Viroporin-mediated Membrane Permeabilization

Abstract

Enterovirus nonstructural 2B protein is involved in cell membrane permeabilization during late viral infection. Here we analyze the pore forming activity of poliovirus 2B and several of its variants. Solubilization of 2B protein was achieved by generating a fusion protein comprised of poliovirus 2B attached to a maltose-binding protein (MBP) as an N-terminal solubilization partner. MBP-2B was assayed using large unilamellar vesicles as target membranes. This fusion protein was able to assemble into discrete structures that disrupted the permeability barrier of vesicles composed of anionic phospholipids. The transbilayer aqueous connections generated by MBP-2B were stable over time, allowing the passage of solutes of molecular mass under 1,000 Da. Oligomerization was investigated using fluorescence resonance energy transfer. Our data indicate that MBP-2B aggregation occurs at the membrane surface. Moreover, MBP-2B binding to membranes promoted the formation of SDS-resistant tetramers. We conclude that MBP-2B forms oligomers capable of generating a tetrameric aqueous pore in lipid bilayers. These findings are the first evidence of viroporin activity shown by a protein from a naked animal virus. Enterovirus nonstructural 2B protein is involved in cell membrane permeabilization during late viral infection. Here we analyze the pore forming activity of poliovirus 2B and several of its variants. Solubilization of 2B protein was achieved by generating a fusion protein comprised of poliovirus 2B attached to a maltose-binding protein (MBP) as an N-terminal solubilization partner. MBP-2B was assayed using large unilamellar vesicles as target membranes. This fusion protein was able to assemble into discrete structures that disrupted the permeability barrier of vesicles composed of anionic phospholipids. The transbilayer aqueous connections generated by MBP-2B were stable over time, allowing the passage of solutes of molecular mass under 1,000 Da. Oligomerization was investigated using fluorescence resonance energy transfer. Our data indicate that MBP-2B aggregation occurs at the membrane surface. Moreover, MBP-2B binding to membranes promoted the formation of SDS-resistant tetramers. We conclude that MBP-2B forms oligomers capable of generating a tetrameric aqueous pore in lipid bilayers. These findings are the first evidence of viroporin activity shown by a protein from a naked animal virus. Enteroviruses are nonenveloped animal viruses belonging to the Picornaviridae family. Infection by species of theEnterovirus genus leads to significant alterations in cell membranes, including morphological changes and functional modifications (for review, see Refs. 1Carrasco L. Adv. Virus Res. 1995; 45: 61-112Google Scholar and 2Carrasco L. Guinea R. Irurzun A. Barco A. Semler B. Wimmer E. The Picornaviruses. American Society for Microbiology Press, Washington, D.C.2002: 337-354Google Scholar). Of the cellular alterations induced by viral infection, enhancement of plasma membrane permeability is among the most relevant. Increased membrane permeability occurs at two well defined times during the viral infection cycle: at an early stage, when virus particles enter cells, and late during infection, when most viral products are being synthesized (1Carrasco L. Adv. Virus Res. 1995; 45: 61-112Google Scholar, 2Carrasco L. Guinea R. Irurzun A. Barco A. Semler B. Wimmer E. The Picornaviruses. American Society for Microbiology Press, Washington, D.C.2002: 337-354Google Scholar, 3Carrasco L. Perez L. Irurzun A. Lama J. Martinez-Abarca F. Rodriguez P. Guinea R. Castrillo J.L. Sanz M.A. Ayala M.J. Carrasco L. Sonenberg N. Wimmer E. Regulation of Gene Expression in Animal Viruses. Plenum Publishing Corp., London1993: 283-305Google Scholar). Late membrane leakiness, occurring from the 3rd h postinfection, requires viral gene expression. Initially it involves the diffusion of ions and small molecules, but not macromolecules, through the plasma membrane (4Carrasco L. Smith A.E. Nature. 1976; 264: 807-809Google Scholar, 5Carrasco L. Nature. 