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PubMed2004

E. coli-expressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability.

The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction. We report here the application of the E. coli expression system in the production of recombinant NV capsid proteins. In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E. coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E. coli-expressed VA387 proteins did not form VLPs. Using the E. coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207. Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors. VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus. Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells.

Antigens, Viral↗
PubMed1994

Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution.

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 A resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VP0 to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N-terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N-terminal extension of VP1, as well as portions corresponding to VP4 and the N-terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP0 scissile bond is located some 20 A away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoil-shaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA-dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N-terminal network and in generating stable virions.

Capsid↗
PubMed1994

Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of asymmetric Gag dimers.

The primary functions of most virus capsids are to protect the viral genome in the extra-cellular milieu and deliver it to the host. In contrast, the capsids of fungal viruses, like the cores of all other known double stranded RNA viruses, are not involved in host recognition but do shield their genomes, and they also carry out transcription and replication. Nascent (+) strands are extruded from transcribing virions. The capsids of the yeast virus L-A are composed of Gag (capsid protein; 76 kDa), with a few molecules of Gag-Pol (170 kDa). Analysis of these 420 A diameter shells and those of the fungal P4 virus by cryo-electron microscopy and image reconstruction shows that they share the same novel icosahedral structure. Both capsids consist of 60 equivalent Gag dimers, whose two subunits occupy non-equivalent bonding environments. Stoichiometry data on other double-stranded RNA viruses indicate that the 120-subunit structure is widespread, implying that this molecular architecture has features that are particularly favorable to the design of a capsid that is also a biosynthetic compartment.

Capsid↗
PubMed1995

Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system.

Bacteriophage capsid assembly pathways provide excellent model systems to study large-scale conformational changes and other mechanisms that regulate the formation of macromolecular complexes. These capsids are formed from proheads: relatively fragile precursor particles which mature by undergoing extensive remodeling. Phage HK97 employs novel features in its strategy for building capsids, including assembly without a scaffolding protein, and the formation of a network of covalent cross-links between neighboring subunits in the mature virion. In addition, proteolytic cleavage of the capsid protein from 42 kDa to 31 kDa is essential for maturation. To investigate the structural bases for proteolysis and cross-linking, we have used cryo-electron micrographs to reconstruct the three-dimensional structures of purified particles from four discrete stages in the assembly pathway: Prohead I, Prohead II, Head I and Head II. Prohead I has icosahedral T = 7 packing of blister-shaped pentamers and hexamers. The pentamers are 5-fold symmetric, but the hexamers exhibit an unusual departure from 6-fold symmetry, as if two trimers had undergone a shear dislocation of about 25 A. Proteolytic conversion to Prohead II leaves the outer surface largely unchanged, but a major loss of density from the inner surface is observed, which we infer to represent the excision of the amino-terminal domains of the capsid protein. Upon expansion to the Head I state, the capsid becomes markedly larger, thinner walled, and more polyhedral: moreover, the capsomer shapes change radically; especially notable is the disappearance of the large hexon dislocation. No differences between Head I and the covalently cross-linked Head II could be observed at the current resolution of about 25 A, from which we infer that it is the conformational rearrangements effected by expansion that create the micro-environments needed for the autocatalytic formation of the isodipeptide bonds found in the mature virions ("pseudo-active sites").

Amino Acid Sequence↗
PubMed1995

A novel basis of capsid stabilization by antiviral compounds.

Picornaviruses are inactivated by a family of hydrophobic drugs that bind at an internal site in the viral capsid and inhibit viral uncoating. A basis for the capsid stabilization previously unrecognized is revealed by molecular dynamics simulations of the antiviral drug WIN52084s bound to a hydrophobic pocket of solvated human rhinovirus 14. Isothermal compressibilities of the complex and human rhinovirus 14 without the antiviral drug calculated from density fluctuations show that the presence of WIN52084s increases the compressibility of the viral capsid near the antiviral drug. This counterintuitive result is understandable on the basis of the empirical evidence of thermal melting temperatures and protein-folding entropies of globular proteins. Based on this evidence, we propose that a larger compressibility from drug binding confers greater thermal stability to capsid proteins by increasing the conformational entropy of capsids, thereby diminishing the entropy gain with uncoating. We suggest that compressibility is fundamental to the structural integrity of viral capsids and that examination of compressibility and antiviral activity will provide insights into the disassembly process.

