SARS-CoV 2 accessory protein 7b forms homotetramers in detergent

doi:10.21203/rs.3.rs-1446025/v1

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SARS-CoV 2 accessory protein 7b forms homotetramers in detergent

https://doi.org/10.21203/rs.3.rs-1446025/v1

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A global pandemic is underway caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome, like its predecessor SARS-CoV, contains open reading frames that encode accessory proteins involved in virus-host interactions active during infection and which likely contribute to pathogenesis. One of these accessory proteins is 7b (p7b), with only 44 and 43 residues in SARS-CoV and SAR-CoV-2, respectively. It has one predicted transmembrane domain fully conserved, which suggests a functional role, whereas most variability is contained in the predicted cytoplasmic C-terminus. In SARS-CoV, 7b protein is expressed in infected cells where the transmembrane domain was necessary and sufficient for Golgi localization. Also, anti-p7b antibodies have been found in the sera of SARS-CoV convalescent patients. In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. We show that 7b protein is almost completely α-helical in both SARS viruses and has a single transmembrane domain. In SDS, p7b forms various oligomers, from monomers to tetramers, but only monomers when exposed to reductants DTT or TCEP. Combination of SDS gel electrophoresis and and analytical ultracentrifugation (AUC) in both equilibrium and velocity modes suggests a dimer-tetramer equilibrium, and an equilibrium between monomeric, dimeric and tetrameric forms in the presence of reductant. Inclusion of pentamers or higher oligomers in the SARS-2 7b model worsened the fits. Although sensitivity to reductants suggests the involvement of disulfide-linked dimers, the presence of disulfide bonds was not essential to form tetramers. A preliminary model of this association was generated with Alpha-Fold, which was exposed to a molecular dynamics simulation in presence of a model lipid membrane. However, neither of the two models provide any evident pathway for ions. To confirm this, SARS-2 p7b was studied using Planar Bilayer Electrophysiology. Addition of p7b produced occasional membrane permeabilization that is not consistent with bona fide ion channels made of a tetrameric assembly of α-helices.

accessory protein 7b

SARS-2-CoV

COVID-19

analytical ultracentrifugation

channel activity

coronavirus

Coronaviruses (CoV) are vertebrate pathogens which cause human respiratory diseases that typically affect the respiratory tract and gut. They have been kown to cause common cold symptoms in humans, and a variety of lethal diseases in birds and mammals [1]. However, in 2003, the virus responsible for the severe acute respiratory syndrome (SARS-CoV) [2], referred hereafter as SARS-1, produced a near pandemic with 8,098 infected and 774 deaths, i.e., a 10% mortality rate [3]. Currently, a global pandemic of Coronavirus disease 19, i.e., COVID-19, (https://www.who.int), caused by SARS-CoV-2, hereafter SARS-2 [4], is underway at the time of writing this manuscript, infecting 410 million people and causing more than six million deaths [5]. It is important to urgently explore all possible pharmaceutically accessible therapeutic targets in SARS-2 proteins and host interactions [6]. CoVs belong to the family Coronaviridae, subfamily Coronavirinae, and are distributed into four genera [7]. In CoVs genomes, the first two thirds encode non-structural genes; open reading frames ORF1a and ORF1b produce polyproteins pp1a and pp1ab, which are processed into 16 nonstructural proteins (nsp1 to 16). The last third of the genome hosts the ORFs for structural proteins: spike (S), envelope (E), membrane (M) and nucleoprotein (N), and also other so-called ‘accessory’ proteins, which vary in number and sequence even among CoVs belonging to the same lineage [8–10].

