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PubMed1996

Conserved features in papillomavirus and polyomavirus capsids.

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.

Animals↗
PubMed1996

Mutational analysis of the bacteriophage P4 capsid-size-determining gene.

Satellite phage P4 (11,624 bp) depends on the morphopoietic genes (capsid, tail) and lysis genes of its helper phage P2 (33.5 kb) for its lytic development. In the morphopoietic process, P4 redirects the assembly pathway of large, P2 size, capsids (diameter = 60 nm) to yield smaller, P4 size, capsids (diameter = 45 nm), 1/3 in volume of that of its helper. The P4-specified capsid size determination is dependent on the function of the 27-kDa gpSid. To study the capsid size-determining function, we carried out a mutational analysis of the P4 sid gene. Use of a P4-derived genome of 29.1 kb (P461), which can be packaged only into large, P2 size, capsids allowed us to select P4 Sid- mutants. By DNA sequencing we characterized 25 P4 Sid- mutants, of which 10 contain base pair substitutions and 15 contain deletions. Both types of mutations are clustered in separate locations within the sid gene. Our results suggest that the Sid polypeptide contains three distinct functional domains.

Amino Acid Sequence↗
PubMed1999

Insertional mutagenesis of AAV2 capsid and the production of recombinant virus.

The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system.

Capsid↗
PubMed2002

Baculovirus expression and antigenic characterization of the capsid proteins of three Norwalk-like viruses.

Human caliciviruses (HuCVs) are antigenically diverse. The antigenic relationships among different HuCVs have been difficult to study because HuCVs cannot be passaged in the laboratory. In this study, we describe cloning, sequencing and expression of the viral capsid proteins of three HuCVs that were identified in outbreaks of acute gastroenteritis in Virginia in 1997-1998. Yields of the capsid proteins similar to previously expressed recombinant Norwalk virus were obtained using the baculovirus expression system. Recombinant VA97207 capsid protein (rVA97207) and rVA98387, but not rVA98115, formed virus-like particles (VLPs). All three recombinant capsid antigens detected seroresponses in patients involved in outbreaks of acute gastroenteritis associated with genetically homologous or related HuCVs. The antigenic relationships of the three strains were further characterized using hyperimmune antisera against the three capsid antigens as well as four previously characterized recombinant capsid antigens of Norwalk (rNV), Mexico (rMxV), Hawaii (rHV), and Grimsby viruses (rGrV). VA98387 shared 98% aa identity with GrV; rVA98387 was detected by antisera to GrV. VA98115 shared 87% aa identity with Desert Storm virus and 65% aa identity with prototype Norwalk virus (NV); rVA98115 reacted weakly with NV antisera. VA97207 shared 80% aa identity with Amsterdam and 75% aa identity with Leeds strains and rVA97207 was not detected by any of the heterologous antibodies. In conclusion, VA97207 and VA98115 may belong to CV antigenic types not previously expressed, while VA98387 is a GrV-like virus. Low levels of cross-reactive antibodies were detected between types. Further studies to characterize these antigens and to develop enzyme immune assays (EIAs) for these strains are in progress.

Animals↗
PubMed1992

Mutations that eliminate the requirement for the vertex protein in bacteriophage T4 capsid assembly.

The capsid of bacteriophage T4 is composed of two essential structural proteins, gp23, the major constituent of the capsid, and gp24, a less prevalent protein that is located in the pentameric vertices of the capsid. gp24 is required both to stabilize the capsid and to allow it to be further matured. This requirement can be eliminated by bypass-24 (byp24) mutations within g23. We have isolated, cloned and sequenced several new byp24 mutations. These mutations are cold-sensitive in the absence of gp24, and are located in regions of g23 not known to contain any other mutations affecting capsid assembly. The cold-sensitivity of the byp24 mutations can be reduced by further mutations within g23 (trb mutations). Cloning and sequencing of these trb mutations has revealed that they lie in regions of g23 that contain clusters of mutations that cause the production of high levels of petite and giant phage (ptg mutations). Despite the proximity of the trb mutations to the ptg mutations, none of the ptg mutations has a Trb phenotype. The mutation ptE920g, which is also located near one of the ptg clusters, and which produces only petite and wild-type phage, has been shown to confer a Trb but not a Byp24 phenotype. The relevance of these observations to our understanding of capsid assembly is discussed.

Alleles↗
PubMed1992

The polarity suppression factor of bacteriophage P4 is also a decoration protein of the P4 capsid.

