Pocket Factor Research Articles

Implication for alphavirus host-cell entry and assembly indicated by a 3.5\xc5 resolution cryo-EM structure

Alphaviruses are enveloped RNA viruses that contain several human pathogens. Due to intrinsic heterogeneity of alphavirus particles, a high resolution structure of the virion is currently lacking. Here we provide a 3.5 Å cryo-EM structure of Sindbis virus, using block based reconstruction method that overcomes the heterogeneity problem. Our structural analysis identifies a number of conserved residues that play pivotal roles in the virus life cycle. We identify a hydrophobic pocket in the subdomain D of E2 protein that is stabilized by an unknown pocket factor near the viral membrane. Residues in the pocket are conserved in different alphaviruses. The pocket strengthens the interactions of the E1/E2 heterodimer and may facilitate virus assembly. Our study provides structural insights into alphaviruses that may inform the design of drugs and vaccines.

Hepatitis A Virus Capsid Structure.

Hepatitis A virus (HAV) has been enigmatic, evading detailed structural analysis for many years. Its recently determined high-resolution structure revealed an angular surface without the indentations often characteristic of receptor-binding sites. The viral protein 1 (VP1) β-barrel shows no sign of a pocket factor and the amino terminus of VP2 displays a "domain swap" across the pentamer interface, as in a subset of mammalian picornaviruses and insect picorna-like viruses. Structure-based phylogeny confirms this placement. These differences suggest an uncoating mechanism distinct from that of enteroviruses. An empty capsid structure reveals internal differences in VP0 and the VP1 amino terminus connected with particle maturation. An HAV/Fab complex structure, in which the antigen-binding fragment (Fab) appears to act as a receptor-mimic, clarifies some historical epitope mapping data, but some remain difficult to reconcile. We still have little idea of the structural features of enveloped HAV particles.

IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis.

The HIV capsid is semipermeable and covered in electropositive pores that are essential for viral DNA synthesis and infection. Here, we show that these pores bind the abundant cellular polyanion IP6, transforming viral stability from minutes to hours and allowing newly synthesised DNA to accumulate inside the capsid. An arginine ring within the pore coordinates IP6, which strengthens capsid hexamers by almost 10°C. Single molecule measurements demonstrate that this renders native HIV capsids highly stable and protected from spontaneous collapse. Moreover, encapsidated reverse transcription assays reveal that, once stabilised by IP6, the accumulation of new viral DNA inside the capsid increases >100 fold. Remarkably, isotopic labelling of inositol in virus-producing cells reveals that HIV selectively packages over 300 IP6 molecules per infectious virion. We propose that HIV recruits IP6 to regulate capsid stability and uncoating, analogous to picornavirus pocket factors. HIV-1/IP6/capsid/co-factor/reverse transcription.

Exploring Strategies for Labeling Viruses with Gold Nanoclusters through Non-equilibrium Molecular Dynamics Simulations.

Biocompatible gold nanoclusters can be utilized as contrast agents in virus imaging. The labeling of viruses can be achieved noncovalently but site-specifically by linking the cluster to the hydrophobic pocket of a virus via a lipid-like pocket factor. We have estimated the binding affinities of three different pocket factors of echovirus 1 (EV1) in molecular dynamics simulations combined with non-equilibrium free-energy calculations. We have also studied the effects on binding affinities with a pocket factor linked to the Au102pMBA44 nanocluster in different protonation states. Although the absolute binding affinities are over-estimated for all the systems, the trend is in agreement with recent experiments.3 Our results suggest that the natural pocket factor (palmitic acid) can be replaced by molecules pleconaril (drug) and its derivative Kirtan1 that have higher estimated binding affinities. Our results also suggest that including the gold nanocluster does not decrease the affinity of the pocket factor to the virus, but the affinity is sensitive to the protonation state of the nanocluster, i.e., to pH conditions. The methodology introduced in this work helps in the design of optimal strategies for gold-virus bioconjugation for virus detection and manipulation.

