Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses

1 bPlum Island Animal Disease Center, ARS, USDA, Greenport, NY 11944, USA. cLaboratory of Molecular Biophysics. Department of Physics. University Jaume I, 12071 Castellón, Spain *Corresponding author: E-mail: [email protected] Mutation-induced changes of transmembrane pore size revealed by combined ionchannel conductance and single vesicle permeabilization analyses. Eneko Largoa, Douglas P. Gladueb, Johana Torralbaa, Vicente M. Aguilellac, Antonio Alcarazc, Manuel V. Borcab, and José L. Nievaa* aBiofisika Institute (CSIC, UPV/EHU) and Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao, Spain. This is the accepted manuscript of the article that appeared in final form in Biochimica et Biophysica Acta (BBA) - Biomembranes 1860(5) : 1015-1021 (2018), which has been published in final form at https://doi.org/10.1016/j.bbamem.2018.01.012. © 2018 Elsevier under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 Abstract: Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (> 1nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homooligomerization, suggests prospective strategies to block/attenuate pestiviruses. Key words: pore-forming peptide; membrane permeabilization; ion channel; ER membrane; peptide-lipid interaction 3 1. Introduction Unregulated pore formation in cell membranes results in the alteration of the ionic homeostasis, the loss of electrochemical gradients and ultimately cell death. Secreted pore-forming proteins or peptides targeting membranes constitute molecular weapons produced by organisms of diverse origin, and are also part of ancestral immune systems, which are functional in defending against pathogen invasion [1-6]. Membrane specificity of pore formation can be in addition exploited as the basis for antibiotic and anti-cancer drug development [7-9]. Thus, unravelling the molecular mechanisms that sustain the formation of permeating pores and govern their functioning in lipid bilayers remains a highly relevant research issue [3, 10-13]. Poration of the plasma membrane and the different components of the endomembrane system is also a significant issue in the cell infection cycle of many viruses. Formation by virally encoded pore-forming proteins (designated as “viroporins”[14]), of poorly-selective, ion-conductive channels has been proved instrumental in uncoating, transport and maturation of viral particles, but can also influence spread and pathogenicity [15, 16]. A conspicuous organelle targeted by viroporins is the Endoplasmic Reticulum (ER) [17]. Besides its taking part in the different stages of viral replication and assembly, permeabilization of this organelle can modulate several viral functions and plays an important role in host cell-death [15, 17]. Thus, ER permeabilization by pore-forming viral products is a biologically relevant phenomenon, but studies addressing the mechanisms of pore formation in ER model membranes are to a great extent missing. To address this issue, we have recently modelled the ER permeabilization phenomenon combining: (i) membranes that emulated the ER lipid composition; and 4 (ii) the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 product [18-20]. In the one hand, even though the ER is the main site of synthesis of sterols and complex sphingolipids, the ER membrane displays only low concentrations of these lipid species at localized sites [21]. Accordingly, the overall ER membrane can be fairly modelled by a combination of its main constituent phospholipids: zwitterionic PC and PE plus the anionic PI mixed in a roughly 5:3:2 molar ratio [21]. On the other hand, CSFV p7 is a small, hydrophobic protein of approximately 60-70 amino acids, which displays membrane-porating activity and forms homo-oligomers that mainly localize to the ER [15, 18, 19, 22]. Large Unilamellar vesicle (LUV) permeability assays mapped the p7 porating domain to its C-terminal transmembrane helix, while the addition of the preceding polar segment conferred pH dependence and sensitivity to channel blockers [19]. These observations were further supported by functional measurements of ionconducting channel (IC) activity in ER-like planar lipid membranes [20], which confirmed that a sequence combining the polar segment and the C-terminal helix, designated as p7C, comprises the pore-forming domain of the protein. Here, to gain insights into the mechanisms underlying ER permeabilization by viral products and its potential relation to virulence, we have focused on the effect of point mutations involving strictly conserved residues within the cytosolic loop and transmembrane helix of CSFV p7C [18]. We have followed an unprecedented combined approach to analyze pore formation, namely, IC activity measurements in planar bilayers and determination of the permeabilization degrees of individual vesicles by fluorescence microscopy imaging. Our combinational approach provided evidence sustaining the formation in ER-like membranes of two types of p7C pores with approximate diameters of 1 and 5 nm. Moreover, the observation that a mutation favoring the small pores hampered virus production without affecting ER localization, 5 points to the large pore structures as potential targets for blocking pestivirus propagation and pathogenicity. 