Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition.

Mutations in the predicted C'-C"-D edge of the first immunoglobulin-like domain of the poliovirus receptor were previously shown to eliminate poliovirus binding. To identify capsid residues that expand receptor recognition, 16 poliovirus suppressor mutants were selected that replicate in three different mutant receptor-expressing cell lines as well as in cells expressing the wild-type receptor. Sequence analysis of the mutant viruses revealed three capsid residues that enable poliovirus to utilize defective receptors. Two residues are in regions of the capsid that are known to regulate receptor binding and receptor-mediated conformational transitions. A third residue is located in a highly exposed loop on the virion surface that controls poliovirus host range in mice by influencing receptor recognition. One of the suppressor mutations enables the primate-restricted P1/Mahoney strain to paralyze mice by enabling the virus to recognize a receptor in the mouse central nervous system. Capsid mutations that suppress receptor defects may exert their effect at the binding site or may improve receptor binding by regulating structural transitions of the capsid.     

Inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles.

The entry of animal viruses into cells is associated with permeabilization of the infected cells to protein toxins such as alpha-sarcin (C. Fernández-Puentes and L. Carrasco, Cell 20:769-775, 1980). This phenomenon has been referred to as "the early permeabilization by animal viruses" (L. Carrasco, Virology 113:623-629, 1981). A number of inhibitors of poliovirus growth such as WIN 51711 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile (DEPC) and Ro 09-0410 specifically block the uncoating step of poliovirus but have no effect on attachment or entry of poliovirus particles into cells. These agents are potent inhibitors of the early permeabilization induced by poliovirus to the toxin alpha-sarcin. Thus, the uncoating of poliovirus is required for the permeabilization of cell membranes to proteins. The increased entry of labeled heparin promoted by virus entry is not blocked by these agents, indicating that poliovirus binds to its receptor and is internalized along with heparin in endosomes in the presence of WIN 51711, DEPC, or Ro 09-0410. We conclude that the delivery to the cytoplasm of some molecules that coenter with virion particles does not take place if the uncoating process is hindered.     

Distribution of drug resistance mutations in type 3 poliovirus identifies three regions involved in uncoating functions.

We have previously described the use of an uncoating inhibitor, WIN 51711, to select drug-resistant mutants of the Sabin strain of poliovirus type 3. Two-thirds of the mutants proved to be dependent on the drug for plaque formation because of extreme thermolability (A. G. Mosser and R. R. Rueckert, J. Virol. 67:1246-1254, 1993). Here we report the responsible mutations; all were traced to single amino acid substitutions. Mutations conferring dependence and thermolability occurred in all four capsid proteins (VP1 to VP4), but all were clustered near residue 53 of VP4 at the inner capsid surface. Amino acid substitutions of the remaining non-drug-dependent mutants were mapped to three distinct loci: (i) on or near the inner capsid surface, at VP4 residue 46 or VP1 residue 129, in the vicinity of the drug dependence substitutions; (ii) at residues 192, 194, and 260 in the lining of the VP1 beta barrel, which is the drug-binding site; and (iii) at VP1 residue 105 on the edge of the canyon surrounding the fivefold axis of symmetry, the putative receptor-binding site. All of the mutations increased the eclipse rate of cell-attached virus. Such mutants help identify parts of the capsid that play a role in viral uncoating functions.     

The crystal structure of coxsackievirus A9: new insights into the uncoating mechanisms of enteroviruses

BACKGROUND: Coxsackievirus A9 (CAV9), a human pathogen causing symptoms ranging from common colds to fatal infections of the central nervous system, is an icosahedral single-stranded RNA virus that belongs to the genus Enterovirus of the family Picornaviridae. One of the four capsid proteins, VP1, includes the arginine-glycine-aspartate (RGD) motif within its C-terminal extension. This region binds to integrin alpha v beta 3, the only receptor for CAV9 to be conclusively identified to date. RESULTS: The crystal structure of CAV9 in complex with the antiviral compound WIN 51711 has been solved to 2.9 A resolution. The structures of the four capsid proteins, VP1 to VP4, resemble those of other picornaviruses. The antiviral compound is bound in the VP1 hydrophobic pocket, and it is possible that the pocket entrance contains a second WIN 51711 molecule. Continuous electron density for the VP1 N terminus provides a complete picture of the structure close to the fivefold axis. The VP1 C-terminal portion is on the outer surface of the virus and becomes disordered five-residues N-terminal to the RGD motif. CONCLUSIONS: The RGD motif is exposed and flexible in common with other known integrin ligands. Although CAV9 resembles coxsackie B viruses (CBVs), several substitutions in the areas implicated in CBV receptor attachment suggest it may recognise a different receptor. The structure along the fivefold axis provides new information on the uncoating mechanism of enteroviruses. CAV9 might bind a larger natural pocket factor than other picornaviruses, an observation of particular relevance to the design of new antiviral compounds.     

Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus.

The three-dimensional structure of the Sabin strain of type 3 poliovirus has been determined at 2.4 A resolution. Significant structural differences with the Mahoney strain of type 1 poliovirus are confined to loops and terminal extensions of the capsid proteins, occur in all of the major antigenic sites of the virion and typically involve insertions, deletions or the replacement of prolines. Several newly identified components of the structure participate in assembly-dependent interactions which are relevant to the biologically important processes of viral assembly and uncoating. These include two sites of lipid substitution, two putative nucleotides and a beta sheet formed by the N-termini of capsid proteins VP4 and VP1. The structure provides an explanation for the temperature sensitive phenotype of the P3/Sabin strain. Amino acids that regulate temperature sensitivity in type 3 poliovirus are located in the interfaces between promoters, in the binding site for a lipid substituent and in an assembly-dependent extended beta sheet that stabilizes the association of pentamers. Several lines of evidence indicate that these structural components also control conformational transitions at various stages of the viral life cycle.     

Host range determinants located on the interior of the poliovirus capsid.

The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.     

Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor.

Poliovirus initiates infection by binding to its cell receptor and undergoing a receptor-mediated conformational alteration. To identify capsid residues that control these interactions, we have isolated and characterized poliovirus mutants that are resistant to neutralization by a soluble form of the poliovirus receptor. Twenty one soluble receptor-resistant (srr) mutants were identified which still use the poliovirus receptor to infect cells. All but one srr mutant contain a single amino acid change at one of 13 different positions, either on the surface or in the interior of the virion. The results of binding and alteration assays demonstrate that both surface and internal capsid residues regulate attachment to the receptor and conformational change of the virus. Mutations that reduce alteration also affect receptor binding, suggesting a common structural basis for early events in poliovirus infection.     

Two distinct binding affinities of poliovirus for its cellular receptor

To study the kinetics and equilibrium of poliovirus binding to the poliovirus receptor, we used surface plasmon resonance to examine the interaction of a soluble form of the receptor with poliovirus. Soluble receptor purified from mammalian cells is able to bind poliovirus, neutralize viral infectivity, and induce structural changes in the virus particle. Binding studies revealed that there are two binding sites for the receptor on the poliovirus type 1 capsid, with affinity constants at 20 degrees C of K(D)(1) = 0.67 microm and K(D)(2) = 0.11 microm. The relative abundance of the two binding sites varies with temperature. At 20 degrees C, the K(D)(2) site constitutes approximately 46% of the total binding sites on the sensor chip, and its relative abundance decreased with decreasing temperature such that at 5 degrees C, the relative abundance of the K(D)(2) site is only 12% of the total binding sites. Absolute levels of the K(D)(1) site remained relatively constant at all temperatures tested. The two binding sites may correspond to docking sites for domain 1 of the receptor on the viral capsid, as predicted by a model of the poliovirus-receptor complex. Alternatively, the binding sites may be a consequence of structural breathing, or could result from receptor-induced conformational changes in the virus.     

Model for the differential stabilities of rhinovirus and poliovirus to mild acidic pH, based on electrostatics calculations.

Previous calculations of electrostatic interactions in the rhinovirus capsid have identified a subset of histidine residues, paired with lysine or arginine, that may be involved in pH-induced conformational changes related to viral uncoating. Further calculations with the finite difference method, accounting for the dielectric environment of the ionizable groups, suggest that charge burial in the crystal conformation will prevent protonation of these histidine residues in the pentamer-pentamer interface. Calculations with a modelled pentamer-pentamer interface in which three beta-strands are removed recover mildly acidic pKa values for the histidines. These results are discussed in the context of the structural interactions of these three beta-strands, which form a beta-sheet extension from the rest of the capsid, and with regard to the conformation of the homologous beta-sheet extension in poliovirus, which also possesses homologous histidine-lysine/arginine pairs. A model is developed in which the structural stability of the beta-sheet extension is related to the difference in acid stability of rhinovirus and poliovirus. It is suggested that, for poliovirus prior to cell receptor binding, the beta-sheet extension is stable at pH 3, the pentamer-pentamer interface histidines remain buried, and the virus is acid-stable. Cell receptor binding of poliovirus destabilizes the beta-sheet extension and the acid lability that is proposed to result could be involved in viral uncoating. For rhinovirus it is suggested that the observed conformational change in the absence of cell receptor binding involves a further acidic pH-activated process or conformational fluctuations that rearrange the beta-sheet extension and expose the pentamer-pentamer interface histidine residues to the acidic medium. Sequence analysis and electrostatics calculations reveal an aspartic acid in the beta-sheet extension that may have different pKa values in rhinovirus and poliovirus.     