1978; 272: 694-699Google Scholar). Intense efforts have been made to unveil the molecular mechanisms underlying this process. Some years ago, it was argued that the intracellular accumulation of viral particles and/or massive expression of viral products might be the triggering factors for enhanced permeability and eventual lysis of the cell. Contrary to this idea, cumulative evidence suggests that the sole expression of certain enterovirus proteins might be responsible for the increase in membrane permeability (6Lama J. Carrasco L. J. Biol. Chem. 1992; 267: 15932-15937Google Scholar, 7Aldabe R. Barco A. Carrasco L. J. Biol. Chem. 1996; 271: 23134-23137Google Scholar, 8Van Kuppeveld F.J.M. Hoenderop J.G.J. Smeets R.L.L. Willems P.H.G.M. Kijkman H.B.P.M. Galama J.M.D. Melchers W.J.G. EMBO J. 1997; 16: 3519-3532Google Scholar). Particularly, the individual expression of 2B and 2BC proteins of coxsackie virus B3 and poliovirus has been shown to reproduce several late infection effects, such as changes in plasma membrane permeability (7Aldabe R. Barco A. Carrasco L. J. Biol. Chem. 1996; 271: 23134-23137Google Scholar, 8Van Kuppeveld F.J.M. Hoenderop J.G.J. Smeets R.L.L. Willems P.H.G.M. Kijkman H.B.P.M. Galama J.M.D. Melchers W.J.G. EMBO J. 1997; 16: 3519-3532Google Scholar, 9Doedens J.R. Kirkegaard K. EMBO J. 1995; 14: 894-907Google Scholar), increased cytoplasmic calcium ion levels (8Van Kuppeveld F.J.M. Hoenderop J.G.J. Smeets R.L.L. Willems P.H.G.M. Kijkman H.B.P.M. Galama J.M.D. Melchers W.J.G. EMBO J. 1997; 16: 3519-3532Google Scholar, 10Aldabe R. Irurzun A. Carrasco L. J. Virol. 1997; 71: 6214-6217Google Scholar), inhibition of protein secretion (9Doedens J.R. Kirkegaard K. EMBO J. 1995; 14: 894-907Google Scholar, 11Barco A. Carrasco L. EMBO J. 1995; 14: 3349-3364Google Scholar, 12Van Kuppeveld F.J.M. Melchers W.J.G. Kirkegaard K. Doedens J.R. Virology. 1997; 227: 111-118Google Scholar), and disassembly of the Golgi complex (11Barco A. Carrasco L. EMBO J. 1995; 14: 3349-3364Google Scholar, 13Sandoval I.V. Carrasco L. J. Virol. 1997; 71: 4679-4693Google Scholar). Because of their capacity to alter the permeability barrier of cell membranes, these enteroviral proteins have been proposed to represent the first examples of “viroporins” from naked viruses (7Aldabe R. Barco A. Carrasco L. J. Biol. Chem. 1996; 271: 23134-23137Google Scholar, 8Van Kuppeveld F.J.M. Hoenderop J.G.J. Smeets R.L.L. Willems P.H.G.M. Kijkman H.B.P.M. Galama J.M.D. Melchers W.J.G. EMBO J. 1997; 16: 3519-3532Google Scholar, 9Doedens J.R. Kirkegaard K. EMBO J. 1995; 14: 894-907Google Scholar, 12Van Kuppeveld F.J.M. Melchers W.J.G. Kirkegaard K. Doedens J.R. Virology. 1997; 227: 111-118Google Scholar, 14Barco A. Carrasco L. J. Virol. 1998; 72: 3560-3570Google Scholar, 15Van Kuppeveld F.J.M. Galama J.M.D. Zoll J. Van den Hurk P.J.J.C. Melchers W.J.G. J. Virol. 1996; 70: 3876-3886Google Scholar). Enterovirus 2B proteins are hydrophobic overall and rather small (∼100 amino acids). Hydrophobicity within the 2B sequence appears to be distributed across two main domains (2Carrasco L. Guinea R. Irurzun A. Barco A. Semler B. Wimmer E. The Picornaviruses. American Society for Microbiology Press, Washington, D.C.2002: 337-354Google Scholar, 15Van Kuppeveld F.J.M. Galama J.M.D. Zoll J. Van den Hurk P.J.J.C. Melchers W.J.G. J. Virol. 1996; 70: 3876-3886Google Scholar): a first stretch (amino acids 32–55 in poliovirus), which is partially amphipathic as a cationic α-helix at the N terminus (amino acids 34–49 in poliovirus), and a second hydrophobic region, which might constitute a transmembrane domain (TMD) 1The abbreviations used are: ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid sodium salt; DOPG, dioleoylphosphatidylglycerol; DPPG, dipalmitoylphosphatidylglycerol; DPX, p-xylenebis(pyridinium)bromide; FRET, fluorescence resonance energy transfer; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; IANBD, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine; Kx , apparent mol fraction partition coefficient; LUV(s), large unilamellar vesicle(s); MBP, maltose-binding protein; N-NBD-PE, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; POPG, 1-palmitoyl-2-oleoylphosphatidylglycerol; PS, phosphatidylserine; SAPG, 1-stearoyl-2-arachidonoylphosphatidylglycerol. (amino acids 61–81 in poliovirus). This distribution seems to be functionally meaningful because mutations altering either first domain amphipathicity or second domain hydrophobicity have been shown to interfere with the ability of 2B to increase permeability (7Aldabe R. Barco A. Carrasco L. J. Biol. Chem. 1996; 271: 23134-23137Google Scholar, 8Van Kuppeveld F.J.M. Hoenderop J.G.J. Smeets R.L.L. Willems P.H.G.M. Kijkman H.B.P.M. Galama J.M.D. Melchers W.J.G. EMBO J. 1997; 16: 3519-3532Google Scholar, 12Van Kuppeveld F.J.M. Melchers W.J.G. Kirkegaard K. Doedens J.R. Virology. 