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1.
Four different soluble components of adenovirus type 9 (Rosen's group II) were identified. These were a complete hemagglutinin (HA), an incomplete HA, components carrying group-specific complement-fixing (CF) antigen, and components identified only by their hemagglutination-inhibition (HI) antibody consuming capacity and antigen activity in CF tests with an antiserum against complete HA. The complete HA sedimented relatively rapidly. It was composed of 12 pentons (vertex capsomers plus projections) aggregated into the form of a pentagonal dodecahedron. The length of the projections was about 12 to 13 mmu. Thus they appeared longer than the corresponding structures of types 3 and 11, but shorter than those of types 4 and 5. The rate of sedimentation of complete HA of type 9 was intermediate to those of the complete HA of types 3 and 11. The incomplete HA sedimented together with components carrying group-specific CF antigen, but could be separated from those by anion-exchange chromatography. Two different antigens were present in incomplete HA. One could absorb a group-specific hemagglutination-enhancing antibody, and was sensitive to treatment with trypsin. The other antigen could absorb the type-specific HI antibody and was not destroyed by trypsin. In addition to the incomplete HA, a separate population of more slowly sedimenting components showed a capacity to absorb HI antibody. These components could also be identified in CF tests when an antiserum against complete HA was applied. The incomplete HA, group-specific CF antigen, and slowly sedimenting HI antibody absorbing components are suggested to represent isolated penton, hexon, and fiber components, respectively. 相似文献
2.
The dedecon and hexon components of adenovirus types 8 and 9 have been extensively purified for use in establishing the basis of the cross-reaction between these types. Dodecons, the complete hemagglutinins, were purified 304- to 362-fold by fluorocarbon extraction, calcium phosphate batch chromatography, and ion-exchange column chromatography. Hexons, the group complement-fixation (CF) antigens, were purified 230- to 240-fold by erythrocyte adsorption, ion-exchange chromatography, and exclusion chromatography. Component antisera prepared in rabbits were tested in reciprocal fashion with crude virus and dodecon and hexon components. By hemagglutination-inhibition (HI), the dodecons of types 8 and 9 demonstrated the same predominantly one-sided relationship characteristic of the crude antigens. Some neutralizing activity was associated with both dodecons and hexons of each type. However, combining anti-dodecon and anti-hexon sera or producing antisera against the combined dodecon-hexon components resulted in neutralizing titers which were identical to titers obtained with antisera against the crude virus harvests. Dedecons of each type appear to share at least one antigenic determinant with hexons of the same type, and this determinant may reside on the vertex capsomere. Hexons possess group- and type-specific determinants, as shown by CF, neutralization, and immunodiffusion tests, and may exhibit some minor relationship between types 8 and 9. The results with the purified components are consistent with the predominantly one-sided antigenic relationship between types 8 and 9 in the conventional HI tests and the largely type-specific relationship by neutralization tests. 相似文献
3.
Comparative hemagglutination-enhancement (HE) tests demonstrated diversified patterns of antigenic specificities both in the fiber and vertex capsomer part of pentons of human adenovirus types 3, 11 (subgroup I), 9, 15 (II), 1, 2, 4, 5, 6 (III), and 12. All fibers contained a type-specific antigen. Subgroup II and III fibers, in addition, contained specificities both unique for each subgroup and also common to the two subgroups. Fibers of serotypes 4 and 12 displayed a somewhat deviating behavior. All vertex capsomers tested shared a group-specific part. This was the only antigenic specificity demonstrable for serotype 12. Maximal penton HE titers of all sera were reached in tests with incomplete hemagglutinin of type 11. In addition, maximal HE activity of sera against individual serotypes also was recorded against pentons of other members of the same subgroup. Antigen characteristics of vertex capsomers of type 4 indicated a closer relationship to subgroup I than to subgroup III. The toxin activity of pentons was more sensitive to trypsin treatment than their capacity to function as incomplete hemagglutinin. Homotypic antipenton sera, unabsorbed or absorbed with homotypic fibers to remove antibodies against this component, and, to a varying extent, also heterotypic antipenton sera could neutralize toxin activity. Antifiber sera could neutralize toxin activity of pentons carrying short fibers (10 nm, type 3) but not of those carrying long fibers (28 to 31 nm, type 2). It is concluded that toxin activity is carried by a specific part of vertex capsomers and that cell detachment can be brought about via a direct contact between this component and cell membranes. Fiber-mediated attachment does not seem to be necessary for this biological activity to become expressed. 相似文献
4.
