Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion

The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn(2+) ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.     

The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 A resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.     

Visualization of the externalized VP2 N termini of infectious human parvovirus B19

The structures of infectious human parvovirus B19 and empty wild-type particles were determined by cryoelectron microscopy (cryoEM) to 7.5-Å and 11.3-Å resolution, respectively, assuming icosahedral symmetry. Both of these, DNA filled and empty, wild-type particles contain a few copies of the minor capsid protein VP1. Comparison of wild-type B19 with the crystal structure and cryoEM reconstruction of recombinant B19 particles consisting of only the major capsid protein VP2 showed structural differences in the vicinity of the icosahedral fivefold axes. Although the unique N-terminal region of VP1 could not be visualized in the icosahedrally averaged maps, the N terminus of VP2 was shown to be exposed on the viral surface adjacent to the fivefold β-cylinder. The conserved glycine-rich region is positioned between two neighboring, fivefold-symmetrically related VP subunits and not in the fivefold channel as observed for other parvoviruses.     

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.     

Conformational changes in adeno-associated virus type 1 induced by genome packaging

Adeno-associated virus (AAV) is frequently used as a vector for gene therapy. The viral capsid consists of three structural proteins (VP1, VP2, and VP3) that have a common C-terminal core (VP3), with N-terminal extensions of increasing length in VP2 and VP1. The capsid encloses a single-stranded genome of up to 4.7 kb, which is packaged into empty capsids. The N-terminal extension of VP1 carries a phospholipase domain that becomes accessible during infection in the endosomal pathway. We have used cryo-electron microscopy and image reconstruction to determine subnanometer-resolution structures of recombinant AAV1 that has packaged different amounts of a 3. 6-kb recombinant genome. The maps show that the AAV1 capsid undergoes continuous conformational changes upon packaging of the genome. The rearrangements occur at the inner capsid surface and lead to constrictions of the pores at the 5-fold symmetry axes and to subtle movements of the β-sheet regions of the capsid proteins. In fully packaged particles, the genome forms stem-like features that contact the inner capsid surface at the 3-fold symmetry axes. We think that the reorganization of the inner surface has an impact on the viral life cycle during infection, preparing the externalization of phospholipase domains through the pores at the 5-fold symmetry axes and possibly genome release.     

Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells.

Rotaviruses are triple-layered particles that contain four major capsid proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP3. We have cloned each of the rotavirus genes coding for a major capsid protein into the baculovirus expression system and expressed each protein in insect cells. Coexpression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The coexpression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Coexpression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of nonneutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs. The production of VP2/4/6 particles indicated that VP4 interacts with VP6. Cell binding assays performed with each of the VLPs indicated that VP4 is the viral attachment protein. Chimeric particles containing VP7 from two different G serotypes also were obtained. The ability to express individual proteins or to coexpress different subsets of proteins provides a system with which to examine the interactions of the rotavirus structural proteins, the role of individual proteins in virus morphogenesis, and the feasibility of a subunit vaccine.     

Three-dimensional structure of the rotavirus haemagglutinin VP4 by cryo-electron microscopy and difference map analysis.

The three-dimensional structure of the rotavirus spike haemagglutinin viral protein 4 (VP4) has been determined to a resolution of 26 A by cryo-electron microscopy and difference analysis of intact virions and smooth (spikeless) particles. Native and spikeless virions were mixed prior to cryo-preservation so that both structures could be determined from the same micrograph, thereby minimizing systematic errors. This mixing strategy was crucial for difference map analysis since VP4 only accounts for approximately 1% of the virion mass. The VP4 spike is multi-domained and has a radial length of approximately 200 A with approximately 110 A projecting from the surface of the virus. Interactions between VP4 and cell surface receptors are facilitated by the bi-lobed head, which allows multi-site interactions, as well as the uniform distribution of the VP4 heads at maximum radius. The bi-lobed head is attached to a square-shaped body formed by two rods that have a slight left-handed helical twist. These rods merge with an angled, rod-like domain connected to a globular base approximately 85 A in diameter. The anchoring base displays pseudo 6-fold symmetry. This surprising finding may represent a novel folding motif in which a single polypeptide of VP4 contributes similar but non-equivalent domains to form the arms of the hexameric base. The VP4 spike penetrates the virion surface approximately 90 A and interacts with both outer (VP7) and inner (VP6) capsid proteins. The extensive VP4-VP7 and VP4-VP6 interactions imply a scaffolding function in which VP4 may participate in maintaining precise geometric register between the inner and outer capsids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T = 3 capsid protein leads to larger T = 4 capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.     

