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1.
Polyomavirus was purified from infected mouse cell lysates under mild physiological conditions. When analyzed in a sucrose gradient, a major virus peak (240S) was identified. This sucrose-isolated virus could be divided into two populations based on its stability to CsCl gradient centrifugation. Members of the unstable population were shown to eject their DNA cores when subjected to CsCl gradient centrifugation, forming empty capsids, whereas the stable population was unaffected by the same CsCl treatment. Formaldehyde fixation of the 240S virus particles stabilized the virions and prevented ejection of DNA and generation of empty capsids. When formaldehyde-fixed 240S virus was examined with the electron microscope, only full virions were observed. These results indicate that polyoma capsids are not preformed in vivo, but instead are generated when infected cell lysates are subjected to harsh CsCl purification procedures. 相似文献
2.
Polyadenylated RNA isolated from the cytoplasm of mouse 3T6 cells 28 h after infection with polyoma virus has been isolated and translated in vitro. Polyoma capsid proteins VP1 and VP2 have been identified in the cell-free product by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide fingerprinting. Polyoma mRNA species have been isolated by preparative hybridization to purified viral DNA immobilized on cellulose nitrate filters and shown to code for both VP1 and VP2. These experiments establish conditions for the isolation of late polyoma mRNA and the cell-free synthesis of polyoma capsid proteins and indicate that the active mRNA species are at least partially virus coded. 相似文献
3.
Differential adsorption of polyoma virions and capsids to mouse kidney cells and guinea pig erythrocytes. 总被引:1,自引:8,他引:1
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Adsorption of 125I-labeled polyoma virions and capsids to the surface of mouse kidney cells (MKC) and guinea pig erythrocytes was examined. Purified polyoma capsids lack the ability to compete with polyoma virions for specific binding sites on the surface of MKC. These same capsids were, however, able to block virion adsorption to guinea pig erythrocytes. UV-inactivated virions blocked cellular receptors on MKC and thus inhibited infectious virions from infecting the cells. Capsids were unable to inhibit virion infection of MKC. Adsorption of polyoma virions to MKC and infection of these cells were found to be independent of the ability of the virions to agglutinate guinea pig erythrocytes. 相似文献
4.
Characterization of the mRNA''s for the polyoma virus capsid proteins VP1, VP2, and VP3. 总被引:19,自引:12,他引:7
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Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA. 相似文献
5.
Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro. 总被引:4,自引:4,他引:0
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The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5. 相似文献
6.
Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA. 总被引:19,自引:0,他引:19
The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active. 相似文献
7.
Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein. 总被引:11,自引:6,他引:5
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Treatment of purified polyoma virions with 6 M guanidine-hydrochloride and 0.01 M beta-mercaptoethanol resulted in the immediate loss of both hemagglutinating and plaque-forming ability. Gel filtration through Sepharose CL-6B beads allowed separation of the dimer, VP1, VP2, VP3, and histone proteins VP4-7 in highly purified form. Renaturation of the purified VP1 protein resulted in the formation of subunits that were morphologically, biophysically, and immunologically similar to native virion capsomeres. 相似文献
8.
9.
Herpes simplex virus capsids assembled in insect cells infected with recombinant baculoviruses: structural authenticity and localization of VP26. 总被引:13,自引:11,他引:2
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B L Trus F L Homa F P Booy W W Newcomb D R Thomsen N Cheng J C Brown A C Steven 《Journal of virology》1995,69(11):7362-7366
Recently, recombinant baculoviruses have been used to show that expression of six herpes simplex virus type 1 genes results in the formation of capsid-like particles. We have applied cryoelectron microscopy and three-dimensional image reconstruction to establish their structural authenticity to a resolution of approximately 2.7 nm. By comparing capsids assembled with and without the expression of gene UL35, we have confirmed the presence of six copies of its product, VP26 (12 kDa), around each hexon tip. However, VP26 is not present on pentons, indicating that the conformational differences between the hexon and penton states of the major capsid protein, VP5, extend to the VP26 binding site. 相似文献
10.