Antiviral Agents↗
PubMedDate not supplied

The effects of radiation damage on the structure of frozen hydrated HSV-1 capsids.

Radiation damage imposes stringent limits on the information content of electron micrographs of biological specimens. In this study, we have investigated its effects on frozen, hydrated specimens and three-dimensional reconstructions calculated from cryomicrographs using capsids of herpes simplex virus as a model system. Multiple-exposure series of micrographs of both B-capsids (which contain no DNA) and C-capsids (which are fully packaged) were recorded and reconstructions were calculated from the first exposures, corresponding to a cumulative electron dose of 6-7 e-/A2, and from later exposures (25-40 e-/A2). Experimental procedures were standardized to ensure that perceived changes in the micrographs and reconstructions would be attributable to radiation damage alone. The effects of the higher doses in both the micrographs and the reconstructions were expressed as a progressive blurring of the finer details, corresponding to a delocalization of structure in the ice-embedded specimens. The resolutions of the reconstructions were quantified according to a form of the Fourier ring correlation coefficient criterion, according to which the first-exposure reconstructions had resolutions of 30-36 A. The fifth-exposure B-capsid reconstruction had comparable nominal resolution, although it exhibited progressively lower correlations at higher spatial frequencies. Qualitatively similar changes in the series of C-capsid reconstructions were observed although they were more pronounced, presumably because these micrographs had lower contrast and signal-to-noise ratios. We infer that the observed changes in the images and reconstructions and the concomitant loss in contrast in the immediate vicinity of the capsid surface may reflect radiation-induced perturbation of molecular structure and/or the release of peptide fragments. Nevertheless, the observed changes are relatively subtle, at least at the operational resolution of this study; overall, our results support earlier indications (M. F. Schmid et al. J. Struct. Biol. 108, 62-68, 1992) that prospects are quite good for tilt-series reconstructions from cryoelectron micrographs, including six to eight views of the same specimen.

Capsid↗
PubMed1998

Structure of the human cytomegalovirus B capsid by electron cryomicroscopy and image reconstruction.

The three-dimensional structure of B capsids of the beta-herpesvirus human cytomegalovirus (HCMV) was investigated at a resolution of 3.5 nm from electron cryomicrographs by image processing and compared with the structure obtained for the alpha-herpesvirus herpes simplex virus type 1 (HSV-1). The main architectural features of the HSV-1 and HCMV capsids are similar: the T = 16 icosahedral lattice consists of 162 capsomers, composed of two distinct morphological units, 12 pentamers and 150 hexamers, with triplex structures linking adjacent capsomers at positions of local threefold symmetry. The main differences in the HSV-1 and HCMV capsids are found in the diameter of the capsids (125 and 130 nm, respectively); the hexamer spacing and relative tilt (center-to-center hexon spacing at outer, edge, 17.9 and 15.8 nm, respectively); the morphology of the tips of the hexons (similar in length but 33% thinner in HCMV); and the average diameter of the scaffold (44 and 76 nm, respectively). By analogy with HSV-1, the mass on the HCMV hexon tip is attributed to the smallest capsid protein (HCMV gene UL48/49). The differences in capsid structure are discussed in relation to the ability of the HCMV structure to package a genome some 60% larger than that of HSV-1.

Capsid↗
PubMed1994

Molecular characterization and expression of the capsid protein of a Norwalk-like virus recovered from a Desert Shield troop with gastroenteritis.