Specific for SARS-CoVs are eight ORFs that encode accessory proteins, namely ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b [11, 12]. Although these proteins have been considered not essential for viral replication in vitro [13–15], several of these have been found to be involved in virus-host interactions during infection in vivo [13, 16]. Accessory proteins may confer biological advantages to the virus in the natural host, and contribute to pathogenesis [11]. In SARS-1, protein 7b was predicted to be translated from a second ORF of SARS-CoV sgRNA7 [17] and expression was confirmed in infected Vero cells [18]. Although no experiments have been performed to detect the expression of 7b in tissue samples from SARS patients, the presence of anti-p7b antibodies in SARS convalescent patient sera indicates that the 7b protein is likely expressed in vivo [19], and is likely incorporated into purified virions [18]. In SARS-1, protein 7b is 44 amino acids long and has a highly hydrophobic polypeptide predicted to span the membrane, with a luminal N-terminus and a cytoplasmic C-terminus [20]. Similar to p7a, the 7b protein is localized throughout the Golgi compartment in both SARS infected cells and in cells transfected with 7b cDNA [18]. It is incorporated to the SARS-1 virion as one of the structural proteins, but it is not detected on the cell surface of the transfected cells [20]. The transmembrane domain of p7b was found to be both necessary and sufficient for its Golgi localization, and was attributed to residues 21–23 and 27–30 [21]. SARS-1 ORF7b is not essential for viral replication in vitro and in vivo [15, 18, 22], but a 45-nt deletion in the transmembrane domain of ORF7b was observed in a prototype virus (strain Frankfurt-I) isolated during the 2003 SARS outbreak [23] with replicative advantage in some cells [24], suggesting an attenuating role for 7b protein. Also, studies using siRNA specific for SARS-CoV sgRNA7 showed silencing of the expression of 7a, 7b, 8a and 8b [25], indicating that p7a/p7b (and p8a/p8b) may play certain roles during the replication cycle of SARS-1. It has been shown that the 7b can induce apoptosis in infected cells [26], but the significance of this in the viral life cycle is not clear [27].

The current global pandemic is caused by SARS-2 which, like its SARS-1 predecessor, also encodes accessory proteins. The sequence of 7b protein in SARS-2 is one residue shorter than the one in SARS-1, 43 residues long, and their predicted transmembrane domain is fully conserved (Fig. 1), suggesting a functional role. Overall, sequence identity is 88%, where > 90% of the variability is contained in the predicted cytoplasmic C-terminus. Recently, SARS-2 p7b has been shown to mediate apoptosis in cells mediated by tumor necrosis factor-α (TNF-α)[28]. However, no structural details have been reported on 7b protein. It has been shown that SARS-2 7b forms oligomers in gels, and a pentameric model similar to phospholamban was proposed, but with no experimental data [29]. Herein, we have focused most of the discussion on p7b from SARS-2 7b, since it is the most current and relevant form, although some experiments have been performed on SARS-1 7b for comparison.

In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. Oligomeric size was determined using analytical ultracentrifugation (AUC) in the sedimentation equilibrium (SE) and sedimentation velocity (SV) modes, whereas possible channel activity was tested using planar lipid bilayers. Finally, we suggest a preliminary model for the interaction of the 7b monomers obtained by a molecular dynamics simulation performed in presence of a lipid membrane.

Peptide purification and reconstitution.

The 43-residue long SARS-2 7b protein was obtained as a crude peptide, synthesized with amidated C-terminus and free N-terminus (Genscript, USA). SARS-1 7b protein was synthesized in-house with amidated C-terminus and free N-terminus using microwave-assisted solid-phase fluorenylmethyloxycarbonyl (FMOC) chemistry using an Odyssey Microwave peptide synthesizer (CEM corporation). The protein was cleaved from the resin with trifluoroacetic acid (TFA) and lyophilized. The peptides were dissolved in TFA (10 µL) followed by dilution with acetonitrile to a final concentration of 5 mg/mL. The solution was injected into a C4-300 Å reverse-phase high-performance liquid chromatography (RP-HPLC) column (Phenomenex, Cheshire, UK) connected to a HPLC system (Shimadzu, Japan). The solvents used were solvent A: water with 0.1% TFA (v/v), and solvent B: isopropanol/acetonitrile (4:1 v/v) with 0.1% TFA (v/v). The peptide was eluted with a linear gradient from 30–75% of solvent B. Pooled fractions were lyophilized and the purity of the samples was checked by MALDI-TOF MS. The transmembrane domain (p7b-TM) was synthesized and purified in the same way.

Reconstitution in membranes.

Reconstitution of p7b in lipid membranes was performed first by mixing the lyophilized protein in TFE with 20x LPR_m (molar lipid-to-protein ratio) of DMPC lipid or ‘ERGIC lipid mixture’ (POPC : POPE : bovine PI : POPS : Cholesterol in molar ratio 45:20:13:7:15) in chloroform. Lipids were purchased from Avanti Polar Lipids (Alabaster, US). The mixture was dried under a N₂ stream and incubated in vacuum overnight before resuspension in water by vortexing and freeze-thawing. Reconstitution of the transmembrane domain (7b-TM) was achieved by mixing ethanol-dissolved lipid and peptide. The solvent was then evaporated with N₂ gas and the sample was rehydrated in water.

