Introduction

Severe Acute Respiratory Syndrome (SARS) is a viral respiratory illness caused by the SARS coronavirus (SARS-CoV) that affected 8,098 people worldwide, provoking 774 deaths1. Similarly to other enveloped viruses, SARS-CoV enters cells through fusion of its viral membrane with a host cell membrane. This fusion process is mediated by the spike (S) glycoprotein, a 1,255-amino acid type I transmembrane protein2 that assembles into trimers on the virion surface to form the characteristic spike structure of the SARS-CoV. These spikes are essential for the infection of the host cell and are responsible for both binding to cellular receptors (via S1 subunit)3,4 and fusion of viral and target cell membranes (via S2 subunit)18. Both peptides insert into but not significantly disturb (our data and data in references 15, 22 and 23) the highly-ordered, zwitterionic outer leaflet of the plasma membrane bilayer91. This binding process bridges viral and cell membranes and thus facilitates trimerization of other S2 subunits. Trimers are, in general, the fusion-active oligomeric state of class I fusion proteins16. As a result, a trimeric extended prehairpin conformation is formed. At this point, it is important to emphasize that the actual conformational state of the SARS-CoV fusion peptides remains elusive. SARSFP and SARSIFP adopt, respectively, a V-shaped and a linear helical conformation in dodecylphosphatidylcholine micelles94, but have a high tendency to aggregate and to form intramolecular β-sheets and extended β-strands stabilized by intermolecular interactions in models of lipid bilayers as well as to adopt, in small fractions, α-helical and unordered structures22,23. In the context of the intact protein, however, the structure and oligomerization state of those peptide segments still need to be addressed, although it has been proposed that membrane-bound self-associated peptides may provide the major driving force for trimerization of the whole protein18,75. Peptide binding, conformational change and possibly aggregation into the membrane may trigger S2 refolding into a trimeric hairpin conformation. As a result, a six-helix bundle would form, bringing not only viral and target membranes into close proximity, but also the internal fusion peptide (SARSIFP) and the pretransmembrane (SARSPTM) domain of the S2 subunit95. Due to the high hydration repulsion of the closely apposed lipid bilayers and the requirement for bending membranes to minimize areas of strong interbilayer repulsion16,96, displacement of water molecules from the membrane surface and changes in membrane curvature seem to be the prerequisites for allowing close intermembrane contact and subsequent formation of the high-energy hemifusion intermediate state. Interaction of SARSFP and SARSIFP with PE or with negatively charged lipids contained either in the plasma (via nonendocytic pathway) or in the endosome (via endocytic pathway) membranes may be important for the formation of point-like protrusions or for stabilization of the hemifusion stalk91. Since anionic phospholipids are mostly located in the inner leaflet of the membrane, it would be possible that the action of lipid flippases and scramblases could be endorsed by the peptide perturbation on the outer leaflet of the plasma membrane97. The major effects of the peptides at the pre-fusion state could be the following: induction of positive curvature on PE-rich membranes, as indicated by our DSC data; and membrane dehydration and induction of bending moment on the outer leaflet of bilayers comprised of anionic lipids, as suggested by our ESR data. The latter effects may also be responsible for triggering stalk formation, which is further stabilized by exposure of PE on the outer leaflet to SARIFP. Hemifusion could be further facilitated by membrane interaction of a loop peptide segment located in between HR1 and HR2 domains74 and by a possible heteroligomerization of SARSIFP with SARSPTM95. Juxtaposition of SARSIFP and SARSPTM leads to a synergistic and cooperative action of both peptides that causes membrane destabilization and further peptide insertion73. Exposure of SARSFP to the inner leaflet of the merged viral and cell membranes could have a great impact in the post-fusion state. Indeed, SARSFP could act by promoting positive curvature and stabilizing the high positively-curved inner leaflet that characterizes the porous state, thus facilitating pore formation (our DSC data). However, the molecular details of the above processes still need to be investigated. Overall, the two putative fusion peptides from SARS-CoV S protein may help to regulate membrane fusion by acting in the early and late stages of the membrane fusion process.