1997; 227: 111-118Google Scholar,14Barco A. Carrasco L. J. Virol. 1998; 72: 3560-3570Google Scholar, 15Van Kuppeveld F.J.M. Galama J.M.D. Zoll J. Van den Hurk P.J.J.C. Melchers W.J.G. J. Virol. 1996; 70: 3876-3886Google Scholar) and with viral RNA replication (12Van Kuppeveld F.J.M. Melchers W.J.G. Kirkegaard K. Doedens J.R. Virology. 1997; 227: 111-118Google Scholar, 16Van Kuppeveld F.J.M. Galama J.M.D. Zoll J. Melchers W.J.G. J. Virol. 1995; 69: 7782-7790Google Scholar). In an effort to gain further insight into the cytolytic processes that occur during enteroviral infection, we explored the molecular mechanism used by poliovirus 2B protein to disrupt the permeability barrier of phospholipid vesicles. Large unilamellar vesicles (LUVs) encapsulating different solutes in their aqueous compartments were employed to monitor the kinetics of solute release and entry into the membrane-bound aqueous compartments. This model system allowed the definition of several requirements for pore formation by 2B in terms of individual lipids, protein doses, and limiting size of the solutes able to cross the membrane. The experimental data presented here indicate that poliovirus 2B may assemble into tetrameric aqueous pores of diameter in the range of 5–10 Å in anionic bilayers. Our experimental approach and findings contribute to a better knowledge of the structural and functional features of viroporins (1Carrasco L. Adv. Virus Res. 1995; 45: 61-112Google Scholar). N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (N-NBD-PE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), dipalmitoylphosphatidylglycerol (DPPG), and 1-stearoyl-2-arachidonoylphosphatidylglycerol (SAPG) were purchased from Avanti Polar Lipids (Birmingham, AL). 5-((((2-Iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) andN,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), 8aminonaphthalene-1,3,6-trisulfonic acid sodium salt (ANTS), and p-xylenebis(pyridinium)bromide (DPX) were from Molecular Probes (Junction City, OR). Plasmids encoding maltose binding protein (MBP)-2B fusion proteins were constructed by poliovirus into were by as R. B. Res. 16: Scholar). were by were as fusion proteins from using as the was used to of with which was further at an of was The were induced with and at for an h by were in a with and disrupted by at The was at for at to cellular The was to an proteins were with with were to proteins from the were by in by were to the of M.J. Scholar) in The lipid of the were by Scholar). The diameter of vesicles was as by using a The size not with MBP-2B at of the of into the was by the J. Scholar). ANTS, DPX, and were by the by in a with were to in a were by the at and the at was the and the The of to fluorescence of the vesicles at was as the fluorescence of The of permeabilization was from is the fluorescence the of is the fluorescence of the is the fluorescence the of The of release was fluorescence a were at and used within of was for vesicles at for at of aqueous induced by MBP-2B was and into the of these into the was assayed by as J. 1998; Scholar). of solutes into vesicles was through of the in the of membranes to the by and R. Scholar). with mol were with protein for at was to the The of permeabilization was from the of This was using and to the fluorescence the of to and is the fluorescence of vesicles with and with MBP-2B protein two within the 2B sequence and This allowed the of 2B with and and were to of 2B at a and in the at The was by the of The fraction was by a with protein were by using a system with a of Da. was as L. E. N. Scholar). and were at and for the were and was used to the apparent mol fraction partition were by the in fluorescence from with lipid was in a with and at and and of of Kx were by the experimental to the is the lipid is the apparent or lipid at which the protein fraction is is the of K. 