We show that the product of the polarity suppression (psu) gene from bacteriophage P4 associates with P4 capsids. This association can occur when Psu is (i) provided in vivo from the P4 genome or from a plasmid or (ii) provided in vitro by mixing viable phage particles with Psu protein. Psu is unable to associate with the larger capsid of P4's helper phage P2. Discrimination of the P4 and P2 capsids by Psu appears to be independent of the presence of the P4 genome in the capsid, since P2 size capsids filled with P4 DNA cannot accommodate Psu association. P4 psu particles devoid of Psu are less stable than P4 particles carrying Psu. These results indicate that, in addition to its antitermination activity at Rho-dependent terminators, Psu is also a decoration protein that stabilizes the P4 capsids.

Amino Acid Sequence↗
PubMed2005

Diversity and identity of mechanical properties of icosahedral viral capsids studied with elastic network normal mode analysis.

We analyze the mechanical properties and putative dynamical fluctuations of a variety of viral capsids comprising different sizes and quasi-equivalent symmetries by performing normal mode analysis using the elastic network model. The expansion of the capsid to a swollen state is studied using normal modes and is compared with the experimentally observed conformational change for three of the viruses for which experimental data exist. We show that a combination of one or two normal modes captures remarkably well the overall translation that dominates the motion between the two conformational states, and reproduces the overall conformational change. We observe for all of the viral capsids that the nature of the modes is different. In particular for the T=7 virus, HK97, for which the shape of the capsid changes from spherical to faceted polyhedra, two modes are necessary to accomplish the conformational transition. In addition, we extend our study to viral capsids with other T numbers, and discuss the similarities and differences in the features of virus capsid conformational dynamics. We note that the pentamers generally have higher flexibility and propensity to move freely from the other capsomers, which facilitates the shape adaptation that may be important in the viral life cycle.

Bacteriophages↗
PubMed2005

Domain study of bacteriophage p22 coat protein and characterization of the capsid lattice transformation by hydrogen/deuterium exchange.

Viral capsids are dynamic structures which undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Limited proteolysis and biochemical experiments indicated that the coat protein consists of two domains connected by a flexible loop. To investigate the properties and roles of the sub-domains, we have cloned them and initiated structure and function studies. The N-terminal domain, which is made up of 190 amino acid residues, is largely unstructured in solution, while the C-terminal domain, which consists of 239 amino acid residues, forms a stable non-covalent dimer. The N-terminal domain adopts additional structure in the context of the C-terminal domain which might form a platform on which the N-terminal domain can fold. The local dynamics of the coat protein in both procapsids and mature capsids was monitored by hydrogen/deuterium exchange combined with mass spectrometry. The exchange rate for C-terminal domain peptides was similar in both forms. However, the N-terminal domain was more flexible in the empty procapsid shells than in the mature capsids. The flexibility of the N-terminal domain observed in the solution persisted into the procapsid form, but was lost upon maturation. The loop region connecting the two domains exchanged rapidly in the empty procapsid shells, but more slowly in the mature capsids. The global stabilization of the N-terminal domain and the flexibility encoded in the loop region may be a key component of the maturation process.

Amino Acid Sequence↗
PubMed2006

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.

Active Transport, Cell Nucleus↗
PubMed2006

Bacteriophage T4 capsid: a unique platform for efficient surface assembly of macromolecular complexes.

We report the first description of a macromolecular complex display system using bacteriophage T4. Decorated with two dispensable outer capsid proteins, Hoc (155 copies) and Soc (810 copies), the 120 nm x 86 nm T4 capsid particle offers a unique binding site-rich platform for surface assembly of hetero-oligomeric complexes. To display the 710 kDa anthrax toxin complex, two bipartite functional fusion proteins, LF-Hoc and LFn-Soc, were constructed. Using a defined in vitro binding system, sequential assembly was performed by first attaching LF-Hoc and/or LFn-Soc to hoc-soc- phage, saturating the Hoc and Soc binding sites. Trypsin-nicked PA63 was then assembled into heptamers through specific interaction with the capsid-exposed LFn domain. EF was then attached to the unoccupied sites of PA63 heptamers, completing the assembly of the tripartite anthrax toxin. Negative electron microscopy showed decoration of each capsid with a layer of heptameric PA63 rings. Up to 229 anthrax toxin complexes, equivalent to a total of 2400 protein molecules and a mass of about 133 MDa (2.7 times the mass of capsid shell), were anchored on a single particle, making it the highest density display reported on any virus. The phage T4 capsid lattice provides a stable biological platform allowing maximum display of large hetero-oligomeric complexes in vitro and offers insights for developing novel vaccines, analysis of protein-protein interactions, and structure determination of complexes.