Infrared spectroscopy.

FTIR spectra were recorded on a Nicolet Nexus 560 spectrometer (Madison, USA) purged with N₂ and equipped with a MCT/A detector cooled with liquid nitrogen. Attenuated total reflection (ATR) spectra were measured with a 25-reflections ATR accessory from Graseby Specac (Kent, UK) and a wire grid polarizer (0.25 mM, Graseby Specac). Approximately 100 µL of sample in water at 20:1 LPR molar ratio were applied onto a trapezoidal (50 x 2 x 20 mm) Ge internal reflection element (IRE). A dry, or D₂O saturated, N₂ stream flowing through the ATR compartment was used to remove bulk water or to achieve D₂O exchange, respectively. A total of 200 interferograms collected at a resolution of 4 cm^− 1 were averaged for every sample and processed with one-point zero filling and Happ-Genzel apodisation. The % of amino acids embedded in the membrane was obtained from an amide hydrogen-deuterium exchange experiment, where the lipid/protein film was subjected to a flow of D₂O saturated nitrogen for 30 min. The area of the amide II (N-H bending, centered at ~ 1550 cm^− 1) and amide I (C = O stretching, centered at ~ 1655 cm^− 1) bands was obtained by peak integration from 1510 cm^− 1 to 1590 cm^− 1 and 1600 cm^− 1 to 1700 cm^− 1 The fraction of non-exchanged residues was determined as described previously [30].

Gel electrophoresis.

The peptide samples were solubilized in NuPAGE sample buffer, with or without reductant, 5 mM TCEP (Tris(2-carboxyethyl)-phosphine) or dithiothreitol (DTT), and run on a 13.5% Bis-Tris gel following the NuPAGE protocol (Invitrogen). The gel was stained with Coomassie blue G-250.

Analytical Ultracentrifugation (AUC).

AUC sedimentation velocity (AUC-SV) experiments were performed using a Beckman ProteomeLab XL-I analytical ultracentrifuge. p7b protein samples were reconstituted in detergent (5 mM myristyl sulfobetaine (C14SB, Sigma), 50 mM Tris pH 7.3, 100 mM NaCl), with or without addition of 2 mM TCEP and in presence of 29.4% (v/v) D₂O to eliminate the contribution of detergent buoyancy. The samples were centrifuged at 50,000 rpm in epon 2-sector centrepiece AUC cells with quartz windows. Absorbance profile at 280 nm was collected every 10 min for 15 hours. Sedimentation profile were analysed in SEDFIT using c(s) model [31] and plotted with GUSSI [32]. The S-values corresponding to monomer, dimer, or tetramer of full length 7b in C14SB micelles were predicted considering the properties of detergent, protein and buffer composition. The molecular weight (MW), aggregation number and specific volume of C14SB detergent was 363.6 Da, 83–130 (www.anatrace.com) and 0.965–0.978 mL/g (based on our density matching data), respectively. Using the sequence of SARS2-7b, the MW is 5180 Da and the specific volume is 0.7702 mL/g (calculated using Sednterp software). The buffer was 50 mM Tris, 100 mM NaCl and 29.4% D₂O, with density ρ = 1.0353 g/mL and viscosity, η = 1.0997 cP (calculated using Sednterp software). Assuming the lowest estimate of detergent bound, the MW of the complex (M_C) and the mass fractions of the detergent (δ_D) can be calculated as in Table 1, where ν_C is the specific volume of the complex. However, assuming the highest estimate of C14SB ν_D and highest aggregation number, the sedimentation coefficients were 0.13 S, 0.43 S and 0.99 S. Therefore, the range of predicted S values is shown in the last column of Table 1.

Table 1

Prediction of range of S values for monomers, dimers and tetramers.
	M_C (Da)	δ_D	ν_C (mL/g)	MW Mb (Da)	Diameter (nm)	S (s)	S range
Monomer	5,180 + 30,179 = 35,359	5.826	0.936	1095	4.7	0.37	0.13–0.37
Dimer	10,360 + 30,179 = 40,539	2.913	0.915	2136	4.9	0.69	0.43–0.70
Tetramer	20,720 + 30,179 = 50,899	1.456	0.886	4210	5.24	1.29	0.99–1.29

Table 1. Prediction of S values for the oligomers indicated on the left. The values are shown as an example assuming lowest ν_D and aggregation number for C14SB micelles, whereas the last column (bold) includes the range of S values obtained considering also the largest values of ν_D and aggregation number.