Association of endonuclease activity with serotypes belonging to the three subgroups of human adenoviruses. 总被引:4,自引:3,他引:1
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Endonuclease activity has been detected in association with highly purified virions, pentons, and/or dodecons of adenovirus types 2, 3, 5, 9, 12, 15, and 16. Only single-strand scissions in substrate DNA were detected. The nuclease activity was detected by a highly sensitive ethidium bromide fluorimetric assay procedure. 相似文献
5.
Soluble components of type 8 adenovirus have been studied. Four different components were isolated by anion-exchange chromatography and purified by further chromatographic procedures, by zonal centrifugation, and by erythrocyte absorption and elution. The four components exhibited the following characteristics. (i) Fiber antigen was trypsin-resistant and functioned as incomplete hemagglutinin (agglutinated rat and human erythrocytes only in the presence of certain types of adenovirus antisera). (ii) The penton was trypsin-sensitive, exerted a cytotoxic effect, and also showed incomplete hemagglutination, being active in the presence of a majority of heterotypic adenovirus antisera studied. (iii) The group-specific hexon antigen reacted in complement fixation reaction and gel precipitation with sera prepared against other types of adenoviruses, besides showing characteristics indicating the presence of a type-specific antigen component. (iv) The soluble complete hemagglutinin was trypsin-sensitive, displayed cytotoxic effect, adsorbed easily to human and rat erythrocytes, and could be eluted from them by means of receptor-destroying enzyme. The three hemagglutinins of adenovirus type 8 proved to be highly unstable, and their demonstration was only successful by using a large quantity of freshly prepared concentrated virus material. Considering these conditions, a method was developed for their concentration and purification. 相似文献
6.
Erling Norrby 《Journal of virology》1969,4(5):657-662
Antisera against isolated capsid components of intermediate adenovirus strains, types 3-16 (the San Carlos agent) and 15-9 and of "parental" prototype strains were compared in neutralization tests, hemagglutination-inhibition (HI) tests employing soluble and virion-associated hemagglutinin as antigens, and by electron microscopy. Hexons of the intermediate strains were found to be similar to, but not identical to, those of the prototype strains with which a cross-reaction occurred in neutralization tests (types 3 and 15). In contrast, fibers of intermediate strains displayed characteristics relating them to the corresponding components of prototype strains to which a relationship has been found in HI tests. Fibers (and possibly even pentons) of types 9 and 15-9 appeared to be identical, whereas fibers of types 3-16 and 16 displayed antigen specificities of both common and unique nature. 相似文献
7.
Antisera against hexons of serotypes 2, 4, 5, and 6 (subgroup III), and 15 (subgroup II) were absorbed with purified hexons of various serotypes representing the different subgroups of human adenoviruses. Group, subgroup, and type specificities of hexons could be distinguished. The subgroup specificity of type 4 hexons resembled that of hexons of subgroup I members (types 3, 11, and 16). Antihexon sera gave a type-specific inhibition of virion-associated hemagglutinin. The inhibiting activity of different sera was found to be inversely related to the length of fibers of the serotype concerned. Virions of serotypes carrying fibers shorter than about 20 nm (types 3, 4, 9, 11, and 15) were readily inhibited, whereas those of serotypes with longer fibers (types 2 and 6) were inhibited only by relatively large amounts of antibody measured in terms of homotypic complement fixation activity. The reciprocal cross-neutralization between serotypes 4 and 16 was studied separately. Hexons of both serotypes each carried a type-specific component and, in addition, a unique antigen specificity common to the two types. This common antigen specificity was interpreted to be available to a larger extent at the surface of virions (and probably also isolated hexons) of type 4 than of type 16. These results suggest an explanation for the predominantly one-sided character of the cross-neutralization between types 4 and 16. 相似文献
8.