Conclusions

Our main findings were: (1) SARS fusion peptides increase the ordering of the headgroup and acyl chain regions of MLVs containing negatively-charged, but not zwitterionic phospholipids; (2) Membrane fusion promoters induce similar effects on the head group ordering than do the fusion peptides, whereas membrane fusion inhibitors cause opposing effects; (3) Changes in the order parameters of the lipids are generally greater for the more fusogenic SARSFP peptide than for SARSIFP; (4) Both peptides promote dehydration of PG-containing membranes and this effect is well correlated with the increased head group ordering; and (5) DSC data support a hypothesis that SARSFP induces positive curvature on DiPoPE vesicles, whereas SARSIFP promotes opposing stresses on the intrinsic negative curvature of DiPoPE depending on the ionic strength.

Peptide-induced chain-packing energy and membrane surface ordering of the outer leaflet of negatively charged lipid bilayers promote partial membrane dehydration and could generate bending moment, as suggested by our ESR studies. Both effects may induce negative curvature and decrease the hydration repulsion of apposed bilayers. Possible peptide involvement on the formation of the pre-fusion point-like protrusions and intermediate hemifusion stalk as well as on the stabilization of the fusion pore state suggest that the SARS fusion peptides might play important roles in the whole membrane fusion process. Taken together, our findings suggest that the SARS fusion peptides have the ability to change the physicochemical properties of model membranes depending on the lipid composition and on the ionic strength. Therefore, they can act in the early and late stages of the membrane fusion process, conferring them a functional plasticity that might be important to help overcome the high kinetic barrier involved in the SARS-CoV-induced membrane fusion.

Methods

Materials

N-terminally acetylated and C-terminally amidated SARSFP (770MWKTPTLKYFGGFNFSQIL788) and SARSIFP (873GAALQIPFAMQMAYRF888) peptides were either purchased from GenScript (Piscataway Township, NJ) or manually synthesized according to the standard Fmoc solid-phase peptide synthesis method on a Rink-Amide resin98. The details of peptide synthesis are described in Vicente et al.99. Purification was performed as described in Supplementary section SI1.

The phospholipids 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC), 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DiPoPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), and the spin labels 1-palmitoyl-2-stearoyl(n-doxyl)-sn-glycero-3-phosphocholine (n-PCSL, where n = 5 and 16), 1,2-dioleoyl-sn-glycero-3-phospho(tempo)choline (DOPTC), and 1,2-dipalmitoyl-sn-glycero-3-phospho(tempo)choline (DPPTC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Cholesterol (Chol) and linoleic acid (LA) were obtained from Sigma-Aldrich (St. Louis, MO). All reagents were used without further purification.

Sample preparation

Phospholipids (1.6 mg for DSC and 0.5 mg for ESR) and spin labels (0.5 mol% for CW ESR and 1 mol% for pulsed ESR) either in chloroform or chloroform/methanol 1:1 (v/v) stock solutions were mixed in a glass tube. After dried under a N2 flow, the lipid film was ultracentrifuged under vacuum overnight to remove traces of solvent. For CW ESR experiments, the sample was hydrated in 20 mM potassium phosphate buffer, pH 7.4, sonicated in a bath type sonicator for a few seconds and maintained at a temperature above the main phase transition of the lipid for at least two hours for complete hydration. Samples were then subjected to at least six freeze-thaw cycles. A measured volume of SARSFP or SARSIFP stock solutions in dimethyl sulfoxide (DMSO) was added to the preformed multilamellar lipid dispersions. For DSC experiments, peptides dissolved in either acetonitrile/water 1:1 (v/v) or in DMSO solutions were diluted into buffer and added to the lipid film for hydration. Samples were vortexed for few seconds, maintained at a temperature above the phase transition for each lipid during at least 30 min, and subjected to six freeze-thaw cycles. The amount of phospholipid (final lipid concentration of 10 mg/ml for ESR and 2 mg/ml for DSC) and peptides used provided a 20:1 lipid/peptide molar ratio for most of the experiments. It is worth mentioning that the same amount of DMSO or acetonitrile/water 1:1 added in the peptide-containing samples was also used in the peptide-free samples as controls for the ESR and DSC experiments. The control samples were prepared using the same protocol as those of the peptide-containing samples. For DiPoPE/peptide samples, peptides and lipids dissolved in chloroform/methanol 1:1 (v/v) stock solutions were mixed in a glass tube, dried to a lipid film under N2 gas and lyophilized overnight. Samples were hydrated in 20 mM sodium phosphate buffer, pH 7.4, with or without 150 mM sodium chloride, and freeze-thaw cycled six times below the liquid crystalline-to-inverted hexagonal (Lα-HII) phase transition temperature (TH) of the lipid. DiPoPE concentration was 10 mg/ml and peptide concentration varied from 0.2 to 0.5 mol% (500:1 and 200:1 lipid/peptide molar ratio, respectively). For ESEEM experiments, POPC/POPG 7:3 mol/mol and peptides (20:1 lipid/peptide molar ratio) were prepared as above, but hydrated in 20 mM sodium phosphate, 150 mM NaCl D2O buffer, pD = 7.4 (actual pH measurement). Peptide concentration was confirmed spectrophotometrically by using the theoretical molar extinction coefficients of 6,990 M−1cm−1 for SARSFP and 1,490 M−1cm−1 for SARSIFP.

