Polymorphism in the assembly of polyomavirus capsid protein VP1.

Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.     

Assembly properties of the human immunodeficiency virus type 1 CA protein

During retroviral maturation, the CA protein oligomerizes to form a closed capsid that surrounds the viral genome. We have previously identified a series of deleterious surface mutations within human immunodeficiency virus type 1 (HIV-1) CA that alter infectivity, replication, and assembly in vivo. For this study, 27 recombinant CA proteins harboring 34 different mutations were tested for the ability to assemble into helical cylinders in vitro. These cylinders are composed of CA hexamers and are structural models for the mature viral capsid. Mutations that diminished CA assembly clustered within helices 1 and 2 in the N-terminal domain of CA and within the crystallographically defined dimer interface in the CA C-terminal domain. These mutations demonstrate the importance of these regions for CA cylinder production and, by analogy, mature capsid assembly. One CA mutant (R18A) assembled into cylinders, cones, and spheres. We suggest that these capsid shapes occur because the R18A mutation alters the frequency at which pentamers are incorporated into the hexagonal lattice. The fact that a single CA protein can simultaneously form all three known retroviral capsid morphologies supports the idea that these structures are organized on similar lattices and differ only in the distribution of 12 pentamers that allow them to close. In further support of this model, we demonstrate that the considerable morphological variation seen for conical HIV-1 capsids can be recapitulated in idealized capsid models by altering the distribution of pentamers.     

An Interaction between Glutathione and the Capsid Is Required for the Morphogenesis of C-Cluster Enteroviruses

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.     

Picornaviral capsid assembly: similarity of rhinovirus and enterovirus precursor subunits.

Cytoplasmic extracts of rhinovirus 1A-infected HeLa cells, pulsed 15 min with [3H]leucine, contained a 13S subunit which was rich in the capsid precursor, peptide 92. After a 30-min chase, most of the capsid-related protein sedimented in a 14S peak that contained equimolar amounts of the capsid peptides epsilon, alpha, and gamma, and some residual chain 92. The 14S subunit could be dissociated at pH 4.8 into 6S subunits containing only epsilon, alpha, and gamma chains in equal proportions, indicating that the 14S subunit is an oligomer of (epsilon gamma alpha) protomers. These subunits resemble subunits previously identified in the assembly of enteroviruses. These observations support the idea that rhinovirus assembly is basically similar to that of enteroviruses. Comparative studies on the peptide stoichiometry of the virion and the capsid precursor subunits indicate that rhinovirus 1A can contain as many as 11 immature protomers per virion.     

Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: the evolution of pentameric vertex proteins in icosahedral viruses

Many large viral capsids require special pentameric proteins at their fivefold vertices. Nevertheless, deletion of the special vertex protein gene product 24 (gp24) in bacteriophage T4 can be compensated by mutations in the homologous major capsid protein gp23. The structure of such a mutant virus, determined by cryo-electron microscopy to 26 angstroms, shows that the gp24 pentamers are replaced by mutant major capsid protein (gp23) pentamers at the vertices, thus re-creating a viral capsid prior to the evolution of specialized major capsid proteins and vertex proteins. The mutant gp23* pentamer is structurally similar to the wild-type gp24* pentamer but the insertion domain is slightly more distant from the gp23* pentamer center. There are additional SOC molecules around the gp23* pentamers in the mutant virus that were not present around the gp24* pentamers in the wild-type virus.     

Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells.

Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.     

Synthesis and assembly of hepatitis A virus-specific proteins in BS-C-1 cells.

To determine the mechanism for the delayed and inefficient replication of the picornavirus hepatitis A virus in cell culture, we studied the kinetics of synthesis and assembly of virus-specific proteins by metabolic labeling of infected BS-C-1 cells with L-[35S]methionine and L-[35S]cysteine. Sedimentation, electrophoresis, and autoradiography revealed the presence of virions, provirions, procapsids, and 14S (pentameric) subunits. Virions and provirions contained VP1, VP0, VP2, and VP3; procapsids contained VP1, VP0, and VP3; and pentamers contained PX, VP0, and VP3, as previously shown by immunoblotting (D.A. Anderson and B.C. Ross, J. Virol. 64:5284-5289, 1990). Under single-cycle growth conditions label was found in 14S subunits immediately after labeling from 15 to 18 h postinfection (p.i.); however, a proportion of labeled polyprotein was not cleaved and assembled into pentamers for a further 18 h. When analyzed at 72 h p.i., incorporation of label which flowed into virions was detected from 3 h p.i., with maximal uptake levels being observed from 12 to 15 h p.i. Viral antigen, infectious virus, and viral RNA were determined in parallel, with coincident peaks in these variables being observed 12 h after the period of maximum label uptake. It was also found that the lag between the synthesis of the viral polyprotein and assembly of viral particles was the same after labeling from either 12 to 15 or 27 to 30 h p.i. despite increased levels of viral RNA during this period, suggesting that factors additional to the level of RNA are involved in the restriction of viral replication. Sedimentation and immunoblot analysis revealed an additional protein of approximately 100 kDa containing both VP1- and VP2-reactive sequences and sedimenting slightly more slowly than 14S pentamers, which may represent intact P12A assembled into pentamers as has been reported for the P1 of some other picornaviruses (S. McGregor and R. R. Rueckert, J. Virol. 21:548-553, 1977). The results of this study suggest that cleavage of the hepatitis A virus polyprotein to produce pentamers is protracted (though not rate limiting) early in infection, while the assembly of pentamers into higher structures is a rapid process once sufficient viral RNA is produced for encapsidation.     