Three-dimensional localization of pORF65 in Kaposi's sarcoma-associated herpesvirus capsid
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Of the six herpesvirus capsid proteins, the smallest capsid proteins (SCPs) share the least sequence homology among herpesvirus family members and have been implicated in virus specificity during infection. The herpes simplex virus-1 (HSV-1) SCP was shown to be horn shaped and to specifically bind the upper domain of each major capsid protein in hexons but not in pentons. In Kaposi's sarcoma-associated herpesvirus (KSHV), the protein encoded by the ORF65 gene (pORF65) is the putative SCP but its location remains controversial due to the absence of such horn-shaped densities from both the pentons and hexons of the KSHV capsid reconstructions. To directly locate the KSHV SCP, we have used electron cryomicroscopy and three-dimensional reconstruction techniques to compare the three-dimensional structure of KSHV capsids to that of anti-pORF65 antibody-labeled capsids. Our difference map shows prominent antibody densities bound to the tips of the hexons but not to pentons, indicating that KSHV SCP is attached to the upper domain of the major capsid protein in hexons but not to that in pentons, similar to HSV-1 SCP. The lack of horn-shaped densities on the hexons indicates that KSHV SCP exhibits structural features that are substantially different from those of HSV-1 SCP. The location of SCP at the outermost regions of the capsid suggests a possible role in mediating capsid interactions with the tegument and cytoskeletal proteins during infection. 相似文献
9.
Cells infected with herpes simplex virus type 1 (HSV-1) were conventionally embedded or freeze substituted after high-pressure freezing and stained with uranyl acetate. Electron tomograms of capsids attached to or undergoing envelopment at the inner nuclear membrane (INM), capsids within cytoplasmic vesicles near the nuclear membrane, and extracellular virions revealed the following phenomena. (i) Nucleocapsids undergoing envelopment at the INM, or B capsids abutting the INM, were connected to thickened patches of the INM by fibers 8 to 19 nm in length and < or =5 nm in width. The fibers contacted both fivefold symmetrical vertices (pentons) and sixfold symmetrical faces (hexons) of the nucleocapsid, although relative to the respective frequencies of these subunits in the capsid, fibers engaged pentons more frequently than hexons. (ii) Fibers of similar dimensions bridged the virion envelope and surface of the nucleocapsid in perinuclear virions. (iii) The tegument of perinuclear virions was considerably less dense than that of extracellular virions; connecting fibers were observed in the former case but not in the latter. (iv) The prominent external spikes emanating from the envelope of extracellular virions were absent from perinuclear virions. (v) The virion envelope of perinuclear virions appeared denser and thicker than that of extracellular virions. (vi) Vesicles near, but apparently distinct from, the nuclear membrane in single sections were derived from extensions of the perinuclear space as seen in the electron tomograms. These observations suggest very different mechanisms of tegumentation and envelopment in extracellular compared with perinuclear virions and are consistent with application of the final tegument to unenveloped nucleocapsids in a compartment(s) distinct from the perinuclear space. 相似文献
10.