Three-dimensional structure of a mouse-adapted type 2/type 1 poliovirus chimera.

The crystal structure of V510, a chimeric type 2/type 1 poliovirus, has been determined at 2.6 A resolution. Unlike the parental Mahoney strain of type 1 poliovirus, V510 is able to replicate in the mouse central nervous system, due entirely to the replacement of six amino acids in the exposed BC loop of capsid protein VP1. Significant structural differences between the two strains cluster in a major antigenic site of the virus, located at the apex of the radial projection which surrounds the viral five-fold axis. Residues implicated in the mouse-virulence of poliovirus by genetic studies are located in this area, and include the residues which are responsible for stabilizing the conformation of the BC loop in V510. Despite evidence that this area is not involved in receptor binding in cultured primate cells, the genetic and structural observations suggest that this area plays a critical role in receptor interactions in the mouse central nervous system. These results provide a structural framework for further investigation of the molecular determinants of host and tissue tropism in viruses.     

Molecular dynamics simulations of human rhinovirus and an antiviral compound

The human rhinovirus 14 (HRV14) protomer, with or without the antiviral compound WIN 52084s, was simulated using molecular dynamics and rotational symmetry boundary conditions to model the effect of the entire icosahedral capsid. The protein asymmetrical unit, comprising four capsid proteins (VP1, VP2, VP3, and VP4) and two calcium ions, was solvated both on the exterior and the interior to fill the inside of the capsid. The stability of the simulations of this large system (~800 residues and 6,650 water molecules) is comparable to more conventional globular protein simulations. The influence of the antiviral compound on compressibility and positional fluctuations is reported. The compressibility, estimated from the density fluctuations in the region of the binding pocket, was found to be greater with WIN 52084s bound than without the drug, substantiating previous computations on reduced viral systems. An increase in compressibility correlates with an entropically more favorable system. In contrast to the increase in density fluctuations and compressibility, the positional fluctuations decreased dramatically for the external loops of VP1 and the N-terminus of VP3 when WIN 52084s is bound. Most of these VP1 and VP3 loops are found near the fivefold axis, a region whose mobility was not considered in reduced systems, but can be observed with this simulation of the full viral protomer. Altered loop flexibility is consistent with changes in proteolytic sensitivity observed experimentally. Moreover, decreased flexibility in these intraprotomeric loops is noteworthy since the externalization of VP4, part of VP1, and RNA during the uncoating process is thought to involve areas near the fivefold axis. Both the decrease in positional fluctuations at the fivefold axis and the increase in compressibility near the WIN pocket are discussed in relationship to the antiviral activity of stabilizing the virus against uncoating.     

Human rhinovirus 14 complexed with antiviral compound R 61837.

The binding of the antirhinoviral agent R 61837 to human rhinovirus 14 has been examined by X-ray crystallographic methods. The compound R 61837 binds in the same pocket (underneath the canyon floor) as the "WIN" antirhinoviral agents. It does not penetrate as far into the pocket but causes similar conformational changes in the virus capsid. The movement of residues 1217 to 1221 of viral protein 1 (in the "FMDV loop") is more pronounced for R 61837 than for WIN compounds. Although both R 61837 and WIN antiviral agents partially fill the same hydrophobic pocket, atomic binding interactions differ, showing that considerable diversity in the nature of antiviral agents is possible.     