2.Materials and Methods 2.1 Materials – Synthetic peptides p7C-wt, p7C-FH, and p7C-KK (sequences displayed in Fig 1A) were produced as previously described [18, 19]. Phosphatidylcholine (PC), phospahtidylethanolamine (PE), phospahtidylethanolamineN-(lissamine rhodamine B sulfonyl) (Rho-PE), and phosphatidylinositol (PI) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). The 8-aminonaphtalene1,3,6-trisulfonic acid sodium salt (ANTS), p-xylenebis(pyridinium)bromide (DPX), phenol 4-[5-(4-methyl-1-piperazinyl)[2,5'-bi-1H-benzimidazol]-2'-yl]- trihydrochloride (Hoechst 33258), and Alexa Fluor 488 were obtained from Molecular Probes (Junction City, OR, USA). Rabbit polyclonal antibody against GFP (FL) conjugated with horseradish peroxidase (HRP) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Plasmid containing mCh-Sec61 beta was a gift from Gia Voeltz (Addgene plasmid # 49155) [23]. 2.2 Monolayer penetration assays – Penetration into lipid monolayers was measured to compare the capacity of p7 peptides for inserting into membranes that mimic the ER [19]. In brief, maximal changes in surface pressure were monitored as a function of initial surface pressure (π0) in a fixed-area circular trough (μTrough S system, Kibron, Helsinki) measuring 2 cm in diameter and with a volume of 1.25 ml. The aqueous phase consisted of 1 ml of 5 mM NaOAc, 100 mM NaCl (pH 5.0). Lipids, dissolved in chloroform, were spread over the surface and the desired π0 was attained by changing the amount of lipid applied to the air-water interface. For allowing incorporation into 6 the monolayer, peptides were injected into the aqueous subphase with a Hamilton microsyringe. 2.3 Vesicle permeability assays – ER-like large unilamellar vesicles (LUV) were prepared according to the extrusion method [24]. Vesicle permeabilization was assayed by monitoring the release to the medium of encapsulated fluorescent ANTS (ANTSDPX assay) [25]. LUV containing 12.5 mM ANTS, 45 mM DPX, 20 mM NaCl and 5 mM Hepes were obtained by separating the unencapsulated material by gel-filtration in a Sephadex G-75 column that was eluted with 5 mM Hepes and 100 mM NaCl (pH 7.4). Internal and external osmolarities were measured in a cryoscopic osmometer (Osmomat 030, Gonotec, Berlin, Germany) and adjusted by adding NaCl. Fluorescence measurements were performed in an SLM Aminco 8100 spectrofluorimeter (Spectronic Instruments, Rochester, NY) by setting the ANTS emission at 520 nm and the excitation at 355 nm. A cutoff filter (470 nm) was placed between the sample and the emission monochromator. The baseline leakage (0%) corresponded to the fluorescence of the vesicles at time 0, while 100% leakage was the fluorescence value obtained after addition of Triton X-100 (0.5% v/v). 2.4 Planar lipid membranes formation –. Two monolayers were made from 5 mg/ml pentane solutions of lipid mixture buffered with 5 mM NaOAc with 150 mM KCl at both sides of Teflon chambers partitioned by a 15 m thick Teflon film with 70-100 m diameter orifices. Planar lipid bilayers were formed by monolayer apposition on the orifices previously treated with a 1% solution of hexadecane in pentane. Protein and peptides dissolved in DMSO were supplemented to the lipid solutions prior to monolayer formation only in one of the chamber sides, the cis side. Bilayer formation was directly detected and its thickness can be estimated by capacitance measurements. 7 2.5 Channel conductance measurements – An electric potential was applied using Ag/AgCl electrodes in 2 M KCl, 1.5% agarose bridges assembled within standard 250 µl pipette tips. Potential is defined as positive when it is higher at the side of the protein addition (the cis side), while the trans side is set to ground. An Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) in the voltage-clamp mode was used for measuring the current and applying potential. The membrane chamber and the head stage were isolated from external noise sources with a double metal screen (Amuneal Manufacturing Corp., Philadelphia, PA). For each sample, at least 50 different traces were typically recorded (recording time for each trace was 200 s). A rough first estimation of pore diameter considered a cylindrical neutral pore. Thus, channel conductance G can be written in terms of solution conductivity  , and pore dimensions, according to the following equation: 2 4  D GL (1) where L and D stand for length and diameter, respectively. 