Structure of the herpes simplex virus capsid: effects of extraction with guanidine hydrochloride and partial reconstitution of extracted capsids. 总被引:42,自引:36,他引:6
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Viral B capsids were purified from cells infected with herpes simplex virus type 1 and extracted in vitro with 2.0 M guanidine hydrochloride (GuHCl). Sodium dodecyl sulfate-polyacrylamide gel analyses demonstrated that extraction resulted in the removal of greater than 95% of capsid proteins VP22a and VP26 while there was only minimal (less than 10%) loss of VP5 (the major capsid protein), VP19, and VP23. Electron microscopic analysis of extracted capsids revealed that the pentons and the material found inside the cavity of B capsids (primarily VP22a) were removed nearly quantitatively, but extracted capsids remained otherwise structurally intact. Few, if any, hexons were lost; the capsid diameter was not greatly affected; and its icosahedral symmetry was still clearly evident. The results demonstrate that neither VP19 nor VP23 could constitute the capsid pentons. Like the hexons, the pentons are most likely composed of VP5. When B capsids were treated with 2.0 M GuHCl and then dialyzed to remove GuHCl, two bands of viral material were separated by sucrose density gradient ultracentrifugation. The more rapidly migrating of the two consisted of capsids which lacked pentons and VP22a but had a full complement of VP26. Thus, VP26 must have reassociated with extracted capsids during dialysis. The more slowly migrating band consisted of torus-shaped structures approximately 60 nm in diameter which were composed entirely of VP22a. These latter structures closely resembled torus-shaped condensates often seen in the cavity of native B capsids. The results suggest a similarity between herpes simplex virus type 1 B capsids and procapsids of Salmonella bacteriophage P22. Both contain an internal protein (VP22a in the case of HSV-1 B capsids and gp8 or "scaffolding" protein in phage P22) that can be extracted in vitro with GuHCl and that is absent from mature virions. 相似文献
11.
Herpes simplex virus-1 (HSV-1) virions are large, complex enveloped particles containing a proteinaceous tegument layer connected to an icosahedral capsid. The major capsid protein, VP5 (149 kDa), makes up both types of capsomere, pentons and hexons. Limited trypsin digestion of VP5 identified a single stable 65 kDa fragment which represents a proposed protein folding nucleus. We report the 2.9 A crystal structure of this fragment and its modeling into an 8.5 A resolution electron cryomicroscopy map of the HSV-1 capsid. The structure, the first for any capsid protein from Herpesviridae, revealed a novel fold, placing herpesviruses outside any of the structurally linked viral groupings. Alterations in the geometrical arrangements of the VP5 subunits in the capsomeres exposes different residues, resulting in the differential association of the tegument and VP26 with the pentons and hexons, respectively. The rearrangements of VP5 subunits required to form both pentavalent and hexavalent capsomeres result in structures that exhibit very different electrostatic properties. These differences may mediate the binding and release of other structural proteins during capsid maturation. 相似文献
12.
The Pattern of Tegument-Capsid Interaction in the Herpes Simplex Virus Type 1 Virion Is Not Influenced by the Small Hexon-Associated Protein VP26
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Dong-Hua Chen Joanita Jakana David McNab Joyce Mitchell Z. Hong Zhou Matthew Dougherty Wah Chiu Frazer J. Rixon 《Journal of virology》2001,75(23):11863-11867
Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. H. Zhou, D. H. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons. 相似文献
13.
Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus. 总被引:5,自引:2,他引:3
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P T Wingfield S J Stahl D R Thomsen F L Homa F P Booy B L Trus A C Steven 《Journal of virology》1997,71(12):8955-8961
VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26. 相似文献
14.