1998; Scholar). in and at membrane were through fluorescence resonance energy from to was by the L. E. N. Scholar), in the and of the were in a with at and of and The of energy was to L. 1978; Scholar) from the fluorescence of the in the and of in the vesicles as shown in Oligomerization in the membranes was using of protein with lipid for at were and the of MBP-2B oligomers was using 2B is a hydrophobic its 2B that were for and stable in aqueous to be using several Solubilization of 2B in requires the of of in the which with in a system not the different for the of aqueous 2B fusion was as the the of its for anionic membranes In MBP-2B were in and the fusion protein as an aqueous In a further effort to 2B from these fusion were to with MBP-2B by to 2B in and the of the 2B to 2B involved the membrane-bound MBP-2B with the this induced of the lipid the of the of MBP-2B allowed of the activity of 2B in a for the in which we the features of MBP-2B with membranes and it be that under that The data shown in the ability of MBP-2B to composed of anionic of and in vesicles most of the of this to the may be by increased fluorescence because of the of J. Scholar). The size of these solutes is in the range of that of the used for cell permeability (6Lama J. Carrasco L. J. Biol. Chem. 1992; 267: 15932-15937Google Scholar, 7Aldabe R. Barco A. Carrasco L. J. Biol. Chem. 1996; 271: 23134-23137Google Scholar, 9Doedens J.R. Kirkegaard K. EMBO J. 1995; 14: 894-907Google Scholar, 12Van Kuppeveld F.J.M. Melchers W.J.G. Kirkegaard K. Doedens J.R. Virology. 1997; 227: 111-118Google Scholar,14Barco A. Carrasco L. J. Virol. 1998; 72: 3560-3570Google Scholar). we here that experimental evidence for 2B at the plasma membrane has been for coxsackie virus 2B in and (8Van Kuppeveld F.J.M. Hoenderop J.G.J. Smeets R.L.L. Willems P.H.G.M. Kijkman H.B.P.M. Galama J.M.D. Melchers W.J.G. EMBO J. 1997; 16: 3519-3532Google Scholar). In poliovirus the protein has been at intracellular membranes (7Aldabe R. Barco A. Carrasco L. J. Biol. Chem. 1996; 271: 23134-23137Google Scholar, 13Sandoval I.V. Carrasco L. J. Virol. 1997; 71: 4679-4693Google Scholar), and the that membrane permeabilization is responsible for enhanced permeability of the plasma membrane not be the kinetics of the aqueous of into the with may be that a was the mol with is with a permeabilization by structures attached to the lipid not in the different vesicles in the permeabilization in vesicles at mol to the apparent mol fraction partition for this system of the protein was with lipid vesicles under these of at such protein in the membrane suggests that lysis was by a lysis was not a of massive of protein to the membrane. the as 2B we explored the of 2B The changes in the protein sequence of the MBP-2B proteins generated were to the ability of 2B to cell membranes. The the cationic amphipathic plasma membrane permeability as well as the capacity of 2B to disrupt intracellular (8Van Kuppeveld F.J.M. Hoenderop J.G.J. Smeets R.L.L. Willems P.H.G.M. Kijkman H.B.P.M. Galama J.M.D. Melchers W.J.G. EMBO J. 1997; 16: 3519-3532Google Scholar, 12Van Kuppeveld F.J.M. Melchers W.J.G. Kirkegaard K. Doedens J.R. Virology. 1997; 227: 111-118Google Scholar). with these this MBP-2B of the release of the solute to the shown in this amino acid in 2B the and the of permeabilization by and within the hydrophobic at the of the amphipathic has been shown to interfere with 2B ability to membrane permeability in (7Aldabe R. Barco A. Carrasco L. J. Biol. Chem. 1996; 271: 23134-23137Google Scholar). This the and the of permeabilization by and In a within the second hydrophobic domain of 2B A. Carrasco L. J. Virol. 1998; 72: 3560-3570Google Scholar) the permeabilization levels the of permeabilization was by further that second domain hydrophobicity in the 2B protein Kuppeveld F.J.M. Galama J.M.D. Zoll J. Melchers W.J.G. J. Virol. 