Single-molecule conformational dynamics of viroporin ion channels regulated by lipid-protein interactions

Classic swine fever is a highly contagious and often fatal viral disease that is caused by the classical swine fever virus (CSFV). Protein p7 of CFSV is a prototype of viroporin, a family of small, highly hydrophobic proteins postulated to modulate virus-host interactions during the processes of virus entry, replication and assembly. It has been shown that CSFV p7 displays substantial ion channel activity when incorporated into membrane systems, but a deep rationalization of the size and dynamics of the induced pores is yet to emerge. Here, we use high-resolution conductance measurements and current fluctuation analysis to demonstrate that CSFV p7 channels are ruled by equilibrium conformational dynamics involving protein-lipid interactions. Atomic force microscopy (AFM) confirms the existence of a variety of pore sizes and their tight regulation by solution pH. We conclude that p7 viroporin forms subnanometric channels involved in virus propagation, but also much larger pores (1–10 nm in diameter) with potentially significant roles in virus pathogenicity. Our findings provide new insights into the sources of noise in protein electrochemistry and demonstrate the existence of slow complex dynamics characteristic of crowded systems like biomembrane surfaces

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

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 (>1 nS) 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 homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses

The P. aeruginosa effector Tse5 forms membrane pores disrupting the membrane potential of intoxicated bacteria

The type VI secretion system (T6SS) of Pseudomonas aeruginosa injects effector proteins into neighbouring competitors and host cells, providing a fitness advantage that allows this opportunistic nosocomial pathogen to persist and prevail during the onset of infections. However, despite the high clinical relevance of P. aeruginosa, the identity and mode of action of most P. aeruginosa T6SS-dependent effectors remain to be discovered. Here, we report the molecular mechanism of Tse5-CT, the toxic auto-proteolytic product of the P. aeruginosa T6SS exported effector Tse5. Our results demonstrate that Tse5-CT is a pore-forming toxin that can transport ions across the membrane, causing membrane depolarisation and bacterial death. The membrane potential regulates a wide range of essential cellular functions; therefore, membrane depolarisation is an efficient strategy to compete with other microorganisms in polymicrobial environments.We gratefully acknowledge the Laboratories of Dr. Daniel Ladant (Institut Pasteur, Paris) and Dr. Victor de Lorenzo (Centro Nacional de Biotecnologia, Madrid) for the plasmids received (pKTop and pSEVA plasmids, respectively). Also, we would like to acknowledge the Laboratory of Dr. Joseph Mougous for the P. aeruginosa strains received. The technical assistance from Cristina Civantos and Adrian Ruiz is also very much appreciated. We acknowledge the FGCZ for the mass spectrometry analyses and the technical support (Functional Genomics Center Zurich (FGCZ), University/ETH Zurich). D.A.-J. acknowledges support by the MINECO Contracts CTQ2016-76941-R and PID2021-127816NB-I00, Fundacion Biofisica Bizkaia, the Basque Excellence Research Centre (BERC) programme, and IT709-13 and IT1745-22 of the Basque Government, and Fundacion BBVA. A.G.-M. acknowledges the financial support received from the Spanish Ministry of Universities and the Grants for the requalification of the Spanish university system for 2021-2023, financed by the European Union-Next Generation EU-Margarita Salas Modality. A.A. acknowledges support from the Spanish Ministry of Science and Innovation (Project 2019-108434GB-I00 funded by MCIN/AEI/10.13039/501100011033), Generalitat Valenciana (project AICO/2020/066) and Universitat Jaume I (project UJI-B2018-53). M.Q.-M. acknowledges support from the Spanish Ministry of Science and Innovation (Project IJC2018-035283-I funded by MCIN/AEI/10.13039/501100011033) and Universitat Jaume I (project UJI-A2020-21). P.B acknowledges the financial support received from the Spanish Ministry of Science and Innovation through the Ramon y Cajal Programme (contract RYC2019-026551-I)

Molecular recognition of the native HIV-1 MPER revealed by STED microscopy of single virions

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions

Classical Swine Fever Virus p7 Protein Interacts with Host Protein CAMLG and Regulates Calcium Permeability at the Endoplasmic Reticulum