Roles of Triplex and Scaffolding Proteins in Herpes Simplex Virus Type 1 Capsid Formation Suggested by Structures of Recombinant Particles
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Ali Saad Z. Hong Zhou Joanita Jakana Wah Chiu Frazer J. Rixon 《Journal of virology》1999,73(8):6821-6830
Typical herpes simplex virus (HSV) capsids contain seven proteins that form a T=16 icosahedron of 1,250-A diameter. Infection of cells with recombinant baculoviruses expressing two of these proteins, VP5 (which forms the pentons and hexons in typical HSV capsids) and VP19C (a component of the triplexes that connect adjacent capsomeres), results in the formation of spherical particles of 880-A diameter. Electron cryomicroscopy and computer reconstruction revealed that these particles possess a T=7 icosahedral symmetry, having 12 pentons and 60 hexons. Among the characteristic structural features of the particle are the skewed appearance of the hexons and the presence of intercapsomeric mass densities connecting the middle domain of one hexon subunit to the lower domain of a subunit in the adjacent hexon. We interpret these connecting masses as being formed by VP19C. Comparison of the connecting masses with the triplexes, which occupy equivalent positions in the T=16 capsid, reveals the probable locations of the single VP19C and two VP23 molecules that make up the triplex. Their arrangement suggests that the two triplex proteins have different roles in controlling intercapsomeric interactions and capsid stability. The nature of these particles and of other aberrant forms made in the absence of scaffold demonstrates the conformational adaptability of the capsid proteins and illustrates how VP23 and the scaffolding protein modulate the nature of the VP5-VP19C network to ensure assembly of the functional T=16 capsid. 相似文献
15.
We have tested the two principal theories which explain the previous finding that small amounts of type-specific antibody to the adenovirus hexon can neutralize infectivity, whereas even large amounts of cross-reactive antibody do not. a) It has been suggested that the type-specific determinants are especially prominent in the virion. We have therefore measured the capacity of whole virus to bind appropriate antibodies, using a sensitive radioimmunoprecipitation (RIP) system. In fact, virions bound type-specific and cross-reactive antibodies impartially. Moreover, they bound both much less effectively than did free hexon or disrupted virus, suggesting that many of each kind of determinant are inaccessible in virions. b) It has been suggested that the type-specific determinants are confined to those hexons located next to the pentons, and that they are the targets for neutralizing antibody. We have therefore studied the antigenicity of peripentonal and nonamer hexons isolated from virions, and found that each possessed both kinds of determinants. Furthermore, these were present in the same proportion as in hexons purified from the soluble antigens in infected cells ("free hexons"). We concluded that the mechanism of neutralization by antibody is complicated, and that the type-specific determinants exposed on the virion must play a crucial role. 相似文献
16.
Three-dimensional localization of the smallest capsid protein in the human cytomegalovirus capsid
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The smallest capsid proteins (SCPs) of the human herpesviruses differ substantially in size and sequence and are thought to impart some unique aspects of infection to their respective viruses. We used electron cryomicroscopy and antibody labeling to show that the 8-kDa SCP of human cytomegalovirus is attached only to major capsid protein subunits of the hexons, not the pentons. Thus, the SCPs of different herpesviruses illustrate that a protein can evolve significantly in sequence, structure, and function, while preserving its role in the architecture of the virus by binding to a specific partner in a specific oligomeric state. 相似文献
17.
Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented. 相似文献
18.
19.
Genetic Analysis of Adenovirus Type 2 II. Preliminary Phenotypic Characterization of Temperature-Sensitive Mutants 总被引:9,自引:9,他引:0
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The properties of temperature-sensitive mutants of adenovirus type 2 representing 12 complementation groups were studied. All mutants were normal with respect to adsorption as measured by viral inclusion formation and viral DNA synthesis as shown by velocity sedimentation in alkaline sucrose gradients. One mutant, however, formed viral inclusions of altered morphology at the nonpermissive temperature. The synthesis of the major capsid proteins was examined by immunodiffusion. On this basis, the complementation groups could be arranged as follows: (i) one group was negative for all three proteins; (ii) three groups failed to synthesize penton bases; (iii) eight groups were positive for hexons, pentons, and fibers. The assembly of virus particles at 39 C was examined by equilibrium sedimentation in CsCl; three groups were found defective, whereas two of the penton-negative groups were positive for virion production. Tests of the thermolability of virions at 50 C revealed eight groups labile whereas the remainder were insensitive to heat inactivation. None of five mutants inoculated in newborn rats induced tumors, although three of them were capable of in vitro transformation. 相似文献
20.
Structure of isolated nucleocapsids from venezuelan equine encephalitis virus and implications for assembly and disassembly of enveloped virus
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Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is approximately 11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation. 相似文献