1995; 69: 7782-7790Google Scholar) the of permeabilization by and the by In the data from the of MBP-2B to the functional of the hydrophobic domains within 2B the of The permeabilization a for anionic in the membrane made of were not by with and/or not in permeabilization either not the and species in cell membranes, the was for vesicles made of PI, was a for the activity of the the was by the of by MBP-2B the or different in 2B activity was assayed in vesicles made from of different and of The data shown in indicate that MBP-2B was to with with in In vesicles made of with a acid to that of were by the is of that most of lipid were to in to these it seems that the of with a of was to an 2B activity in of lipid to the changes in of induced at a of are to of two are to the changes in of induced at a of are to of two are in a The in were to the and size of the structures by to be to be able to conclude that the of discrete the complex not from to in the lipid the pore passage of membrane is not to be as a of the of the protein; and has to be a size for able to from the vesicles. MBP-2B vesicles was in the shown in A. The that the ability of MBP-2B to vesicles was with the of with vesicles inhibition was using that the is induced by the of MBP-2B to the anionic membranes. The of the MBP-2B was by solute with solute entry The these was that the formation of discrete the entry of solutes the pores have vesicles were by with of of solutes into these was from the of by In this an in as by the in fluorescence not of the in the membrane R. Scholar, A. J.L. J. J. Res. Scholar). We this to represent a of solute entry and in vesicles the of The data in indicate that vesicles allowed the entry of Moreover, when solute entry and were as a of the protein processes were shown to occur to a These findings are with the in the membranes of aqueous connections that allowed diffusion of the the molecular mass of the solutes induced by several were in including and of vesicles with was made of the was that with was of The of the is of the of Å Scholar). this pore size within the range of that to an used in cell permeability In the of the structures and the size of the of MBP-2B forming an aqueous pore in the membrane. MBP-2B and membranes, and among MBP-2B protein were by of the and processes not 2B pore forming This was by the of permeabilization induced by and MBP-2B not The MBP-2B fusion protein two in the 2B and These are within the second hydrophobic domain and the region, The of the to changes in the a of the of 2B into the of the membrane. The fluorescence of was in aqueous and in the of vesicles The of vesicles in significant enhancement of the The increased fluorescence in was by of the to the hydrophobic of the This is with the into the membrane of at of the most within the second hydrophobic Of under these is that the of species in the was the binding were to the Kx for the of MBP-2B into membranes the an apparent Kx of was that under the of of was with vesicles. In in fluorescence was at lipid of to in the of membranes. These findings that the protein not to these vesicles and the of activity in vesicles The in the allowed of the aggregation of membrane-bound MBP-2B by of this we a lipid at which the of protein was as the energy and was used as the fluorescence In the of of with an energy was In energy was in of vesicles or when proteins were with vesicles. the mol used in the the energy protein This may be from the The for the is Å Scholar). distribution to the in a for be in at the used be for distributed the significant levels of energy for the that 2B within the membrane. the of MBP-2B within membranes, the of the protein was in the and of vesicles shown in the protein in a with a molecular mass with that of the MBP-2B The of vesicles induced the formation of two molecular mass of and to and of In the of MBP-2B were and of be to were not in when was with vesicles not of the MBP-2B species as a of and to the of the in B. were for binding and a of processes lipid these data that binding to membranes induced the formation of MBP-2B being the molecular mass are a of viral proteins functional is their capacity to cell membrane during virus infection. In this viroporins the release of viral (1Carrasco L. Adv. Virus Res. 1995; 45: 61-112Google Scholar, 2Carrasco L. Guinea R. Irurzun A. Barco A. Semler B. Wimmer E. The Picornaviruses. American Society for Microbiology Press, Washington, D.C.2002: 337-354Google Scholar, 3Carrasco L. Perez L. Irurzun A. Lama J. Martinez-Abarca F. Rodriguez P. Guinea R. Castrillo J.L. Sanz M.A. Ayala M.J. Carrasco L. Sonenberg N. Wimmer E. Regulation of Gene Expression in Animal Viruses. Plenum Publishing Corp., London1993: 283-305Google Scholar). were functional that viroporin activity in are and hydrophobic to in aqueous to aqueous using 2B or several fusion we that the was to MBP-2B to This MBP-2B complex for the pore forming activity of 2B in a been proposed to for the increased membrane permeability by 2B expression (2Carrasco L. Guinea R. Irurzun A. Barco A. Semler B. Wimmer E. The Picornaviruses. American Society for Microbiology Press, Washington, D.C.2002: 337-354Google Scholar, 12Van Kuppeveld F.J.M. Melchers W.J.G. Kirkegaard K. Doedens J.R. Virology. 