Norwalk virus (NV) infection was recently found to be associated with gastroenteritis in U.S. military troops stationed in Saudi Arabia during the 1990 Desert Shield Operation. We identified a Norwalk-like virus in the stools of two military personnel with gastroenteritis by ELISA and IEM. By RT-PCR and sequence analysis, the nucleotide sequence of part of the polymerase region of each of these two "Desert Shield" strains (DSV275 and DSV395) was found to be 73% identical to the corresponding region of NV. In addition, one of the strains (DSV395), which underwent sequence analysis of approximately 2900 consecutive bases, had a genomic organization characteristic of the Caliciviridae. Comparison of the DSV395 amino acid sequence of the capsid region with that of three other viruses in the Norwalk group (Norwalk, Southampton, and Toronto viruses) showed amino acid identity of 47-68%. Consensus sequence analysis of these capsid proteins identified two regions of conserved amino acids that flanked an area of variable amino acids. In addition, the proteins corresponding to the capsid regions of DSV395 and NV were expressed in an in vitro translation system. Immunoprecipitation studies using the expressed capsid proteins and paired DSV395 or NV infection sera indicated the presence of shared antigenic sites between the capsid proteins of DSV395 and NV. However, hyperimmune sera specific for the self-assembled recombinant NV capsid protein did not react with DSV stool antigen in an ELISA, suggesting that there may also be unique antigenic sites not shared between DSV395 and NV.

Amino Acid Sequence↗
PubMed1995

Most of the VP1 unique region of B19 parvovirus is on the capsid surface.

B19 parvovirus is pathogenic in man and a vaccine is desirable. In convalescence after acute infection, the dominant humoral immune response is directed to the minor capsid protein called VP1, which differs from the major capsid protein by an additional NH2-terminal 227 amino acids. We have previously shown that this unique region contains multiple linear neutralizing epitopes. We produced seven recombinant B19 capsids that contained progressively truncated VP1 unique region sequences, each fused to a Flag peptide (AspTyrLysAspAspAspAspLys) at the NH2-terminus. Capsids containing normal VP2 and truncated Flag-VP1 proteins and, in some cases, only truncated Flag-VP1 chimeric proteins, were analyzed by ELISA, affinity chromatography, and electron microscopy using anti-Flag monoclonal antibody. All regions examined showed binding to anti-Flag antibody in multiple assays, indicating that most of the VP1 unique region is external to the capsid and accessible to antibody binding. These results have implications for the design of a B19 parvovirus vaccine and the use of empty capsids for presentation of heterologous protein antigens.

Amino Acid Sequence↗
PubMed1996

Antigenic mapping of the recombinant Norwalk virus capsid protein using monoclonal antibodies.

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.

Animals↗
PubMed1998

Crystallization and preliminary X-ray analysis of the dsDNA bacteriophage HK97 mature empty capsid.

HK97 is a temperate dsDNA bacteriophage of Escherichia coli that is structurally similar to phage lambda, with an icosahedral head of triangulation (7) number 7. Although the capsids of several large dsDNA phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available. We have grown crystals of mature but empty bacteriophage HK97 capsids that diffract to at least 3.5 A using synchrotron radiation. The HK97 Head II crystals are the first capsid crystals from a dsDNA bacteriophage that diffract X-rays to high resolution. A capsid precursor (prohead) was made in vivo by expressing capsid proteins in E. coli. This prohead was purified, converted to Head II in vitro, and used to grow crystals. The empty Head II has the same form as the mature HK97 capsid, but without DNA. The crystals were grown in a mixture of ammonium sulfate and PEG 8000, directly in an X-ray capillary to minimize crystal handling. The unit cell is monoclinic, with dimensions a = 580 A, b = 625 A, c = 790 A, beta = 90.0 degrees and two particles per unit cell.

Bacteriophages↗
PubMed1999

A 110-kDa nuclear shuttle protein, nucleolin, specifically binds to adeno-associated virus type 2 (AAV-2) capsid.