2.6 Single vesicle permeabilization –.For the single vesicle approach, Giant Unilamellar Vesicles (GUVs) made of PC:PE:PI:Rho-PE (50:30:20:0.1 mole ratio) were prepared according to the electroformation method as described in previous works [26, 27]. Confocal fluorescence microscopy images of individual GUVs were obtained in a commercial Nikon D Eclipse TE2000-U fluorescence microscope (Nikon Instruments, Tokyo, Japan). Image processing and analyses were carried out with ImageJ (rsb.info.nih.gov/ij/). Extents of permeabilization were calculated after incubation with Alexa Fluor 488 and 0.2 μM of p7 peptides from the ratio of fluorescence intensity inside and outside each vesicle. Exposure of GUVs to peptide for 8 30 min or 2 hours rendered essentially the same proportion of vesicles permeabilized, and the same levels of permeabilization per vesicle, therefore indicating that the system was at equilibrium at the times selected for performing the measurements (usually 30 min). 2.7 Cell expression of recombinant p7 –.For cell expression of GFP-p7 fusion proteins, 293T cells (2 x 105 cells) were co-transfected with plasmids encoding GFP-p7 fusions and mCh-Sec61 beta (1 µg each) using calcium phosphate [17]. Under these conditions 70-80% of the cells in the culture were successfully transfected. At 36 h post-transfection, cells were fixed with 4% formaldehyde in phosphate saline buffer (PBS) and incubated with Hoechst dye. Confocal images were acquired on a Leica TCS SP5 II microscope (Leica Microsystems GmbH, Wetzlar, Germany), using a x63 water-immersion objective. For the oligomerization assays, 293T cells (1.5 x 106 cells) were transfected with 10 µg of plasmid encoding GFP-p7 constructs. At 36 h posttransfection, cells were collected in cold PBS, sonicated for 1 min on ice with a probe tip sonicator (MSE Soniprep 150, MSE, UK) and dissolved in SDS-PAGE loading buffer. Oligomers were detected by immunoblot analysis using anti-GFP antibody. 2.8 Construction of CSFV mutants –. A full-length infectious clone of the virulent Brescia strain (pBIC) [28] was used as a template to obtain all cDNA as described [18]. pBICv-p7-KK (p7 residues 39KK40 to EE) and pBICv-p7-FH (p7 residues 46FH47 to AA) constructs containing desired mutations in the genomic area encoding for p7 protein were obtained using the QuickChange XL Site-Directed Mutagenesis kit (Stratagene, San Diego, CA). Presence of virus infected cells was detected by immunoperoxidase staining utilizing the CSFV monoclonal antibody WH303 (mAb 9 WH303) [29] and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). 16 EE mutation at the cytosolic loop revealed a more diffuse intracellular fluorescent labeling, a pattern resembling cells expressing the GFP control. Accordingly, this latter construct did not co-localize significantly with mCh-Sec61 (Fig 5B). To test the oligomeric state of the expressed proteins, SDS-PAGE analyses were subsequently performed on extracts of the transfected cells (Fig 5C). Immunoblots probed with the anti-GFP antibody showed bands consistent with the formation of highorder homo-oligomers in cells expressing the GFP-p7 and GFP-p7-FH constructs (arrow). Even though a band consistent with the formation of homo-oligomers was also found in cells expressing the GFP-p7-KK construct, the band corresponding to the higher-order homo-oligomers was absent in these samples. From the results displayed in Figures 5A-C the following conclusion can be drawn: whereas the FH x AA mutation does not alter the biogenesis of the viroporin p7, the KK x EE mutation appears to interfere with insertion into the ER membrane and oligomerization therein. Finally, to evaluate the effect of the amino acid substitutions 39KK40-EE and 46FH47AA in the replication of CSFV, two recombinant CSF viruses containing the corresponding amino acid substitutions in the pore-forming domain of p7 were produced using the cDNA infectious clone of the Brescia strain (BICv) as a template. Mutated cDNA infectious clones pBICv-p7-KK and pBICv-p7-FH were developed by site directed mutagenesis. Infectious RNA was in vitro transcribed from each mutated full-length cDNA and used to transfect SK6 cells [18]. Infectious virus was rescued from transfected cells by day 4 post-transfection