Pocket factors are unlikely to play a major role in the life cycle of human rhinovirus

Human rhinovirus 14 (HRV14) is a member of the rhinovirus genus, which belongs to the picornavirus family, which includes clinically and economically important members, such as poliovirus, foot-and-mouth disease virus, and endomyocarditis virus. Capsid stability plays an important role in the viral infection process, in that it needs to be stable enough to move from cell to cell and yet be able to release its genetic material upon the appropriate environmental cues from the host cell. It has been suggested that certain host cell molecules, "pocket factors," bind to the WIN drug-binding cavity beneath the canyon floor and provide transient stability to a number of the picornaviruses. To directly test this hypothesis, HRV14 was mutated in (V1188M, C1199W, and V1188M/C1199W) and around (S1223G) the drug-binding pocket. Infectivity, limited proteolysis, and matrix-assisted laser desorption ionization analyses indicate that filling the drug-binding pocket with bulky side chains is not deleterious to the viral life cycle and lends some stabilization to the capsid. In contrast, studies with the S1223G mutant suggest that this mutation at least partially overcomes WIN drug-mediated inhibition of cell attachment and capsid breathing. Finally, HRV16, which is inherently more stable than HRV14 in a number of respects, was found to "breathe" only at 37 degrees C and did not tolerate stabilizing mutations in the drug-binding cavity. These results suggest that it is the drug-binding cavity itself and not the putative pocket factor that is crucial for the capsid dynamics, which is, in turn, necessary for infection.     

Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding.

Upon attachment to susceptible cells, poliovirus and a number of other picornaviruses undergo conformational transitions which result in changes in antigenicity, increased protease sensitivity, the loss of the internal capsid protein VP4, and a loss of the ability to attach to cells. These conformationally altered particles have been characterized by using a number of sequence-specific probes, including two proteases, a panel of antiviral monoclonal antibodies, and a panel of antisera against synthetic peptides which correspond to sequences from the capsid protein VP1. With these probes, cell-altered virus is clearly distinguishable from native and heat-inactivated virions. The probes also demonstrate that the cell-induced conformational change alters the accessibility of several regions of the virus. In particular, the amino terminus of VP1, which is entirely internal in the native virion, becomes externalized. Unlike native and heat-inactivated virus, cell-altered virions are able to attach to liposomes. The exposed amino terminus of VP1 is shown to be responsible for liposome attachment. We propose that during infection the amino terminus of VP1 inserts into endosomal membranes and thus plays a role in the mechanism of cell entry.     

Virus capsid dissolution studied by microsecond molecular dynamics simulations

Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.     

Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus.

Empty capsids of foot-and-mouth disease virus (FMDV) type A22 Iraq 24/64, whose structure has been solved by X-ray crystallography, are unusual for picornaviruses since they contain VP2 and VP4, the cleavage products of the protein precursor VP0. Both the N terminus of VP1 and the C terminus of VP4, which pack together close to the icosahedral threefold symmetry axis where three pentamers associate, are more disordered in the empty capsid than they are in the RNA-containing virus. The ordering of these termini in the presence of RNA strengthens interactions within a single protomer and between protomers belonging to different pentamers. The disorder in the FMDV empty capsid forms a subset of that seen in the poliovirus empty capsid, which has VP0 intact. Thus, VP0 cleavage confers stability on the picornavirus capsid over and above that attributable to RNA encapsidation. In both FMDV and poliovirus empty capsids, the internal disordering uncovers a conserved histidine which has been proposed to be involved in the cleavage of VP0. A comparison of the putative active sites in FMDV and poliovirus suggests a structural explanation for the sequence specificity of the cleavage reaction.     

Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.     

A mutation in VP4 defines a new step in the late stages of cell entry by poliovirus.

During the entry of poliovirus into cells, a conformational transition occurs within the virion that is dependent upon its binding to the cell surface receptor. This conformational rearrangement generates an altered particle of 135S, results in the extrusion of capsid protein VP4 and the amino terminus of VP1 from the virion interior, and leads to the acquisition of membrane-binding properties by the 135S particle. Although the subsequent fate of VP4 is unknown, its apparent absence from purified 135S particles has long suggested that VP4 is not directly involved during virus entry. We report here the construction by site-specific mutagenesis of a nonviable VP4 mutant that upon transfection of the cDNA appears to form mature virus particles. These particles, upon interaction with the cellular receptor, undergo the 135S conformational transition but are defective at a subsequent stage in virus entry. The results demonstrate that the participation of VP4 is required during cell entry of poliovirus. In addition, these data indicate the existence of additional stages in the cell entry process beyond receptor binding and the transition to 135S particles. These post-135S stages must include the poorly understood processes by which nonenveloped viruses cross the cell membrane, uncoat, and deliver their genomes into the cytoplasm.     

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.     

Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating

Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins'' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.