to a fusion peptide region, but is in fact down-stream of what is now considered the bona fide fusion peptide (based on the SARS-CoV fusion peptide) [66]. However, M1058 is adjacent to the HR1 domain and so may modulate fusion in some capacity.…”

“…The FP is one of the most important components of the S2 domain. The FP is the primary regulator of virus-cell membrane fusion and its function is controlled by a series of molecular events, including structural changes due to protease activation, pH changes, and ion binding [66,67]. While there are several molecular and structural events necessary for the FP to be exposed and to fulfill its function, activation of the S protein by cellular proteases is perhaps the most critical of those events.…”