A 110-kDa protein was copurified with adeno-associated virus type 2 (AAV-2) virions after CsCl density gradient isopycnic centrifugation. Amino acid sequence of peptides derived from this protein after tryptic digestion, monoclonal antibody production, and Western blot analysis showed that the copurified protein was the major nucleolar phosphoprotein, human nucleolin. Virus overlay assays demonstrated that AAV-2 capsid specifically bound to the human nucleolin, and immunoprecipitation studies confirmed the in vitro binding of nucleolin and intact AAV-2 capsids but not denatured viral proteins. Double-immunofluorescence staining of infected cells showed that AAV capsid and nucleolin were colocalized in both cytoplasm and nucleus. In addition, when cytoplasmic and nuclear fractions were extracted from AAV-infected KB cells at different time points postinfection, immunoprecipitation data and Western blotting showed that AAV capsid formation and nucleolin interact specifically and share their subcellular localization in infected cells. With the known functions of nucleolin in the synthesis of rRNA and ribosome assembly, binding to single-stranded DNA, and acting as a shuttle between cytoplasm and nucleolus, our data showing that AAV-2 capsid binds specifically to nucleolin both in vitro and in vivo suggest a key role of nucleolin in AAV-2 replication, particularly in capsid assembly.

Animals↗
PubMed2000

Rubella virus capsid protein induces apoptosis in transfected RK13 cells.

Rubella virus is an enveloped positive-strand RNA virus that can cause mild to severe birth defects or death in an infected fetus. RV induction of programmed cell death, demonstrated in cell culture, has been implicated in the pathogenesis. The timing of apoptosis, 48 h p.i., suggested that accumulation of RV structural proteins might induce cell death in infected cells. Expression of RV structural proteins, capsid, envelope glycoproteins E1 and E2, in transiently transfected RK13 cells was as potent an inducer of cell death as RV infection. Immunofluorescence microscopy revealed that RV structural protein transfected cells exhibited the condensed nuclei typical of apoptotic cell death. Transfection with the capsid protein construct, but not E2 and E1, resulted in as much cell death as joint expression of all three RV structural proteins. Capsid required a membrane-anchoring domain to induce cell death, but a heterologous polypeptide fused to the capsid membrane anchor did not cause apoptosis. Deletion mutants demonstrated that the apoptosis-inducing activity resides in the N-terminal 170 amino acids of capsid. Though apoptosis-inducing capsid constructs appear to have an ER sub-cellular localization, disruption of the ER calcium storage capacity does not correlate with cell death. Mechanisms consistent with these results are discussed.

Animals↗
PubMed2001

Effect of mutations in Gag on assembly of immature human immunodeficiency virus type 1 capsids in a cell-free system.

Studies of HIV-1 capsid formation in a cell-free system revealed that capsid assembly occurs via an ordered series of assembly intermediates and requires host machinery. Here we use this system to examine 12 mutations in HIV-1 Gag that others studied previously in intact cells. With respect to capsid formation, these mutations generally produced the same phenotype in the cell-free system as in cells, indicating the cell-free system's high degree of fidelity. Analysis of assembly intermediates reveals that a mutation in the distal region of CA (322 LDeltaS) and truncations proximal to the second cys-his box in NC block multimerization of Gag at early stages in the cell-free capsid assembly pathway. In contrast, mutations in the region of amino acids 56-68 (located in the proximal portion of MA) inhibit assembly at a later point in the pathway. Other mutations, including truncations distal to the first cys-his box in NC and mutations in the distal half of MA (88HDeltaG, 85YDeltaG, Delta104-115, and Delta115-129), do not affect formation of immature capsids in the cell-free system. These data provide new information on the role of different domains in Gag during the early events of capsid assembly.

Amino Acid Sequence↗
PubMed2002

Molecular interactions and viral stability revealed by structural analyses of chemically treated cypovirus capsids.