AUC sedimentation equilibrium (AUC-SE) experiments were performed for 7b and 7b-TM samples in the same instrument and buffer conditions as with AUC-SV samples. For each sample, three concentrations were prepared (30, 55, and 100 µM) and centrifuged at four speeds (23,000, 28,000, 34,500, and 42,000 rpm) in 6-sector epon centerpiece AUC cells with quartz windows. Absorbance at 280 nm was measured after 24 h equilibration at each speed (confirmation of equilibrum profile was obtained after performing scans at 30 min intervals). Once obtained, the sedimentation profiles were tested with various self-association models (SEDPHAT) and plotted in GUSSI [32, 33].

The species population plot was drawn in mole fraction scale by calculating the mole fraction scale association constant K_X as described by Fleming [34] using the expression: \({K}_{X}={K}_{A,app}\times \left[Det\right]\) where K_A,app is the fitted association constant in bulk molar scale, and [Det] is the concentration of micellar detergent in solution. For the monomer-dimer-tetramer equilibrium, the mole fraction of each species in the detergent phase: X₄, X₂, and X₁ (tetramer, dimer, and monomer, respectively) was calculated by solving the expression below for X₁ using the Newton-Raphson method:

\({X}_{4}=\left({K}_{X,24}\right){\left({K}_{X,12}\right)}^{2}{\left({X}_{1}\right)}^{4}\) \({X}_{2}=\left({K}_{X,12}\right){\left({X}_{1}\right)}^{2}\) \(4{X}_{4}+2{X}_{2}+{X}_{1}-{X}_{t}=0\) where K_X,24 and K_X,12 are the mole fraction scale association constant for the dimer-tetramer and monomer-dimer equilibrium, respectively, and X_t is the total protein mole fraction in the detergent phase. For the dimer-tetramer equilibrium, the mole fractions were similarly calculated by solving the following expression for X₂:

\({X}_{4}=\left({K}_{X,24}\right){\left({X}_{2}\right)}^{2}\) \(2{X}_{4}+{X}_{2}-{X}_{t}=0\)

Tetrameric SARS-2 p7b models in a lipid bilayer.

The dimeric model of full length SARS-2 p7b was build using AlphaFold2 [35] server (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb), assuming α-helical struture and parallel alignment of the monomers. The distance between the two sulphur atoms of two TM cysteine residues (Cys12) was set to be close enough to form a disulphide bond. To build the initial structures of the tetramer, two possibilities were considered to orient the two homo-dimers, resulting in two different tetrameric models. The two dimers were separated by 0.85 nm to avoid clashes and placed inside a POPC lipid bilayer. Lipid molecules that formed close contacts with the protein tetramer were removed. Protein parameters were based on the AMBER99SB-ILDN force field [36]. The lipid force field used is the slipid, an all-atomistic force field for biological membranes [37, 38]. The system was solvated with TIP3P [39] water molecules and counterions were added to neutralize the system. Molecular dynamics (MD) simulations were performed using GROMACS [40] 5.1.2 software. The LINCS [41] algorithm was used to constrain bonds between heavy atoms and hydrogen to enable a timestep of 2 fs. A 1.2 nm cutoff was used for Van der Waals interaction and short-range electrostatic interaction calculations, and the Particle Mesh Ewald method was implemented for long range electrostatic calculations. The simulation temperature was maintained at 300 K using a V-rescale thermostat [42] and 1 bar pressure using Parrinello-Rahman [43] barostat. Simulations of 100 ns were performed for both tetramers in the presence of the POPC lipid bilayer.

Electrophysiology in lipid bilayers.