Antigens, Bacterial↗
PubMed2004

The coat protein of Rabbit hemorrhagic disease virus contains a molecular switch at the N-terminal region facing the inner surface of the capsid.

To function adequately, many if not all proteins involved in macromolecular assemblies show conformational polymorphism as an intrinsic feature. This general strategy has been described for many essential cellular processes. Here we describe this structural polymorphism in a viral protein, the coat protein of Rabbit hemorrhagic disease virus (RHDV), which is required during virus capsid assembly. By combining genetic, structure modeling, and cryo-electron microscopy and image processing analysis, we have established the mechanism that allows RHDV coat protein to switch among quasi-equivalent conformational states to achieve the appropriate curvature for the formation of a closed shell. The RHDV capsid structure is based on a T = 3 lattice, containing 180 copies of identical subunits, similar to those of other caliciviruses. The quasi-equivalent interactions between the coat proteins are achieved by the N-terminal region of a subset of subunits, which faces the inner surface of the capsid shell. Mutant coat protein lacking this N-terminal sequence assembles into T = 1 capsids. Our results suggest that the polymorphism of the RHDV T = 3 capsid might bear resemblance to that of plant virus T = 3 capsids.

Amino Acid Sequence↗
PubMed2004

In vitro papillomavirus capsid assembly analyzed by light scattering.

Pentamers of the L1 major capsid protein of human papillomavirus (HPV type 11) were purified after expression in E. coli and analyzed for the kinetics of in vitro capsid self-assembly using multi-angle light scattering (MALS). Pentamers self-assembled into capsid-like structures at a rate that was a function of protein concentration. The kinetics of capsid formation were sigmoidal with a concentration-dependent lag phase, followed by a rapid increase in polymerization. Nucleation size and the rate order of subsequent subunit addition were calculated from the concentration dependence of the extent of capsid formation and the rate of the fast phase, respectively. Assembly was second order with a nucleation size of two pentamers. Thus, we suggest that dimers of pentamers are the nucleus for L1 assembly into capsid-like structures, with rapid sequential addition of single pentamers to the growing shell. Although studied in vitro without accessory factors that may be present in vivo, these data are in contrast with the "five-around-one" assembly nucleus previously proposed for polyomaviruses.

Capsid↗
PubMed2006

Proteolytic mapping of the adeno-associated virus capsid.

The three-dimensional structures of the viral capsid of three AAV serotypes have previously been determined by X-ray crystallography or cryoelectron microscopy. These studies of AAV and similar studies of autonomous parvoviruses have yielded important structural information about the virions in a low-energy conformation. However, there is little information on the structural properties of AAV virions in solution under physiological conditions. We demonstrate that proteolytic digestion of AAV2 virions with trypsin results in cleavage at a specific site on the capsid surface while the capsid remains intact. The products of digestion were mapped using unique antibodies, protein sequencing, mass spectroscopy, and 3D structure modeling to a region on a surface loop that is common to all three AAV2 structural proteins. Empty AAV2 capsids could be distinguished from full (DNA-containing) capsids, having an increased susceptibility of VP2 to trypsin and being digested more rapidly by chymotrypsin. Proteolytic analysis utilizing trypsin or chymotrypsin was also capable of distinguishing AAV2 from AAV1 and AAV5, as seen by differential susceptibility and unique fragment patterns. These data demonstrate a novel approach for studying the structure of AAV capsids in solution and should be valuable in the testing and engineering of AAV vectors for gene transfer.

Amino Acid Sequence↗
PubMed1999

Human anti-JC virus serum reacts with native but not denatured JC virus major capsid protein VP1.

The immunoreactivity of human anti-JC virus (JCV) serum against the major capsid protein VP1 of JCV was analyzed by Western blot, dot blot, and hemagglutination inhibition (HAI) assays. JCV-positive human serum reacted with native but not denatured JCV major capsid protein VP1, as demonstrated by dot blot and Western blot. Rabbit antiserum raised against native JCV capsid had immunoreactivities similar to those of human anti-JCV serum. These results indicate that the antigenecity of native and denatured JCV VP1 is different. In addition, both JCV-positive human serum and rabbit antiserum raised against native JCV capsid protein inhibited the hemagglutination activity of JCV capsid particles. In contrast, rabbit antiserum raised against denatured JCV VP1 did not inhibit hemagglutination. These findings reveal that denaturation may alter the antigenic epitopes of JCV VP1. Therefore, keeping the JCV capsid protein native appears to be essential for serological or other immunological analyses of the virus.

Animals↗
PubMed1996

Assembly of the reovirus outer capsid requires mu 1/sigma 3 interactions which are prevented by misfolded sigma 3 protein in temperature-sensitive mutant tsG453.