1997; 227: 111-118Google Scholar). such model is the structural for several cytolytic 1995; Scholar, 1995; Scholar). to this the amphipathic of 2B might transbilayer oligomers and constitute an aqueous with their hydrophobic the lipid and their the of the but not model that the amphipathic to the membrane might partially into a in a 1995; Scholar, B. J. Biol. 1997; Scholar). The first model the formation of oligomers of a discrete These be able to assemble aqueous pores with a molecular mass for the solutes capable of be of a defined In the second may be of and of solutes may across the membranes, is further is that be protein in the membranes such that may be by a mechanism the by the second The experimental presented here the ability of poliovirus 2B to vesicles at and such that stable discrete structures are and in membranes to SDS-resistant and findings to be with the first the formation by viroporin 2B of a transmembrane aqueous pore that diffusion of small The of MBP-2B allowed of the pore formation requirements in terms of lipid were for 2B The of in the membrane was for binding of MBP-2B This the of in the early of the process. proposed mechanisms for the formation of pores by amphipathic that binding to membranes, aggregation occurs at their Scholar, K. 1998; Scholar, J.L. Res. Scholar, Scholar). the protein a a pore be This mechanism seems to be functional in the of to the the MBP-2B protein binding to membranes. These data that the cationic amphipathic allowing further of the MBP-2B to membrane the from a to a pore requires the into the lipid of protein to as a that might be phospholipid J.L. Res. Scholar, Scholar, F. J. Scholar). The of lipid that the permeabilization rather 2B two 32–55 and 61–81 the 2B in which in the amphipathic were by the with 2B activity In an aqueous these domains might in to a that might the The of hydrophobic the of this and the of the might and pore for the DPPG, DOPG, POPG, are the and Å E. J. Scholar, Scholar). it seems that the different levels of permeability for this are to the different is that better and/or into the membrane in the of F. J. Scholar). of the of the protein in membranes that the might represent the pore forming of of the poliovirus 2B protein been using a system A. F. Wimmer E. J. Virol. 1998; 72: Scholar). In a of the coxsackie virus 2B protein have been further using a system Willems P.H.G.M. Galama J.M.D. Melchers W.J.G. Kuppeveld F.J.M. J. Virol. Scholar). Here we evidence to the formation of membrane-bound 2B of SDS-resistant oligomers is a among This has been for A. F. 1995; Scholar) and A. J. Biol. Chem. Scholar, A.E. Scholar) as well as for R. J. EMBO J. Scholar). In this membrane might to the of hydrophobic 2B that be disrupted within the a that be to assemble stable and functional to the molecular mass for the release of solutes was that of is Å Scholar). data a tetrameric aqueous pore with a diameter in the range of Å as the 2B The model in the different structural by transbilayer in the 2B membrane The amphipathic in to the the of the aqueous pore and the hydrophobic of the and the second an transmembrane the the the the in the that the and the of the protein at the of the membrane. a and pore with for poliovirus have been proposed for the domain J.L. P. R. L. 1997; Scholar, L. R. J.L. J. Biol. Chem. Scholar, J. Biol. Chem. Scholar). is to that structural in proteins of have to products that are capable of the membrane permeability In poliovirus 2B protein might structural such as in several to assemble tetrameric aqueous pores in membranes. The of in to the of 2B with membranes, as well as its of to a better of the membrane permeabilization mechanisms that during enterovirus infection.