Planar bilayers were formed by apposition of two monolayers prepared from a 5 mg/mL solution of pure 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti polar lipids, Inc., Alabaster, AL) in pentane. Lipids were added on a ~ 100 µm diameter orifice in the 15 µm thick Teflon partition that separated two identical chambers [44, 45]. The orifice was pretreated with a 3% solution of hexadecane in pentane. Aqueous solutions consisted of 1 M KCl buffered with 5 mM HEPES at pH = 6. All measurements were performed at room temperature (23 ± 1 ^oC). Current events were observed after adding 0.5-1 µL of a 2.5 mg/mL solution of full-length SARS-2 p7b in acetonitrile: H₂O (1:1 v/v) (ACN 50%) to one side of the chamber (cis side). Additions were performed close to the orifice and then membrane was reformed to promote protein incorporation into the lipid bilayer. Successive additions of protein promoted always the same kind of current events. An electric potential was applied using Ag/AgCl electrodes in 2 M KCl with 1.5% agarose bridges assembled within standard 250 µL pipette tips. The potential was defined as positive when it was higher on the side of protein addition (cis side), whereas the trans side was set to ground. An Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) in the voltage-clamp mode was used to measure the current and the applied potential. Data were filtered by an integrated low pass 8-pole Bessel filter at 10 kHz, digitized at a sampling frequency of 50 kHz with a Digidata 1440A (Molecular Devices, Sunnyvale, CA), and analyzed using pClamp 10.7 software (Molecular Devices, Sunnyvale, CA). The chamber and the head stage were isolated from external noise sources with a double metal screen (Amuneal Manufacturing Corp., Philadelphia, PA).

SARS-2 p7b is mostly α-helical in hydrated ERGIC-like lipid bilayers.

When bulk water was removed, the infrared spectrum of p7b reconstituted in ERGIC-like lipid bilayers showed a highly symmetrical and narrow amide I band centered at 1656 cm^− 1, comparable to the spectrum of p7b-TM (Fig. 2). This corresponds to a completely α-helical conformation. However, hydration of the membranes with D₂O produced a spectrum with a shoulder around 1,630 cm^− 1, suggesting a propensity for β structure formation [46], possibly localized at the C-terminal extramembrane domain. Hydrogen-deuterium (H/D) exchange reduces the area of amide I, while keeping amide I constant. From the ratio between these two areas before and after D₂O addition, we calculated approximately 23 amino acids resistant to H/D exchange, consistent with a predicted single TM domain (Fig. 1). Similar results were obtained with SARS-1 p7b and are not shown.

Oligomerization of SARS-2 p7b in SDS gels.

Since 7b protein has two cysteine residues, one in the TMD and one in the extramembrane domain (Fig. 1), we tested if the pattern of migration in SDS was affected by reductants. Under non-reducing conditions (- TCEP), multiple 7b oligomers, from dimers to tetramers, were observed (Fig. 3, left). Under reducing conditions (+ TCEP), only monomers were detected (Fig. 3, right). A similar pattern was observed for SARS-1 7b, where higher oligomers disappeared in the presence of DTT (Fig. S1). The TMD alone, which has only one cysteine residue, formed only monomers and dimers under the same conditions (Fig. S1). Thus, this confirms the presence of disulfide bonds in 7b protein, which may have an effect on oligomerization.

Sedimentation equilibrium of 7b protein in detergent micelles.

The oligomerization behavior of p7b protein was further examined by using sedimentation equilibrium (SE) where radial distribution profiles were fitted to various self-association models. In this technique, the detergent component of the sample is density-matched using D₂O, and behavior is dependent is dependent exclusively on the molecular weight of the protein complex, not its shape. Sedimentation profiles (Fig. 4A) were fitted with several models. After an exhaustive search, the best fit was obtained with a monomer-dimer-tetramer (1-2-4) equilibrium, but also monomer-tetramer (1–4) and dimer-tetramer (2–4). Since monomers were only observed in presence of TCEP in SDS (Fig. 3), we propose that the 2–4 model as the most likely. In presence of TCEP (Fig. 4B), a similar result was observed. Since monomers are clearly observed in SDS, we chose the 1-2-4 model in this case. The distribution of these species depending on the protein-to-detergent ratio is shown in Fig. 4C, calculated using the protocol described previously [47, 48]. This data indicates that tetramer formation does not require the presence of disulfide-linked dimers, although whether tetramers with or without disulfide bonds are identical is not known.

Table 2

Affinity constants in the equilibrium monomer-dimer-tetramer.
Affinity constants	- TCEP	+ TCEP
K_a 1–2 (M^− 1)	3.867	2.791
K_a 2–4 (M^− 1)	5.034	6.697

Table 2. Decimal logarithm of the association constants for the 1-2-4 model: monomer-dimer (1–2) and dimer-tetramer (2–4) equilibrium for SARS-2 p7b.