DSC Experiments

The effects of the peptides on the thermotropic behavior of the lipid phase transitions were recorded in a VP-DSC MicroCal MicroCalorimeter (Microcal, Northampton, MA, USA) using a heating rate of 33.2 °C/h for DiPoPE and of 23.4 °C/h for the other lipids. Samples were firstly degassed and then equilibrated for 15 minutes at the starting temperature prior to measurements. Analyses of thermograms were performed using Microcal Origin software.

ESR Experiments

CW-ESR experiments were carried out on a Varian E-109 spectrometer operating at 9.5 GHz. Temperature was controlled by a homemade temperature control unit coupled to the spectrometer, whose accuracy is about 0.2 °C. Samples were transferred to glass capillaries (1.5 mm I.D.), which were set into a quartz tube containing a mineral oil bath to help stabilize the sample temperature. The following acquisition parameters were used: center field, 3,362 G; scan width, 80 to 160 G; modulation amplitude, 0.5 or 1.0 G; modulation frequency, 100 kHz; microwave power, 5 or 10 mW; time constant, 128 ms, and acquisition time, 240 s.

Nonlinear least-squares simulations (NLLS) of the CW-ESR spectra were performed using the Multicomponent LabView (National Instruments) software developed by Dr. Christian Altenbach (University of California, Los Angeles, California)24,100. The rotational diffusion rates (R⊥, R∥) and order parameters (S0, S2) were obtained as described in ref. 33 with further details in the section SI2 of supplementary information. Seed values for the magnetic parameters of both 5-PCSL and 16-PCSL were obtained from Earle et al.44 and those of DPPTC were taken from Ge and Freed101. The strategy of the NLLS simulation was performed as described elsewhere33.

Pulsed ESR experiments were performed on a Bruker Elexsys 580 X-band pulsed ESR spectrometer equipped with the Bruker Flexline ER 4118X-MS3 split-ring resonator and the ITC503 Oxford cryogenic system for temperature control. Samples were immersed into liquid nitrogen prior to the measurements at 50 K. Three pulse electron spin echo envelope modulation (ESEEM) experiments were carried out with the π/2 – τ – π/2 – T – π/2 – τ – echo pulse sequence54 and using a four-step phase cycling to suppress unwanted echoes102. The microwave power was adjusted to give 16 ns π/2 pulses and an interpulse delay τ of 236 ns, kept constant in all experiments, was chosen to maximize deuterium modulations at the magnetic field where the echo intensity is maximum. Starting at time delay T = 200 ns, 700 points were recorded with ΔT = 12 ns steps to obtain the three-pulse stimulated echo decays. The integration gate length was 48 ns and the shot repetition time was 1,500 μs. The number of accumulations varied from 20 to 50 depending on the signal-to-noise ratio and on the modulation depth. Data analysis was performed as described in Bartucci et al.62. Briefly, the contribution of the spin relaxation to the ESEEM signal was eliminated by dividing the time-dependent echo amplitudes, V(τ, T), by a bi-exponential decay, 〈V(τ, T)〉, followed by subtraction of unity, as Vnorm(τ, T) = V(τ, T)/〈V(τ, T)〉 − 1. The remained oscillations about zero were apodized with a Hamming window and zero-filled to increase the total number of points to about 4 K. Numerical Fourier transformation was performed and the resultant magnitude spectrum was multiplied by the dwell time ΔT = 12 ns to provide a spectral density in ns units.

Additional Information

How to cite this article: Basso, L. G. M. et al. SARS-CoV fusion peptides induce membrane surface ordering and curvature. Sci. Rep. 6, 37131; doi: 10.1038/srep37131 (2016).

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