Beta-Propiolactone Inactivation of Coxsackievirus A16 Induces Structural Alteration and Surface Modification of Viral Capsids.

Beta-propiolactone (BPL) is an inactivating agent that is widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we present cryo-electron microscopy (cryo-EM) structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids at resolutions of 3.9 Å and 6.5 Å, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N terminus of VP1 capsid protein, and the absence of pocket factor. However, major neutralizing epitopes are very well preserved on these particles. Further biochemical analyses revealed that BPL treatment impairs the abilities of CVA16 particles to bind to the attachment receptor heparan sulfate and to a conformation-dependent monoclonal antibody in a BPL dose-dependent manner, indicating that BPL is able to modify surface-exposed amino acid residues. Taken together, our results demonstrate that BPL treatment may induce alteration of the overall structure and surface properties of a nonenveloped viral capsid, thus revealing a novel mode of action of BPL.IMPORTANCE Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that the BPL concentration affects the binding of inactivated CVA16 particles to their receptor/antibody. Thus, BPL treatment can alter the overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.

Nectin-Like Interactions between Poliovirus and Its Receptor Trigger Conformational Changes Associated with Cell Entry

Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called "pocket factor"), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier.

Hepatitis A virus and the origins of picornaviruses.

Hepatitis A virus (HAV) remains enigmatic, despite some 1.4 million cases worldwide annually1. It differs radically from other picornaviruses, existing in an enveloped form2 and being unusually stable, both genetically and physically3, but has proved difficult to study. We report high-resolution X-ray structures for the mature virus and empty particles. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, while the empty particle harbors only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions which might correspond to receptor binding sites. Peptide scanning data extends the previously reported VP3 antigenic site4, while structure-based predictions5 suggest further epitopes. HAV contains no pocket factor, can withstand remarkably high temperature and low pH, with empty particles being even more robust than full particles. The virus probably uncoats via a novel mechanism, being built differently to other picornaviruses. It utilizes a VP2 ‘domain swap’ characteristic of insect picorna-like viruses6,7 and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between ‘modern’ picornaviruses and the more ‘primitive’ precursor insect viruses, for instance HAV retains the ability to move from cell-to-cell by transcytosis8,9.

The cellular receptor for enterovirus 71.

The cellular receptor for enterovirus 71.

A pH-dependent molecular switch for virion uncoating.

A pH-dependent molecular switch for virion uncoating.

Molecular mechanism of SCARB2-mediated attachment and uncoating of EV71.

Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0087-3) contains supplementary material, which is available to authorized users.

Structure of human enterovirus 71 in complex with a capsid-binding inhibitor

Human enterovirus 71 is a picornavirus causing hand, foot, and mouth disease that may progress to fatal encephalitis in infants and small children. As of now, no cure is available for enterovirus 71 infections. Small molecule inhibitors binding into a hydrophobic pocket within capsid viral protein 1 were previously shown to effectively limit infectivity of many picornaviruses. Here we report a 3.2-Å-resolution X-ray structure of the enterovirus 71 virion complexed with the capsid-binding inhibitor WIN 51711. The inhibitor replaced the natural pocket factor within the viral protein 1 pocket without inducing any detectable rearrangements in the structure of the capsid. Furthermore, we show that the compound stabilizes enterovirus 71 virions and limits its infectivity, probably through restricting dynamics of the capsid necessary for genome release. Thus, our results provide a structural basis for development of antienterovirus 71 capsid-binding drugs.

Hydrophobic partitioning approach to efficient protein separation with magnetic nanoparticles

A novel approach to the hydrophobic partitioning effect on efficient separation of protein such as BSA was demonstrated by the modification of hydrophobic pockets on the surface of silica-coated magnetic nanoparticles with various alkyl groups at various pH levels. The separation efficiency is strongly reflected and can be attained by controlling the size of the hydrophobic pocket and other factors such as the alkyl chain length, the salt concentration, and the pH levels. Furthermore, the adsorption constant ( k ad) was calculated for the hydrophobic partitioning interaction between BSA and the hydrophobic pocket size at various pH levels.