Haagsman HP Diemel RV 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2001,129(1):91-108
Pulmonary surfactant is a barrier material of the lungs and has a dual role: firstly, as a true surfactant, lowering the surface tension; and secondly, participating in innate immune defence of the lung and possibly other mucosal surfaces. Surfactant is composed of approximately 90% lipids and 10% proteins. There are four surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, SP-C and SP-D. Although the sequences and post-translational modifications of SP-B and SP-C are quite conserved between mammalian species, variations exist. The hydrophilic surfactant proteins SP-A and SP-D are members of a family of collagenous carbohydrate binding proteins, known as collectins, consisting of oligomers of trimeric subunits. In view of the different roles of surfactant proteins, studies determining the structure-function relationships of surfactant proteins across the animal kingdom will be very interesting. Such studies may reveal structural elements of the proteins required for surface film dynamics as well as those required for innate immune defence. Since SP-A and SP-D are also present in extrapulmonary tissues, the hydrophobic surfactant proteins SP-B and SP-C may be the most appropriate indicators for the evolutionary origin of surfactant. SP-B is essential for air-breathing in mammals and is therefore largely conserved. Yet, because of its unique structure and its localization in the lung but not in extrapulmonary tissues, SP-C may be the most important indicator for the evolutionary origin of surfactant. 相似文献
11.
Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1. 总被引:6,自引:4,他引:6
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L Montross S Watkins R B Moreland H Mamon D L Caspar R L Garcea 《Journal of virology》1991,65(9):4991-4998
Polyomavirus normally assembles in the nucleus of infected mouse cells. Sf9 insect cells expressing the polyomavirus major capsid protein VP1 were examined by electron microscopy. Capsidlike particles of apparently uniform size were found in the nucleus. Immunogold electron microscopy demonstrated abundant VP1 in the cytoplasm which was not assembled into any recognizable higher-order structure. Cytoplasmic VP1 assembled after the cells were treated with the calcium ionophore ionomycin. Purified VP1 aggregates were shown by negative staining and cryoelectron microscopy to consist predominantly of particles similar to the empty T = 7 viral capsid. Thus, polyomavirus VP1 can assemble in vivo into capsids independent of other viral proteins or DNA. Nuclear assembly may result from increased available calcium in this subcellular compartment. 相似文献
12.
Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures. 相似文献
13.
Both primary mouse kidney and primary mouse embryo cells in culture were used for polyomavirus progeny production. Examination of polyomavirus virion structural integrity revealed that mouse embryo cell progeny contained a threefold greater population of unstable particles when compared with mouse kidney cell progeny. Differences in biological activity between these two progeny virion types were also shown. Mouse kidney cell progeny compared with mouse embryo cell progeny exhibited a 10-fold greater ability to agglutinate guinea pig erythrocytes, a 3-fold lower ability to become internalized into monopinocytotic vesicles, and a 2-fold lower ability to initiate a productive infection based on positive nuclear immunofluorescence when mouse embryo host cell cultures were used. The mouse kidney progeny were also found to bind to host cells less specifically than the mouse embryo cell progeny. When these two progeny virion types were labeled in vivo with 32P and subjected to isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophroesis in the second dimension, differences in the phosphorylation pattern of the major virus-encoded structural protein VP1 species were observed. It was revealed that species D and E of mouse kidney cell progeny were phosphorylated to the same degree, while mouse embryo cell progeny species E and F were phosphorylated equally. These data suggest that the host cells play a role in modulating the biological activity of the virus by affecting the degree and site-specific phosphorylation of the major capsid protein VP1 which may influence the recognition of virus attachment proteins for specific cellular receptors. 相似文献
14.
IQGAP proteins have been identified in a wide spectrum of organisms, ranging from yeast to humans. The most extensively studied family member is the ubiquitously expressed scaffold protein IQGAP1, which participates in multiple essential aspects of mammalian biology. IQGAP1 mediates these effects by binding to and regulating the function of numerous interacting proteins. Over ninety proteins have been reported to associate with IQGAP1, either directly or as part of a larger complex. In this review, we summarise those IQGAP1 binding partners that have been identified in the last five years. The molecular mechanisms by which these interactions contribute to the functions of receptors and their signalling cascades, small GTPase function, cytoskeletal dynamics, neuronal regulation and intracellular trafficking are evaluated. The evidence that has accumulated recently validates the role of IQGAP1 as a scaffold protein and expands the repertoire of cellular activities in which it participates. 相似文献
15.