Cytoplasmic polyhedrosis virus (CPV, genus Cypovirus) is a unique member of the family Reoviridae which lacks the outer protective shells that exist in all other members, yet exhibits unusual stability. We have analyzed the effects of different acidic, basic, detergent, and urea treatments on CPV capsids. The integrity of the CPV capsids was unaffected under high-pH conditions that disrupted the orthoreovirus inner core, consistent with its ability to maintain structural integrity in extremely alkaline environments during infection. However, it was sensitive to low pH, detergents, and urea, similarly to other viruses in this family. The three-dimensional structure comparisons by electron cryomicroscopy of the intact empty CPV capsid with the "spikeless" capsid whose turrets were removed by chemical treatments revealed the interaction footprint of the turret on the capsid shell. The observed structural changes associated with the removal of the turret suggest critical structural roles of the turret in maintaining capsid integrity in addition to its enzymatic activities.

Capsid↗
PubMed1997

Human calicivirus genogroup II capsid sequence diversity revealed by analyses of the prototype Snow Mountain agent.

The Snow Mountain agent (SMA) is the prototype genogroup II and serotype 3 human calicivirus responsible for epidemic outbreaks of acute gastroenteritis. We have cloned the region of the SMA genome that encodes the single capsid protein. The predicted amino acid sequence of the capsid protein is distinct from other calicivirus strains that have been termed SMA-like based on sequence similarity between the RNA polymerase regions and IEM reactivity. In a previous report, a high sequence similarity in a small region of the RNA polymerase between SMA and another strain, OTH-25, suggested that the capsid proteins of OTH-25 and SMA would be very similar. In this report, we show that the capsid proteins of OTH-25 and SMA are more distinct than was predicted by similarity in the RNA polymerase. In addition, phylogenetic analysis of a region of the RNA polymerase and of the N-terminal conserved domain of the capsid protein of 12 human caliciviruses resulted in trees with different topologies, suggesting that recombination has occurred within this group of viruses. Molecular characterization of the prototype calicivirus strains is important in determining the relationships between capsid similarity at the amino acid level, genetic grouping by sequence comparison, and antigenic reactivity.

Amino Acid Sequence↗
PubMed1997

Cleavage of incoming Semliki Forest virus capsid protein within the endocytotic pathway: a feature common to both invertebrate and mammalian cells.

During the initial stages of Semliki Forest virus (SFV) infection in mammalian (baby hamster kidney, BHK) cells, the cleavage of SFV capsid protein could be detected. Analysis of subcellular fractions from SFV-infected BHK cells showed that (a) cleavage of the capsid protein occurred within a prelysosomal compartment of the endocytotic pathway, and (b) following release of the nucleocapsid into the cytoplasm, a 17.5 kD capsid protein fragment could be detected in the subcellular fraction which contained ribosomes. We have previously reported the cleavage of incoming SFV capsid protein in mosquito cells, too. Thus, the proteolytic cleavage of incoming SFV capsid protein is a feature which is common to both invertebrate and mammalian cells. These data further support our hypothesis that the cleavage of incoming capsid protein might provide the conformational change which primes the SFV nucleocapsid for uncoating.

Aedes↗
PubMed1983

Molecular architecture of bacteriophage T7 capsid.

To determine the capsid structure of bacteriophage T7, we have investigated polycapsids, tubular capsid-related structures isolated from lysates of the T7 mutant am16. Biochemical analysis shows polycapsids to be composed of gp10, the major structural protein of the wild-type capsid. The conformational state of gp10 in polycapsids is indistinguishable from that in the mature virus capsid by the criteria of surface charge, buoyant density, and insensitivity to proteolysis by trypsin. Optical diffraction of electron micrographs of negatively stained polycapsids reveals a hexagonal surface lattice of periodicity 12.6 +/- 0.2 nm and is used to analyze the distribution of cylindrical foldings of this lattice into polycapsids (polymorphic variation). These foldings are found to be related to that of the capsid proper through the intrinsic curvature of gp10, each folding having a set of lattice lines whose radius of curvature is close to 29 nm. The fine structure of this surface lattice has been elucidated by digital image processing of electron micrographs. The capsomer is shown unequivocally to be a hexamer of characteristic morphology. By collating these results with earlier observations, we conclude that the structure of the normal T7 capsid is an orthodox icosahedron of triangulation class T = 7, composed of 60 hexamers and 12 pentamers.

Capsid↗