DNA Binding and Condensation Properties of the Herpes Simplex Virus Type 1 Triplex Protein VP19C

Herpesvirus capsids are regular icosahedrons with a diameter of a 125 nm and are made up of 162 capsomeres arranged on a T = 16 lattice. The capsomeres (VP5) interact with the triplex structure, which is a unique structural feature of herpesvirus capsid shells. The triplex is a heterotrimeric complex; one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent capsomeres. VP19C interacts with VP23 and with the major capsid protein VP5 and is required for the nuclear localization of VP23. Mutation of VP19C results in the abrogation of capsid shell synthesis. Analysis of the sequence of VP19C showed the N-terminus of VP19C is very basic and glycine rich. It was hypothesized that this domain could potentially bind to DNA. In this study an electrophoretic mobility shift assay (EMSA) and a DNA condensation assay were performed to demonstrate that VP19C can bind DNA. Purified VP19C was able to bind to both a DNA fragment of HSV-1 origin as well as a bacterial plasmid sequence indicating that this activity is non-specific. Ultra-structural imaging of the nucleo-protein complexes revealed that VP19C condensed the DNA and forms toroidal DNA structures. Both the DNA binding and condensing properties of VP19C were mapped to the N-terminal 72 amino acids of the protein. Mutational studies revealed that the positively charged arginine residues in this N-terminal domain are required for this binding. This DNA binding activity, which resides in a non-conserved region of the protein could be required for stabilization of HSV-1 DNA association in the capsid shell.     

A direct comparison of human papillomavirus type 16 L1 particles reveals a lower immunogenicity of capsomeres than viruslike particles with respect to the induced antibody response

Capsomeres are considered to be an alternative to viruslike particle (VLP)-based vaccines as they can be produced in prokaryotic expression systems. So far, no detailed side-by-side comparison of VLPs and capsomeres has been performed. In the present study, we immunized mice with insect cell-derived human papillomavirus type 16 VLPs and capsomeres. VLPs induced consistently higher antibody titers than capsomeres but the two forms induced similar CD8 T-cell responses after subcutaneous, intranasal, and oral immunization, and at least 20 to 40 times more L1 in the form of capsomeres than in the form of VLPs was needed to achieve comparable antibody responses. These results were confirmed by DNA immunization. The lower immunogenicity of capsomeres was independent of the isotype switch, as it was also observed for the early immunoglobulin M responses. Although there were differences in the display of surface epitopes between the L1 particles, these did not contribute significantly to the differences in the immune responses. capsomeres were less immunogenic than VLPs in Toll-like receptor 4 (TLR4)-deficient mice, suggesting that the lower immunogenicity is not due to a failure of capsomeres to trigger TLR4. We observed better correlation between antibody results from enzyme-linked immunosorbent assays and neutralization assays for sera from VLP-immunized mice than for sera from capsomere-immunized mice, suggesting qualitative differences between VLPs and capsomeres. We also showed that the lower immunogenicity of capsomeres could be compensated by the use of an adjuvant system containing MPL. Taken together, these results suggest that, presumably because of the lower degree of complexity of the antigen organization, capsomeres are significantly less immunogenic than VLPs with respect to the humoral immune response and that this characteristic should be considered in the design of putative capsomere-based prophylactic vaccines.     

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.     

Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.     

Copillaria hepatica: Fine structure of egg shell

The structure of the Capillaria hepatica egg shell was studied with the electron microscope and correlated with light microscope histochemical observations. The shell is composed of fibrous and nonfibrous components, both of which stain for protein. The fibrous component, the major portion of the shell, consists of submicroscopic fibers. The nonfibrous component is located in the outer region of the shell but is not always visible; when present it has a reticulated appearance in electron micrographs. The fibrous component is divided into outer and inner regions. The outer region is composed of radially arranged pillars which are connected at their outer surface by a beam-like network and are anchored at the base to a compact inner region. The inner region consists of a series of concentrically arranged lamellae above which is located a nonlaminated region where the pillar bases originate. At each polar end of the shell is a single opening plugged with a material which contains acid mucopolysaccharide. The fine structure of the body of the plug is unresolvable with the electron microscope; its outer surface is impregnated with electron dense particles. Externally the shell is covered by a 250 Å thick continuous membrane which is in close opposition to the surrounding host tissue.     

The capsid of small papova viruses contains 72 pentameric capsomeres: direct evidence from cryo-electron-microscopy of simian virus 40.