using the pBIC construct encoding for the parental virus BIC with titers reaching almost 107 TCID50/ml. In contrast, after four independent transfection procedures, pBICv-p7-KK and pBICv-p7-FH constructs did not produce 17 infectious viruses. Real-time RT-PCR analysis of total RNA extracted from cells transfected with pBICv-p7-KK and pBICv-p7-FH constructs revealed genomic RNA replication, (data not shown). In addition, immunohistochemistry analysis of transfected cell monolayers was performed using a monoclonal antibody recognizing CSFV structural glycoprotein E2 (Fig. 5D). As expected, cell monolayers transfected with pBIC-p7 showed a large number of cells expressing massive levels of E2 (right-most panel). Conversely, pBICv-p7-FH transfected cells showed the presence of small and isolated foci of cells expressing structural glycoprotein E2, while there was a complete absence of E2 expression in cells transfected with pBICv-p7-KK construct (mid panels). Attempts to rescue infective virus from cell cultures transfected with either pBICv-p7-KK or pBICv-p7-FH were performed by four successive blind passages in fresh monolayers of SK6 cells. No infectious virus could be detected in any of the four passages for either of the constructs tested. Therefore, residues 46FH47 and 39KK40 in the pore-forming domain of p7 appear to be critical for CSFV replication. 18 4. Discussion ER permeabilization is a pivotal event required for the progression of the cell replication cycle of many viruses [15, 17]. The CSFV p7 product has been shown to permeabilize ER-like membranes following a lipid-dependent pattern [18, 19], and to localize in the ER upon expression [22], and hence constitutes an optimal model to study this phenomenon. Following strategies described previously [33, 35], in a preceding work we established a minimal channel structure represented by p7C-wt [19]. Those studies pinpointed the polar, cytosolic loop 33MRDEPIKK40, as a regulatory element that conferred pH and inhibitor sensitivity to the pore, and the C-terminal helix 41WILLLFHAMTNNPVKTITVALLMVSGV67 as the actual pore-forming domain. The p7C-wt peptide combined both elements and was simultaneously competent in conducting ions across lipid bilayers mimicking the ER, and in allowing release of small solutes (ANTS/DPX) from ER-LUVs [20]. Motivated by the observation that the invariant residues 39KK40 and 46FH47 were located within these functional regions [18], we sought to analyze the effect of non-conservative KK x EE and FH x AA double substitutions on the pore-forming activity of p7C-wt. Since the cytosolic loop invests the pore domain of p7 with sensitivity to pH and inhibitors [19, 20], we expected assembly of less regulated pores in membranes be the consequence of substituting the conserved Lys39-Lys40 residues by the negatively charged Glu-Glu dipeptide. However, contrary to our expectations, incorporating the KK x EE mutation in the p7C-KK peptide resulted in defective monolayer penetration and absence of membrane permeabilization (Figs 1-4). These findings suggest that the loop not only regulates permeability but also conditions insertion into ER membranes. 19 In consonance with this limiting defect, the same mutation resulted in an anomalous distribution and defective homo-oligomerization of p7 upon cell expression (Fig. 5AC). Interestingly, the suppressing effect of a cognate mutation in HCV p7 has been related to alterations in membrane-topology [36]. From molecular dynamics simulations, it was inferred that the basic residues sitting in the membrane interface could engage electrostatic interactions with negatively charged lipid polar head groups to properly orient the TM helices [33, 37]. Thus, we infer that perturbing the set of interactions between basic Lys-Lys residues and negatively charged PI at the membrane interface, may also result in the inefficient insertion/pore-formation of CSFV p7. Finally, highlighting the crucial role of pore-forming p7 protein in the CSFV replication cycle, the KK x EE mutation also suppressed production of infectious virus (Fig 5D), an observation in line with previous results obtained for p7 of the BVDV pestivirus [38] and our own Ala-scanning mutagenesis [18]. In contrast, the p7C-FH peptide, displayed less capacity for inserting into monolayers than p7C-wt, but retained the membrane-permeabilizing capacity of the parental sequence measured in ANTS/DPX-LUV assays (Fig. 1). However, complementation of these bulk measurements with IC activity and single ER-GUV permeability determinations revealed functional differences between both sequences (Figs. 2-4). Electrophysiogical recordings disclosed