We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural protein with a viroporin activity, a critical function in the progression of virus infection. We also identified p7 domains and amino acid residues critical for pore formation. Here, we describe how p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Detection of interaction as well as the identification of p7 areas mediating interaction with CAMLG was performed by yeast two-hybrid. p7-CAMLG interaction was further confirmed by confocal microscopy in eukaryotic cells, co-expressing both proteins. Mutant forms of p7 having substituted native residues identified as mediating interaction with CAMLG showed a decreased co-localization compared with the native forms of p7. Furthermore, it is shown that native p7, but not the mutated forms of p7 that fail to interact with CAMLG, efficiently mediates calcium permeability in the ER. Interestingly, viruses harboring some of those mutated forms of p7 have been previously shown to have a significantly decreased virulence in swine.ARS/USDA-University of Connecticut SCA# 58-1940-1-190 and ARS/USDA-University of the Basque Country NACA#8064-32000-056-18S

Molecular Recognition of the Native HIV-1 MPER Revealed by STED Microscopy of Single Virions

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.This study was supported by the Spanish MINECO (BIO2015-64421-R (MINECO/ FEDER UE) to J.L.N.) and the Basque Government (IT838-13 to J.L.N.). P.C., E.R., and S. I. received pre-doctoral fellowships from the Basque Government. P.C. would like to acknowledge the European Biophysical Societies’ Association (EBSA) for receiving an EBSA Bursary for a working visit to a laboratory in an EBSA country. J.C., D.W., and C. E. greatly acknowledge support by the MRC (grant number MC_UU_12010/unit programs G0902418 and MC_UU_12025), the Wellcome Trust (grant 104924/14/Z/14 and Strategic Award 091911 (Micron)), MRC/BBSRC/EPSRC (grant MR/K01577X/1), BBSRC (Deutsche Forschungsgemeinschaft (Research unit 1905 “Structure and function of the peroxisomal translocon”)), the Wolfson Foundation (for initial funding of the Wolfson Imaging Centre Oxford), the EPA Cephalosporin Fund and the John Fell Fund. T.S. is a recipient of a Canada Graduate Scholarship Master’s Award and a Vanier Canada Graduate Scholarship from the Canadian Institutes of Health Research. This work was supported by operating grant NIH-150414 (J.-P.J.) from the Canadian Institutes of Health Research. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program (J.-P.J.). We acknowledge valuable technical assistance from Miguel García-Porra

Molecular Characterization of the Viroporin Function of Foot-and-Mouth Disease Virus Nonstructural Protein 2B

Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains

Focal accumulation of aromaticity at the CDRH3 loop mitigates 4E10 polyreactivity without altering its HIV neutralization profile

Broadly neutralizing antibodies (bnAbs) against HIV-1 are frequently associated with the presence of autoreactivity/polyreactivity, a property that can limit their use as therapeutic agents. The bnAb 4E10, targeting the conserved Membrane proximal external region (MPER) of HIV-1, displays almost pan-neutralizing activity across globally circulating HIV-1 strains but exhibits nonspecific off-target interactions with lipid membranes. The hydrophobic apex of the third complementarity-determining region of the heavy chain (CDRH3) loop, which is essential for viral neutralization, critically contributes to this detrimental effect. Here, we have replaced the aromatic/hydrophobic residues from the apex of the CDRH3 of 4E10 with a single aromatic molecule through chemical modification to generate a variant that preserves the neutralization potency and breadth of 4E10 but with reduced autoreactivity. Collectively, our study suggests that the localized accumulation of aromaticity by chemical modification provides a pathway to ameliorate the adverse effects triggered by the CDRH3 of anti-HIV-1 MPER bnAbs.This study was supported by the following Grants: European Commission (790012 SI H2020-MSCA-IF-2017) (E.R.); US NIAID, NIH grant R01 AI143563 (M.B. Z.); James B. Pendleton Charitable Trust (M.B.Z.); JSPS grant 20H03228 (J. M.M.C.); Spanish MCIU (RTI2018-095624-B-C21; MCIU/AEI/FEDER, UE) (J.L.N.), Basque Government (IT1196-19) (J.L.N.). C.E. acknowledges funding from Medical Research Council (grant number MC_UU_12010/unit programs G0902418 and MC_UU_12025), Wolfson Foundation, Deutsche Forschungsgemeinschaft (Excellence Cluster Balance of the Microverse, Collaborative Research Center 1278 Polytarget), Leibniz Association (Leibniz Campus Infectooptics), Wellcome Institutional Strategic Support Fund, Oxford internal funds (EPA Cephalosporin Fund and John Fell Fund), and support from the Micron Oxford Advanced Bioimaging Unit (Wellcome Trust funding 107457/Z/15/Z). This work was also supported by the Platform Project for Supporting Drug Discovery and Life Science Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED (JP21am0101091). S.I. received a predoctoral fellowship from the BasqueGovernment. P.C. would like to acknowledge the University of the Basque Country (DOCREC18/01), the Basque Government (POS_2018_1_0066) and the European Commission (H2020-MSCA-IF-2019-ST project 892232 FILM-HIV) for funding his position. This research was also supported by the CIFAR Azrieli Global Scholar program (J-P.J.), the Ontario Early Researcher Awards program (J-P.J.), and the Canada Research Chairs program (J-P.J.). Part of the biophysical data presented in this manuscript were collected at the Hospital for Sick Children Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fund