For the TM of 7b (7b-TM), the best fits were 1–2, 1–3 and 2–4, but in presence of TCEP, model 1–2 was better than the other two, possibly due to the higher availability of monomer in this sample (Fig. 5A). The latter suggests that the TM domain alone can drive dimerization even in the absence of disulfide bonds, but tetramerization in p7b-TM (if any) probably requires formation of disulfide-linked dimers via Cys12. The differences between the two cases may be due to an increase in the dimer concentration when disulfide bonds are formed, or to a more favorable interaction mode between the helices in the dimer. Using the full length SARS-1 7b, tetramer formation does not require formation of disulfide bonds (Fig. 5B) and comparison of SARS-1 p7b TM and p7b in presence of TCEP suggest that monomers are more abundant in the first case. Overall, this suggests (i) a significant contribution to higher oligomers (tetramers) stability of the extramembrane domains and (ii) the helix-helix orientation in the dimer may be the same, with or without disulfide bonds.

Sedimentation velocity of 7b protein in detergent micelles.

Although SE is the AUC gold standard to determine molecular weights of membrane proteins, the ambiguity in the models proposed suggested the use of SV to complement the SE data. In the absence of reductant TCEP, SARS-2 p7b produced two bands in the c(s) distribution profile (Fig. 6A), one at 0.5–0.6 S, consistent with the size of a dimer (2-mer) and the other at 1.1 S consistent with a tetrameric form (4-mer), in principle consistent with the SE results (Fig. 4). In presence of TCEP, the feature at 1.1 S disappeared and the lower S species shifted to ~ 0.5 S, still consistent with a dimer. However, we note that this SV data could equally indicate a rapid equilibrium between monomers, dimers and tetramers, which woud have the appearence of intermediate species (dimer) in the c(s) plot [49]. In any case, this indicates that oligomerization does not strictly require disulfide bond formation. The fact that this band is shifted relative to the no-TCEP condition, further suggest sthat it may represent, not a dimer, but a fast equilibrium between the monomer, dimer and monomer [49]. Thus, two possible models emerge, formed either by interaction between (i) two non-disulfide-linked dimers brought together via disulfide interaction, or (ii) two disulfide-linked dimers. A similar pattern of dimers and tetramers were also observed in the case of SARS1 7b (Fig. 6B). Here, the result was slightly different because the larger S band was located in the ‘trimer’ region. As discussed above, this may represent an intermediate species resulting from the fast exchange between dimers and tetramers, reflecting the extramembrane domain differences between these two sequences and further supporting that the extramembrane domain is involved in the formation of the oligomer.

Model Building.

We generated dimers linked by Cys 12 in AlphaFold and neither of these tetrameric models produced a structure compatible with a channel (https://mole.upol.cz/).

Lipid bilayer permeabilization.

Planar membrane electrophysiology was used to test full-length SARS-2 7b protein ability to form channels in lipid bilayers. Addition of the protein diluted in ACN 50% induced only occasional large unstable currents lasting over minutes and presenting stepwise current transitions (Fig. 8). In general, these currents did not produce membrane rupture. p7b-induced current activity was observed at applied voltages ranging from ± 10 to ± 100 mV. Control experiments with ACN 50% alone did not produce any effect on the membrane. The conductance (G = I/V) measured during p7b-induced membrane permeabilization events was of several nanoSiemens, with typical conductance steps of 1–5 nS (Fig. 8). Such high conductances are comparable to those measured in wide porins with diameters of 1–2 nm [50, 51]. However, the instability observed in p7b-induced currents does not resemble that of porins or other channel forming proteins such as SARS-CoV-2 E protein [52], which show quieter currents. An explanation for the appearance of such large transient current levels –maybe related to high protein/lipid ratio, promoting a detergent-like action [53]– is out of the scope of the present work. Thus, the observed p7b-induced bilayer permeabilization evidences that the protein interacts with lipid membranes, but it is hardly compatible with the existence of bona fide ion channels based on a tetrameric assembly with several hydrophobic residues lining the narrow pore.