A temperature-sensitive reovirus mutant, tsG453, whose defect was mapped to major outer capsid protein sigma 3, makes core particles but fails to assemble the outer capsid around the core at non-permissive temperature. Previous studies that made use of electron cryo-microscopy and image reconstructions showed that mu 1, the other major outer capsid protein, but not sigma 3, interact extensively with the core capsid. Although wild-type sigma 3 and mu 1 interact with each other, immunocoprecipitation studies showed that mutant sigma 3 protein was incapable of interacting with mu 1 at the non-permissive temperature. In addition, restrictively-grown mutant sigma 3 protein could not be precipitated by some sigma 3-specific monoclonal antibodies. These observations suggest that in a wild-type infection, specific sigma 3 and mu 1 interactions result in changes in mu 1 conformation which are required to allow mu 1/sigma 3 complexes to condense onto the core capsid shell during outer capsid assembly, and that sigma 3 in non-permissive tsG453 infections is misfolded such that it cannot interact with mu 1.

Capsid↗
PubMed2005

Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl monkey cells.

In this study, we asked if a naturally occurring HIV-1 variant exists that circumvents CypA dependence in human cells. To address this issue, we sought viruses for CypA independence using Debio-025, a cyclosporine A (CsA) analog that disrupts CypA-capsid interaction. Surprisingly, viral variants from the Main group replicate even in the presence of the drug. Sequencing analyses revealed that these viruses encode capsid substitutions within the CypA-binding site (V86P/H87Q/I91V/M96I). When we introduced these substitutions into viruses that normally rely on CypA for replication, these mutants no longer depended on CypA, suggesting that naturally occurring capsid substitutions obviate the need for CypA. This is the first demonstration that isolates from the Main group naturally develop CypA-independent strategies to replicate in human cells. Surprisingly, we found that these capsid substitutions render HIV-1 capable of infecting Owl monkey (OMK) cells that highly restrict HIV-1. OMK cell resistance to HIV-1 is mediated via TRIM-Cyp, which arose from a retrotransposition of CypA into the TRIM5 alpha gene. Interestingly, saturation experiments suggest that the Pro86/Gln87/Val91/Ile96 capsid core is "invisible" to TRIM-Cyp. This study demonstrates that specific capsid substitutions can release HIV-1 from both CypA dependence in human cells and TRIM-Cyp restriction in monkey cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The adenovirus capsid: major progress in minor proteins.

Human adenoviruses have been the subject of intensive investigation since their discovery in the early 1950s: they have served as model pathogens, as probes for studying cellular processes and, more recently, as efficient gene-delivery vehicles for experimental gene therapy. As a result, a detailed insight into many aspects of adenovirus biology is now available. The capsid proteins and in particular the hexon, penton-base and fibre proteins (the so-called major capsid proteins) have been studied extensively and their structure and function in the virus capsid are now well-defined. On the other hand, the minor proteins in the viral capsid, i.e. proteins IIIa, VI, VIII and IX, have received much less attention. Only the last few years have witnessed a sharp increase in the number of studies on their structure and function. Here, a review of the minor capsid proteins is provided, with a focus on new insights into their position and role in the capsid and the opportunities that they provide for improving human adenovirus-derived gene-delivery vectors.

Adenoviruses, Human↗
PubMed2005

Structural studies on recombinant T = 3 capsids of Sesbania mosaic virus coat protein mutants.

When expressed in Escherichia coli, the recombinant coat protein (rCP) of Sesbania mosaic virus (SeMV) was shown to self-assemble into T = 3 capsids encapsidating CP mRNA and 23S rRNA derived from the host. Expression of CP-P53A, in which a conserved proline at position 53 in the beta-annulus was substituted by alanine (CP-P53A), also produced similar capsids. Purified rCP and CP-P53A particles were crystallized and X-ray crystal structures of their mutant capsids were determined to resolutions of 3.6 and 4.1 A, respectively. As in the native viral CP, the CPs in these recombinant capsids adopt the jelly-roll beta-sandwich fold. The amino-terminal residues of the C subunits alone are ordered and form the beta-annulus structure at the quasi-sixfold axes. A characteristic bend in the beta-annulus remains unaffected in CP-P53A. The quasi-threefold interfaces of the capsids harbour calcium ions coordinated by ligands from the adjacent threefold-related subunits in a geometry that is analogous to that observed in the native capsid. Taken together with studies on deletion and substitution mutants of SeMV CP, these results suggest the possibility that the beta-annulus and nucleic acid-mediated interactions may be less important for the assembly of sobemoviruses than previously envisaged.

Calcium↗