Degradación de alcanos mediante Alcanivorax venutensis inmovilizada en hidrogeles adhesivos y biodegradables

188 p.El petróleo y sus derivados son los mayores contaminantes del medio marino. Además puntualmente se producen grandes vertidos accidentales como el que en Noviembre de 2002 produjo un desastre ecológico sin precedentes en la costa norte de la Península Ibérica. En este trabajo se ha desarrollado y se ha ensayado a escala de laboratorio un producto de biorremediación para zonas rocosas contaminadas con restos de hidrocarburos procedentes de vertidos. Se ha utilizado para el producto un soporte polimérico y biodegradable formado por quitosano y goma xantano, en el que se han inmovilizado células de Alcanivorax venustensis. Tanto el soporte como la biomasa se han utilizado en forma liofilizada, facilitando el uso y conservación del producto. Se ha caracterizado el efecto de diferentes parámetros sobre la degradación de alcanos por parte de las células de Alcanivorax venustensis inmovilizadas. También se ha estudiado el posible carácter inducible del proteoma de Alcanivorax venustensis para la degradación de hidrocarburos, encontrando proteínas con una expresión diferencial cuando la biomasa crece en presencia y ausencia de hidrocarburos. Estas proteínas están implicadas en el proceso de biodegradación de los alcanos, y su expresión, es inducible por la presencia de estos en el medio de cultivo durante el crecimiento

Degradación de alcanos mediante Alcanivorax venutensis inmovilizada en hidrogeles adhesivos y biodegradables

188 p.El petróleo y sus derivados son los mayores contaminantes del medio marino. Además puntualmente se producen grandes vertidos accidentales como el que en Noviembre de 2002 produjo un desastre ecológico sin precedentes en la costa norte de la Península Ibérica. En este trabajo se ha desarrollado y se ha ensayado a escala de laboratorio un producto de biorremediación para zonas rocosas contaminadas con restos de hidrocarburos procedentes de vertidos. Se ha utilizado para el producto un soporte polimérico y biodegradable formado por quitosano y goma xantano, en el que se han inmovilizado células de Alcanivorax venustensis. Tanto el soporte como la biomasa se han utilizado en forma liofilizada, facilitando el uso y conservación del producto. Se ha caracterizado el efecto de diferentes parámetros sobre la degradación de alcanos por parte de las células de Alcanivorax venustensis inmovilizadas. También se ha estudiado el posible carácter inducible del proteoma de Alcanivorax venustensis para la degradación de hidrocarburos, encontrando proteínas con una expresión diferencial cuando la biomasa crece en presencia y ausencia de hidrocarburos. Estas proteínas están implicadas en el proceso de biodegradación de los alcanos, y su expresión, es inducible por la presencia de estos en el medio de cultivo durante el crecimiento