two distinct phenotypic traits: co-existence of small and large conductance channels in p7C-wt, and predominance of small apertures in the p7C-FH. Similarly, single-channel recordings of Hepatitis C virus p7 protein by the patch clamp technique revealed distinct conductance values of 35, 57, 120, and 184 pS [33]. It was argued that large conductance levels could represent either insertion of simultaneous channels, or an increase in the number of p7 monomers per channel and hence, a wider current passage. Under our measuring conditions, we observe 20 comparable conductance levels of 120 pS for both p7C-wt and p7C-FH, which would be compatible with channel sizes allowing release of ANTS/DPX, explaining the similar porating capacities observed in the LUV-based assay. However, in the case of the p7C-wt peptide, these structures co-existed with channels giving rise to higher conductance (> 1 nS) whose origin was uncertain. To address the origin of the different stages observed in the membrane permeabilization induced by p7C-wt, the process was imaged at the individual vesicle level by fluorescence microscopy of ER-GUVs (Figs 3 and 4). These objects attain cell dimensions and, when applied to permeability assays, the single GUV approach offers several advantages as compared to measurements in bulk (i.e., LUV-based ANTS/DPX assays). Firstly, owing to the fluorescent lipid label, their surrounding lipid bilayer can be readily observed by confocal microscopy, which allows monitoring its overall stability upon treatment with the permeabilizing agent, i.e., one can discern membrane permeabilization through discrete pores vs. lipid bilayer disaggregation by detergentlike mechanisms [3, 10]. Secondly, GUV membranes can be considered as flat planes devoid of curvature stress, i.e., they mimic the conditions of the planar bilayers used in electrophysiological experiments more closely than LUVs. And thirdly, one can discern solute-encapsulation heterogeneities that may arise in the population of permeabilized vesicles, which go unnoticed in bulk measurements [26]. Explicitly, in the case of totally permeabilized GUVs, the open state of the pore lasts enough as to allow the equilibration of the probe with the external medium. Both, p7Cwt and p7C-FH permeabilized ER-GUVs to alexa-488 and 4 kDa-dextran according to this mechanism (Fig. 4B). However, the number of totally permeabilized ER-GUVs dropped for larger dextrans in the case of p7C-FH, but not in the case of p7C-wt (Fig. 21 4C). This observation suggests that stably opened pores of larger dimensions were only accessible to the wt sequence. On the other hand, the partial permeabilization process seems to be related to transient membrane lesions of the bilayer permeability that do not allow rapid equilibration with the external solution. The fact that the number of ERGUVs partially permeabilized was marginal in the p7C-wt and p7C-FH samples (Fig 4C), underscores the prevalent role of stable channel-pore structures in mediating IC activity and dye influx into GUVs. 22 5. Conclusions In conclusion, our IC activity and GUV permeabilization data provide a clear demonstration for assembling channel-pores with distinct aperture widths by the porating domain of p7 CSFV in ER-mimicking membranes. To our best knowledge, this is the first time that electrophysiological and single-vesicle approaches are combined to solve the permeabilization mechanism of a pore-forming product functioning in the context of ER membranes. Notably, the FH x AA mutation favoring small-size pores abrogated virus production without altering the cell distribution or oligomerization degree of p7 upon expression (Fig. 5). Further research efforts will be required to get insight into the distinct physiological roles of the p7C capacity to form pores of different dimensions, and we foresee that the FH x AA mutation described in this work may provide a useful tool in the context of cell infection by pestiviruses. It is tempting to speculate that manipulating pore-forming function of p7 by mutagenesis may provide in the future new approaches to vaccine and anti-viral development to treat pestivirus infections. 23 Acknowledgements This study was in part supported by the Agricultural Research Service of the US (ARSUSDA Project 8064-32000056-18S to EL and JLN) and the Basque Government (Project IT838-13 to JLN). Financial support from the Ministry of Economy and Competitiveness of Spain (projects no. FIS2013-40473-P and FIS2016-75257-P), and Universitat Jaume I (project no. P1.1B2015-28) is also acknowledged. 24 References: [1] D.M. Ojcius, J.D. 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