The precise role during infection of the group-specific accessory proteins encoded by SARS-CoVs remains incompletely understood. However, since SARS-CoV genomes encode the largest number of accessory proteins among coronaviruses, it is tempting to speculate that they have some important role in the clinical manifestation of infection. SARS-CoV 3a, 6, 7a, and 7b have transmembrane domains [20, 27, 54, 55]. 7a has a high structural similarity to the Ig-like superfamily proteins, although no significant sequence homology [56, 57], whereas 3a forms tetrameric ion channels [58] with a structure in lipid nanodiscs recently solved using cryo-EM [59]. In the present study we have investigated the behavior of 7b in both lipid and detergent environments to assess whether it forms oligomers with ion channel properties.

Overall, the conclusion from our data is that p7b is predominantly α-helical, although the extramembrane domain may form β-strands. This latter domain likely contributes to the stability of the oligomers.

Although oligomers are observed in SDS, this strong detergent and not a suitable environment to study the assembly of α-helices. However, it is clear that no monomers are present in the absence of reductant. This eliminates models involving monomers when fitting SE sedimentation profiles that use the milder detergent C14SB. Therefore, we suggest that the most likely model is an equilibrium between dimers and tetramers. When a reductant is present, only monomers were observed in SDS, and clear changes were also observed in SV experiments. This confirms that disulfide bonds are present in the sample, and that disruption of these bonds affects oligomerization, but does not prevent tetramer formation, since the best model in SE is 1-2-4. Here, the model may be complicated by having two types of dimers (linked or not linked by disulfide bonds), small contribution of trimers or larger oligomers, contribution of TCEP itself and so on. However, we feel it is out of the scope of this paper to characterize further such complex behavior.

Although the involvement, but not the requirement, of disulfide bonds in oligomerization is reminiscent of that of Influenza virus A M2 proton channel, no pore pathway was detected in any of the proposed tetrameric models. In addition, this is supported experimentally since the electrical activity detected in standard bilayers was not consistent with a very narrow tetrameric pore, suggesting that p7b does not form bona fide ion channels. Overall, the presence of DTT-resistant dimer in gel electrophoresis and in SE experiments in presence of reductant suggests that dimerization does not require disulfide bond formation, although the latter may stabilize it. In a similar system, the cysteine residue in the ζζ transmembrane domain was suggested to stabilize the dimeric form, only after formation of proper interface [60]. Overall, the role played by the 7b in the viral life cycle, and during infection of SARS-CoV is unclear. We provide a first glimpse of its oligomerizing behavior.

Ethics approval and consent to participate

Not applicable

Consent for publication

All authors have revised the manuscript prior to submission

Availability of data and materials

The data will be shared on a reasonable request to the corresponding author.

Competing interests

All authors declare no competing interests.

Funding

This study did not have specific funding (J.T.). M.Q. and V.A. acknowledge support from the Government of Spain MCIN/AEI/ 10.13039/501100011033 (project no. 2019-108434GB-I00 AEI/FEDER, UE and IJC2018-035283-I/AEI) and Universitat Jaume I (project no. UJI-A2020-21).

Authors' contributions

WS purified the peptides and performed most experiments, analyzed the data and contributed to writing the manuscript; MQ performed the channel activity experiments and MY performed the simulation. VA contribute to channel analysis and writing the manuscript. JT conceptualized the project, analyzed data and wrote the manuscript. All authors approved the final manuscript.

Acknowledgements

M.Q. and V.A. acknowledge support from the Government of Spain MCIN/AEI/ 10.13039/501100011033 (project no. 2019-108434GB-I00 AEI/FEDER, UE and IJC2018-035283-I/AEI) and Universitat Jaume I (project no. UJI-A2020-21). We thank Siok Wan Gan and Cin Huang Soon for the purification of SARS-1 p7b and p7b-TM.

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SARS-CoV 2 accessory protein 7b forms homotetramers in detergent

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Peptide purification and reconstitution.

Reconstitution in membranes.

Infrared spectroscopy.

Gel electrophoresis.

Analytical Ultracentrifugation (AUC).

Tetrameric SARS-2 p7b models in a lipid bilayer.

Electrophysiology in lipid bilayers.

Results

SARS-2 p7b is mostly α-helical in hydrated ERGIC-like lipid bilayers.

Oligomerization of SARS-2 p7b in SDS gels.

Sedimentation equilibrium of 7b protein in detergent micelles.

Sedimentation velocity of 7b protein in detergent micelles.

Model Building.

Lipid bilayer permeabilization.

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1