An assembly defect as a result of an attenuating mutation in the capsid proteins of the poliovirus type 3 vaccine strain.

The molecular basis of the temperature-sensitive (ts) phenotype of P3/Sabin, the type 3 vaccine strain of poliovirus, was investigated in light of the known correlation between ts and attenuation phenotypes. A phenylalanine at residue 91 of the capsid protein VP3 was a major determinant of both phenotypes, and attenuation and ts could be reverted by the same second-site mutations. The ts phenotype was due to a defect early in the assembly process that inhibited the formation of 14S pentamers, empty capsids, and virions. It was further shown that capsid proteins that were not incorporated into higher-order structures had short half-lives at the nonpermissive temperature.     

Electrostatic interaction between RNA and protein capsid in cowpea chlorotic mottle virus simulated by a coarse-grain RNA model and a Monte Carlo approach

Although many viruses have been crystallized and the protein capsid structures have been determined by x-ray crystallography, the nucleic acids often cannot be resolved. This is especially true for RNA viruses. The lack of information about the conformation of DNA/RNA greatly hinders our understanding of the assembly mechanism of various viruses. Here we combine a coarse-grain model and a Monte Carlo method to simulate the distribution of viral RNA inside the capsid of cowpea chlorotic mottle virus. Our results show that there is very strong interaction between the N-terminal residues of the capsid proteins, which are highly positive charged, and the viral RNA. Without these residues, the binding energy disfavors the binding of RNA by the capsid. The RNA forms a shell close to the capsid with the highest densities associated with the capsid dimers. These high-density regions are connected to each other in the shape of a continuous net of triangles. The overall icosahedral shape of the net overlaps with the capsid subunit icosahedral organization. Medium density of RNA is found under the pentamers of the capsid. These findings are consistent with experimental observations.     

Assembly models for Papovaviridae based on tiling theory

A vital constituent of a virus is its protein shell, called the viral capsid, that encapsulates and hence provides protection for the viral genome. Assembly models are developed for viral capsids built from protein building blocks that can assume different local bonding structures in the capsid. This situation occurs, for example, for viruses in the family of Papovaviridae, which are linked to cancer and are hence of particular interest for the health sector. More specifically, the viral capsids of the (pseudo-) T = 7 particles in this family consist of pentamers that exhibit two different types of bonding structures. While this scenario cannot be described mathematically in terms of Caspar-Klug theory (Caspar D L D and Klug A 1962 Cold Spring Harbor Symp. Quant. Biol. 27 1), it can be modelled via tiling theory (Twarock R 2004 J. Theor. Biol. 226 477). The latter is used to encode the local bonding environment of the building blocks in a combinatorial structure, called the assembly tree, which is a basic ingredient in the derivation of assembly models for Papovaviridae along the lines of the equilibrium approach of Zlotnick (Zlotnick A 1994 J. Mol. Biol. 241 59). A phase space formalism is introduced to characterize the changes in the assembly pathways and intermediates triggered by the variations in the association energies characterizing the bonds between the building blocks in the capsid. Furthermore, the assembly pathways and concentrations of the statistically dominant assembly intermediates are determined. The example of Simian virus 40 is discussed in detail.     

Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity

The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.     

Development of intranuclear inclusions of human polyomavirus JC. Capsid proteins are assembled into virions at the PML nuclear bodies

Human polyomavirus JC (JCV) is a causative agent for progressive multifocal leukoencephalopathy, a fatal demyelinating disorder. The viruses form intranuclear viral inclusions in infected oligodendrocytes. The outer capsid of JCV is thought to be composed of 360 molecules of major capsid protein VP1, and minor capsid proteins VP2 and VP3 in an appropriate ratio. However, the regulatory mechanisms of gene expression for the capsid proteins, their nuclear transport, and formation of viral inclusions are not well understood. We have recently clarified the following regarding the mechanism underlying JCV virion assembly; (i) major and minor capsid proteins are synthesized from messenger RNAs, the expression ratio of which is determined by alternative splicing, (ii) messenger RNAs for the major and minor capsid proteins are polycistronic, and their translation occurs downstream of the regulatory protein, agnoprotein, (iii) major and minor capsid proteins are translocated to the nucleus in a cooperative manner and accumulate at the dot-shaped intranuclear structures called promyelocytic leukemia nuclear bodies (PML-NBs), (iv) efficient viral replication can occur at the PML-NBs, where capsid assembly is likely to be associated with viral DNA replication. PML-NBs are the sites for expression of important nuclear functions for the host cells. The finding that the target of JCV infection is the PML-NB may contribute greatly to our understanding of the mechanism underlying cellular degeneration, which occurs after the formation of intranuclear viral inclusions.     

Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch

Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T=13 lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an amphipathic alpha-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal amphipathic alpha-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the amphipathic alpha-helix in the precursor capsid, as a five-alpha-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.     

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly.

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.     

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.     

Structure of a Novel Shoulder-to-Shoulder p24 Dimer in Complex with the Broad-Spectrum Antibody A10F9 and Its Implication in Capsid Assembly

Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4) in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.     

Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro.

The capsid protein (CA) (p24) of human immunodeficiency virus (HIV) type 1 expressed in Escherichia coli and purified to greater than 90% homogeneity was used to examine assembly in vitro and to probe the nature of interactions involved in the formation of capsid structures. The protein was detected in dimeric and oligomeric forms as indicated by molecular size measurements by gel filtration column chromatography, sedimentation through sucrose, and nondenaturing gel electrophoresis. Chemical cross-linking of CA molecules was observed with several homobifunctional reagents. Oligomer size was dependent on cross-linker concentration and exhibited a nonrandom pattern in which dimers and tetramers were more abundant than trimers and pentamers. Oligomers as large as dodecamers were detected in native polyacrylamide gels. These were stable in solutions of high ionic strength or in the presence of nonionic detergent, indicating that strong interactions were involved in oligomer stabilization. Limited tryptic digestion converted the putative dodecamers to octamers, suggesting that a region involved in CA protein multimerization was exposed in the structure. This region was mapped to the central portion of the protein. The recombinant CA proteins assembled in vitro into long rodlike structures and were disassembled into small irregular spheres by alterations in ionic strength and pH. The observation that assembly and disassembly of purified HIV type 1 CA protein can be induced in vitro suggests an approach for identifying possible control mechanisms involved in HIV viral core assembly.     

In Vitro Assembly of an Empty Picornavirus Capsid follows a Dodecahedral Path

The Picornaviridae are a large family of small, spherical RNA viruses that includes numerous pathogens. The picornavirus structural proteins VP0, VP1, and VP3 are believed to first form protomers, which then form 14S particles and subsequently assemble to form empty and RNA-filled particles. 14S particles have long been presumed to be pentamers. However, the structure of the 14S particles, their mechanism of assembly, and the role of empty particles during infection are all unknown. We established an in vitro assembly system for bovine enterovirus (BEV) by using purified baculovirus-expressed proteins. By Rayleigh scattering, we determined that 14S particles are 488 kDa, confirming they are pentamers. Image reconstructions based on negative-stain electron microscopy showed that 14S particles have 5-fold symmetry, and their structures correlate extremely well with the corresponding pentamer from crystal structures of mature BEV. Purified 14S particles readily assemble in response to increasing ionic strength or temperature to form 5.8-MDa 12-pentamer particles, indistinguishable from native empty particles. Surprisingly, empty particles were sufficiently stable that, under physiological conditions, dissociation is unlikely to be a biologically relevant reaction. This suggests that empty particles are not a storage form of 14S particles, at least for bovine enterovirus, but are either a dead-end product or direct precursor into which viral RNA is packaged by as-yet-unidentified machinery.     

Structures of the major capsid proteins of the human Karolinska Institutet and Washington University polyomaviruses

The Karolinska Institutet and Washington University polyomaviruses (KIPyV and WUPyV, respectively) are recently discovered human viruses that infect the respiratory tract. Although they have not yet been linked to disease, they are prevalent in populations worldwide, with initial infection occurring in early childhood. Polyomavirus capsids consist of 72 pentamers of the major capsid protein viral protein 1 (VP1), which determines antigenicity and receptor specificity. The WUPyV and KIPyV VP1 proteins are distant in evolution from VP1 proteins of known structure such as simian virus 40 or murine polyomavirus. We present here the crystal structures of unassembled recombinant WUPyV and KIPyV VP1 pentamers at resolutions of 2.9 and 2.55 Å, respectively. The WUPyV and KIPyV VP1 core structures fold into the same β-sandwich that is a hallmark of all polyomavirus VP1 proteins crystallized to date. However, differences in sequence translate into profoundly different surface loop structures in KIPyV and WUPyV VP1 proteins. Such loop structures have not been observed for other polyomaviruses, and they provide initial clues about the possible interactions of these viruses with cell surface receptors.