Intermolecular interactions in a two-layered viral capsid that requires a complex symmetry mismatch

The surface of the bluetongue virus core forms a T=13 quasiequivalent icosahedral protein shell with 260 trimers of a single gene product: VP7 protein. Underneath is a smooth layer, made up of VP3 protein, which appears to guide and nucleate the assembly of VP7 trimers. The contacts between the two shells are extensive but nonspecific, and construction of the T=13 icosahedral shell requires polymorphism in the association of the VP7 subunits, each of which has two domains that contribute to trimer formation. We used structural and relative sequence information to guide an investigation of how such a complex structure is achieved during virus assembly and what residues are required to form a stable capsid. Fifteen single or multiple site-specific substitution mutations were introduced into the helical domain of VP7, which is closely associated with the VP3 layer, and the effects on capsid assembly were analyzed. Our data show that both the position and the nature of single residues are critical for the attachment of VP7 to VP3 and that formation of a stable VP7 lattice is not the automatic consequence of trimer formation.     

A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini

The complex infection process of parvoviruses is not well understood so far. An important role has been attributed to a phospholipase A2 domain which is located within the unique N terminus of the capsid protein VP1. Based on the structural difference between adeno-associated virus type 2 wild-type capsids and capsids lacking VP1 or VP2, we show via electron cryomicroscopy that the N termini of VP1 and VP2 are involved in forming globules inside the capsids of empty and full particles. Upon limited heat shock, VP1 and possibly VP2 become exposed on the outsides of full but not empty capsids, which is correlated with the disappearance of the globules in the inner surfaces of the capsids. Using molecular modeling, we discuss the constraints on the release of the globularly organized VP1-unique N termini through the channels at the fivefold symmetry axes outside of the capsid.     

Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6A resolution: implications in virion assembly and immunogenicity

The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6A resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca(2+)-chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca(2+)-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

Expression of Rotavirus Capsid Protein VP6 in Transgenic Potato and Its Oral Immunogenicity in Mice

Murine rotavirus gene six encoding the 41 kDa group specific capsid structural protein VP6 was stably inserted into the Solanum tuberosum genome by Agrobacterium tumefaciens mediated transformation. The molecular mass of plant synthesized VP6 capsid protein determined by immunoblot was similar to the size of both purified virus VP6 monomeric peptides and partially assembled virus-like particles. The amount of VP6 protein synthesized in transgenic potato leaf and tuber was determined by enzyme-linked immunosorbent assay to be approximately 0.01% of total soluble protein. Oral immunization of CD-1 mice with transformed potato tuber tissues containing VP6 capsid protein generated measurable titers of both anti-VP6 serum IgG and intestinal IgA antibodies. The presence of detectable humoral and intestinal antibody responses against the rotavirus capsid protein following mucosal immunization provides an optimistic basis for the development of edible plant vaccines against enteric viral pathogens.     

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus‐like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self‐assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK^? E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:744–748, 2014     

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.     

Nuclear transport of trimeric assembly intermediates exerts a morphogenetic control on the icosahedral parvovirus capsid

The connection between nuclear transport and morphogenesis of a large macromolecular entity has been investigated using the karyophylic capsid of the parvovirus minute virus of mice (MVM) as a model. The VP1 (82 kDa) and VP2 (63 kDa) proteins forming the T = 1 icosahedral MVM capsid at the respective 1:5 molar ratio of synthesis, could be covalently cross-linked with dimethyl suberimidate into two types of oligomeric assemblies, which were present at stoichiometric amounts in infected cell extracts and purified viral particles. The larger species contained VP1 and corresponded in size (200 kDa) to a heterotrimer of one VP1 and two VP2 subunits. The smaller species contained VP2 only and corresponded in size (180 kDa) to a homotrimer. The introduction of bulky residues or the truncation of side-chains involved in multiple interactions at the interfaces between trimers of VPs in the MVM capsid, produced the accumulation of trimeric intermediates that were competent in nuclear translocation but not in capsid assembly. These results indicate that MVM maturation proceeds by cytoplasmic oligomerization of the capsid subunits into two types of trimers, which are the assembly intermediates competent to translocate across the nuclear membrane. Consistent with this conclusion, mutations at basic residues that inactivate a previously identified beta-stranded nuclear localization motif, which notably are not involved in inter or intra-subunit contacts, led to cytoplasmic retention of the two types of trimers, with no evidence for other assembly intermediates. Although a fraction of the VP1-containing trimers were translocated into the nucleus driven by the conventional nuclear transport signal of VP1 N terminus, their further assembly in the absence of the VP2-only trimers yielded large molecular mass amorphous aggregates. Therefore, the nuclear transport stoichiometry of assembly intermediates may exert a morphogenetic quality control on macromolecular complexes like the MVM capsid.