The three-dimensional structure of the simian virus 40 capsid is remarkably similar to the structure of the polyoma empty capsid. This similarity is apparent despite striking differences in the methods used to determine the two structures: image analysis of electron micrographs of frozen-hydrated samples (SV40 virions) and an unconventional x-ray crystallographic analysis (polyoma empty capsids). Both methods have clearly resolved the 72 prominent capsomere units which comprise the T = 7d icosahedral capsid surface lattice. The 12 pentavalent and 60 hexavalent capsomeres consist of pentameric substructures. A pentameric morphology for hexavalent capsomeres clearly shows that the conserved bonding specificity expected from the quasi-equivalence theory is not present in either SV40 or polyoma capsids. Determination of the SV40 structure from cryo-electron microscopy supports the correctness of the polyoma structure solved crystallographically and establishes a strong complementarity of the two techniques. Similarity between the SV40 virion and the empty polyoma capsid indicates that the capsid is not detectably altered by the loss of the nucleohistone core. The unexpected pentameric substructure of the hexavalent capsomeres and the arrangement of the 72 pentamers in the SV40 and polyoma capsid lattices may be characteristic features of all members of the papova virus family, including the papilloma viruses such as human wart and rabbit papilloma.     

Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.     

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.     

Effect of nucleotides 37 and 38 on cleavage of tRNA~(Phe) precursors

Splicing is required for tRNA maturation when the precursors contain the introns. In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNAPhe precursors with different nucleotides 37 and 38 were prepared by in vitro mutagenesis and cleaved by the purified yeast tRNA-splicing endonuclease. The precursors with purine nudeolides at N37 and N38 were found to be the best substrates for the enzyme. When N37 and N38 were replaced by pyrimidine nucleotides, few precursors could be cleaved by the endonuclease. If one is pyrimidine nucleotide, the other one is purine nudeotide at these positions, the cleavage efficiencies are between the two groups of precursors stated above. The pyrimidine nucleotides at these positions might affect the fine structures of the precursors or the distance between the splicing sites, so that the precursors can not be fixed or anchored on the enzyme well, leading to the poor cutting.     

Architecture of the Adenovirus Capsid.

Smith, Kendall O. (Baylor University College of Medicine, Houston, Tex.), Warren D. Gehle, and Melvin D. Trousdale. Architecture of the adenovirus capsid. J. Bacteriol. 90:254-261. 1965.-The capsids of adenovirus type 2 were fragmented by treatment with low concentrations of sodium lauryl sulfate. The clusters of capsomeres resulting from this treatment displayed characteristic patterns. Some of these clusters, each consisting of nine capsomeres, interlocked so as to form the triangular facets of the viral icosahedron. There is some evidence which suggests that the capsomeres are connected to each other by filamentous structures located near their bases. Connections between capsomeres along the edges and at the vertices of the triangular facets were the first to break when particles were treated with sodium lauryl sulfate. Further treatment broke connections between other capsomeres. These data provide additional information concerning the capsomere arrangements and the fine structure of adenoviruses.     

Structure of the scaffold in bacteriophage lambda preheads removal of the scaffold leads to a change of the prehead shell

Small angle X-ray scattering was performed on unprocessed and processed preheads, intermediates in the morphogenesis of bacteriophage λ heads. Unprocessed preheads possess an internal structure (scaffold), necessary for efficient assembly of closed shells. Processed preheads, formed after removal of the scaffold, are able to pack and cut the viral DNA in vitro. Our data show that the scaffold fills out the inside of the shell in an almost (but not completely) homogeneous fashion; structures of the scaffold with the bulk of the mass in a small core inside the shell can be excluded. Unprocessed preheads are larger than processed ones. A change in shell architecture takes place upon transition from unprocessed to processed prehead; the shell becomes roughened up. Shrinking of the shell as well as roughening up can be triggered by accidental partial degradation of the scaffold. The lattice constant of type A polyheads is in agreement with the lattice constant derived from our icosahedral models of the shell, indicating a close relationship between processed preheads and type A polyheads. This observation, together with the type of subunit clustering found, leads us to propose a simple model for the interaction of prehead shell and protein pD, which stabilizes phage DNA after packaging.     

Effect of nucleotides 37 and 38 on cleavage of tRNAPhe precursors

Splicing is required for tRNA maturation when the precursors contain the introns. In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNA^Phe precursors with different nucleotides 37 and 38 were prepared by in vitro mutagenesis and cleaved by the purified yeast tRNA-splicing endonuclease. The precursors with purine nudeolides at N₃₇ and N₃₈ were found to be the best substrates for the enzyme. When N₃₇ and N₃₈ were replaced by pyrimidine nucleotides, few precursors could be cleaved by the endonuclease. If one is pyrimidine nucleotide, the other one is purine nudeotide at these positions, the cleavage efficiencies are between the two groups of precursors stated above. The pyrimidine nucleotides at these positions might affect the fine structures of the precursors or the distance between the splicing sites, so that the precursors can not be fixed or anchored on the enzyme well, leading to the poor cutting.     