Crystal structure of CD155 and electron microscopic studies of its complexes with polioviruses

When poliovirus (PV) recognizes its receptor, CD155, the virus changes from a 160S to a 135S particle before releasing its genome into the cytoplasm. CD155 is a transmembrane protein with 3 Ig-like extracellular domains, D1-D3, where D1 is recognized by the virus. The crystal structure of D1D2 has been determined to 3.5-A resolution and fitted into approximately 8.5-A resolution cryoelectron microscopy reconstructions of the virus-receptor complexes for the 3 PV serotypes. These structures show that, compared with human rhinoviruses, the virus-receptor interactions for PVs have a greater dependence on hydrophobic interactions, as might be required for a virus that can inhabit environments of different pH. The pocket factor was shown to remain in the virus during the first recognition stage. The present structures, when combined with earlier mutational investigations, show that in the subsequent entry stage the receptor moves further into the canyon when at a physiological temperature, thereby expelling the pocket factor and separating the viral subunits to form 135S particles. These results provide a detailed analysis of how a nonenveloped virus can enter its host cell.

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.

Structure of swine vesicular disease virus: mapping of changes occurring during adaptation of human coxsackie B5 virus to infect swine.

The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-A resolution by X-ray crystallography to gain information about the role of sequence changes that occurred as this virus evolved from the parental human pathogen coxsackievirus B5 (CVB5). These amino acid substitutions can be clustered in five distinct regions: (i) the antigenic sites, (ii) the hydrophobic pocket of the VP1 beta-sandwich, (iii) the putative CAR binding site, (iv) the putative heparan sulfate binding site, and (v) the fivefold axis. The VP1 pocket is occupied by a branched pocket factor, apparently different from that present in the closely related virus CVB3 and in other picornaviruses. This finding may be relevant for the design of new antiviral compounds against this site. Density consistent with the presence of ions was observed on the fivefold and threefold axes. The structure also provided an accurate description of the putative receptor binding sites.

Human rhinovirus type 16: mutant V1210A requires capsid-binding drug for assembly of pentamers to form virions during morphogenesis.

Our laboratory has previously reported isolation of human rhinovirus type 16 (HRV16) mutants which depend on WIN 52035 to grow. A rapid rise of progeny virus infectivity occurred when drug was added late in growth cycles, suggesting that the drug-dependence lesion was at the step of virus assembly (W. Wang et al., J. Virol. 72:1210-1218, 1998). Here, we report that capsid subunits, 5S protomers and 14S pentamers, of a drug-dependent mutant were produced normally in the absence of drug, but mutant 80S empty capsids and 150S provirions were not formed, maturation cleavage of provirions (VP0 --> VP2 + VP4) did not occur, and the unassembled mutant capsid subunits were degraded with a half-life of 15 min. Drug was not required by mutant virus for attachment, uncoating, RNA synthesis and protein synthesis, and polyprotein processing except maturation cleavage. The requirement of drug for assembly of mutant pentamers to form provirions and the rapid assembly of preformed subunits (synthesized in the absence of drug) after drug addition suggested that after native pentamers (P5) have been formed they must be converted to an assembly active state (P5(*)), possibly by a conformational change induced by the binding of drug. We propose that pocket factor plays the same role in wild-type virus. In addition, we also report the construction and the properties of a full-length cDNA clone of HRV16, pR16.11, which produces in vitro transcripts with infectivity similar to that of virion RNA. This cDNA clone is available at the American Type Culture Collection.

Identification of the pocket factors in a picornavirus.