Bacillus anthracis CapD, belonging to the gamma-glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan

Several examples of bacterial surface-structure anchoring have been described, but they do not include polyglutamate capsule. Bacillus anthracis capsule, which is composed only of poly-gamma- d-glutamate, is one of the two major virulence factors of the bacterium. We analysed its anchoring. We report that the polyglutamate is anchored directly to the peptidoglycan and that the bond is covalent. We constructed a capD mutant strain, capD being the fourth gene of the capsule biosynthetic operon. The mutant bacilli are surrounded by polyglutamate material that is not covalently anchored. Thus, CapD is required for the covalent anchoring of polyglutamate to the peptidoglycan. Sequence similarities suggest that CapD is a gamma-glutamyltranspeptidase. Furthermore, CapD is cleaved at the gamma-glutamyltranspeptidase consensus cleavage site, and the two subunits remain associated, as necessary for gamma-glutamyltranspeptidase activity. Other Gram-positive gamma-glutamyltranspeptidases are secreted, but CapD is located at the Bacillus surface, associated both with the membrane and the peptidoglycan. Polyglutamate is hydrolysed by CapD indicating that it is a CapD substrate. We suggest that CapD catalyses the capsule anchoring reaction. Interestingly, the CapD(-) strain is far less virulent than the parental strain.     

Crystallographic structure of the T=1 particle of brome mosaic virus

T=1 icosahedral particles of amino terminally truncated brome mosaic virus (BMV) protein were created by treatment of the wild-type T=3 virus with 1M CaCl2 and crystallized from sodium malonate. Diffraction data were collected from frozen crystals to beyond 2.9 A resolution and the structure determined by molecular replacement and phase extension. The particles are composed of pentameric capsomeres from the wild-type virions which have reoriented with respect to the original particle pentameric axes by rotations of 37 degrees , and formed tenuous interactions with one another, principally through conformationally altered C-terminal polypeptides. Otherwise, the pentamers are virtually superimposable upon those of the original T=3 BMV particles. The T=1 particles, in the crystals, are not perfect icosahedra, but deviate slightly from exact symmetry, possibly due to packing interactions. This suggests that the T=1 particles are deformable, which is consistent with the loose arrangement of pentamers and latticework of holes that penetrate the surface. Atomic force microscopy showed that the T=3 to T=1 transition could occur by shedding of hexameric capsomeres and restructuring of remaining pentamers accompanied by direct condensation. Knowledge of the structures of the BMV wild-type and T=1 particles now permit us to propose a tentative model for that process. A comparison of the BMV T=1 particles was made with the reassembled T=1 particles produced from the coat protein of trypsin treated alfalfa mosaic virus (AlMV), another bromovirus. There is little resemblance between the two particles. The BMV particle, with a maximum diameter of 195 A, is made from distinctive pentameric capsomeres with large holes along the 3-fold axis, while the AlMV particle, of approximate maximum diameter 220 A, has subunits closely packed around the 3-fold axis, large holes along the 5-fold axis, and few contacts within pentamers. In both particles crucial linkages are made about icosahedral dyads.     

Towards the preparative and large-scale precision manufacture of virus-like particles

Virus-like particles (VLPs) are of interest in vaccination, gene therapy and drug delivery, but their potential has yet to be fully realized. This is because existing laboratory processes, when scaled, do not easily give a compositionally and architecturally consistent product. Research suggests that new process routes might ultimately be based on chemical processing by self-assembly, involving the precision manufacture of precursor capsomeres followed by in vitro VLP self-assembly and scale-up to required levels. A synergistic interaction of biomolecular design and bioprocess engineering (i.e. biomolecular engineering) is required if these alternative process routes and, thus, the promise of new VLP products, are to be realized.     

Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. (c) 1993 John Wiley & Sons, Inc.     

Myristylated polyomavirus VP2: role in the life cycle of the virus.

The double-stranded genome of the small DNA tumor virus, polyomavirus, is enclosed in a capsid composed of a major protein, VP1, which associates as pentameric capsomeres into an icosahedral structure, and two minor proteins, VP2 and VP3, whose functions and positions within the structure are unknown. The N-terminal glycine of the VP2 coat protein has been shown to be cotranslationally acylated with myristic acid. To study the function of this modification and the role of VP2 in the life cycle of polyomavirus, the N-terminal glycine, critical to the myristylation consensus sequence, has been altered to a glutamic acid or a valine residue by site-directed oligonucleotide mutagenesis. The glycine----glutamic acid mutant DNA has been further studied. When transfected into cells permissive for the polyomavirus full lytic life cycle, this mutant DNA replicated at levels comparable to those of wild-type viral DNA, and small amounts of nonrevertant (mutant) virus could be harvested from the cultures. The virus particles viewed by electron microscopy appeared slightly distorted, but the ratio of full to empty particles was similar to that produced in a wild-type viral infection. Mutant virus was capable of reinfecting permissive cells but with a considerably reduced efficiency.