Bovine enterovirus (BEV), along with other enteroviruses and the rhinoviruses, has a hydrophobic pocket within structural protein VP1. In the crystal structures of these viruses there is electron density commensurate with a non-protein molecule within the pocket. These molecules, termed pocket factors, have been shown to stabilise the capsid and their removal from the pocket is a necessary prerequisite to uncoating. The pocket factors have been proposed, from the electron densities and uncoating studies, to be short chain fatty acids. In order to identify the pocket factor of BEV, we have grown and purified the virus in an identical manner to that used for the crystal structure determination and have performed a lipophilic extraction. Palmitic acid, C(16:0), was the most abundant accounting for 40.8% by mass of the lipophilic extract (39.3 mol%). Myristic acid C(14:0), was next most abundant at 18.5% by mass (20.0 mol%). In addition, we have identified other fatty acids in smaller proportions. We have therefore shown that BEV contains saturated fatty acid pocket factors of varying chain length. We have also compared the profile of the fatty acyl chain composition of BEV with those for uninfected BHK-21 cell plasma membrane and endoplasmic reticulum extracts.

Complexes of poliovirus serotypes with their common cellular receptor, CD155.

Structures of all three poliovirus (PV) serotypes (PV1, PV2, and PV3) complexed with their cellular receptor, PV receptor (PVR or CD155), were determined by cryoelectron microscopy. Both glycosylated and fully deglycosylated CD155 exhibited similar binding sites and orientations in the viral canyon for all three PV serotypes, showing that all three serotypes use a common mechanism for cell entry. Difference maps between the glycosylated and deglycosylated CD155 complexes determined the sites of the carbohydrate moieties that, in turn, helped to verify the position of the receptor relative to the viral surface. The proximity of the CD155 carbohydrate site at Asn105 to the viral surface in the receptor-virus complex suggests that it might interfere with receptor docking, an observation consistent with the properties of mutant CD155. The footprints of CD155 on PV surfaces indicate that the south rim of the canyon dominates the virus-receptor interactions and may correspond to the initial CD155 binding state of the receptor-mediated viral uncoating. In contrast, the interaction of CD155 with the north rim of the canyon, especially the region immediately outside the viral hydrophobic pocket that normally binds a cellular "pocket factor," may be critical for the release of the pocket factor, decreasing the virus stability and hence initiating uncoating. The large area of the CD155 footprint on the PV surface, in comparison with other picornavirus-receptor interactions, could be a potential limitation on the viability of PV escape mutants from antibody neutralization. Many of these are likely to have lost their ability to bind CD155, resulting in there being only three PV serotypes.

1P021回転対称性境界条件によるライノウイルス外殻蛋白質の安定性の計算

1P021回転対称性境界条件によるライノウイルス外殻蛋白質の安定性の計算

Structure of human rhinovirus serotype 2 (HRV2)

Human rhinoviruses are classified into a major and a minor group based on their binding to ICAM-1 or to members of the LDL-receptor family, respectively. They can also be divided into groups A and B, according to their sensitivity towards a panel of antiviral compounds. The structure of human rhinovirus 2 (HRV2), which uses the LDL receptor for cell attachment and is included in antiviral group B, has been solved and refined at 2.6 Å resolution by X-ray crystallography to gain information on the peculiarities of rhinoviruses, in particular from the minor receptor group. The main structural differences between HRV2 and other rhinoviruses, including the minor receptor group serotype HRV1A, are located at the internal protein shell surface and at the external antigenic sites. In the interior, the N termini of VP1 and VP4 form a three-stranded β-sheet in an arrangement similar to that present in poliovirus, although myristate was not visible at the amino terminus of VP4 in the HRV2 structure. The βE-βF loop of VP2, a linear epitope within antigenic site B recognized by monoclonal antibody 8F5, adopts a conformation considerably different from that found in the complex of 8F5 with a synthetic peptide of the same sequence. This either points to considerable structural changes impinged on this loop upon antibody binding, or to the existence of more than one single conformation of the loop when the virus is in solution. The hydrophobic pocket of VP1 was found to be occupied by a pocket factor apparently identical with that present in the major receptor group virus HRV16. Electron density, consistent with the presence of a viral RNA fragment, is seen stacked against a conserved tryptophan residue.