HIV-1 assembly depends on its structural protein, Gag, which after synthesis on ribosomes, traffics to the late endosome/plasma membrane, associates with HIV Env glycoprotein, and forms infectious virions. While Env and Gag migrate to lipid microdomains, their stoichiometry and specificity of interaction are unknown. Pseudotyped viral particles can be made with one viral core surrounded by heterologous envelope proteins. Taking advantage of this property, we analyzed the association of HIV Env and Ebola glycoprotein (GP), with HIV-1 Gag coexpressed in the same cell. Though both viral glycoproteins were expressed, each associated independently with Gag, giving rise to distinct virion populations, each with a single glycoprotein type. Confocal imaging demonstrated that Env and GP localized to distinct lipid raft microdomains within the same cell where they associated with different virions. Thus, a single Gag particle associates "quantally" with one lipid raft, containing homogeneous trimeric viral envelope proteins, to assemble functional virions. 相似文献
16.
Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres. 总被引:3,自引:3,他引:0
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The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, delta NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or delta NCOVP1 were determined to be 0.37 +/- 0.23 microM for VP2 and 0.18 +/- 0.21 microM for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation. 相似文献
17.
Correlation between genetic loci and structural differences in the capsid proteins of polyoma virus plaque morphology mutants. 总被引:10,自引:0,他引:10
Two plaque morphology variants of polyoma virus (A-2 and 208) showed marked differences in agarose gel electrophoresis of the whole particles, isoelectric focusing of the major capsid protein VP1 (45,000 daltons) and three tryptic peptides (A, B and C) of VP1. No major difference in apparent molecular weight on NaDodSO4 gels, amino acid composition or carbohydrate detectable by Schiff staining was revealed between the capsid proteins of the two viruses.Correlations have been made between phenotype, portions of the primary amino acid sequence of VP1 and the physical map of polyoma virus DNA by analysis of this protein from large plaque A-2 virus, minute plaque 208 virus and large plaque 208 virus selected after marker rescue with a fragment of polyoma virus DNA generated by the Hpa II restriction enzyme. The interrelationship of these properties was established by taking advantage of the observations of Miller, Cooke and Fried (1976)that heterozygous markers present on heteroduplex DNA are found in 100% of selected progeny and in only 50% of unselected progeny.All five marker rescued isolates selected for large plaque morphology showed only two A-2-specific characters, the absence of peptide C in tryptic maps of VP1 and the aggregation of VP1 on isoelectric focusing. The other four characters which distinguish A-2 and 208 were present or absent in 40–60% of the five isolates, which is close to the expected 50% for unselected markers. Three of the four A-2-specific characters (the presence of peptide A, absence of peptide B and isoelectric point of VP1) have been found to occur coordinately in the marker rescued isolates. The fourth character (electrophoretic mobility of virus particles in agarose gels) segregated independently.The techniques used in this study should find wide application in correlating primary amino acid sequence, nucleotide sequence and phenotype in other systems. 相似文献
18.
The VP4 capsid protein of poliovirus is N-terminally modified with myristic acid. Within the poliovirus structure, a hydrogen bond is observed between the myristate carbonyl and the hydroxyl side chain of threonine 28 of VP4. This interaction is between two fivefold symmetry-related copies of VP4 and is one of several myristoyl-mediated interactions that appears to structurally link the promoters within the pentamer subunit of the virus particle. Site-specific substitutions of the threonine residue were constructed to investigate the biological relevance of these myristate-protein interactions. Replacement of the threonine with glycine or lysine is lethal, generating nonviable viruses. Substitution with serine or valine led to viable viruses, but these mutants displayed anomalies during virus assembly. In addition, both assembled serine- and valine-substituted virion particles showed reduced infectivity and were more sensitive to thermal inactivation and antibody neutralization. Thus the threonine residue provides interactions necessary for efficient assembly of the virus and for virion stability. 相似文献
19.
Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2. 总被引:3,自引:3,他引:0
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W F Mangel R M Hewick S T Bayley T Wheeler R Harvey M D Waterfield A E Smith 《Journal of virology》1978,25(2):570-578
Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded. 相似文献
20.
VP26 of white spot syndrome virus functions as a linker protein between the envelope and nucleocapsid of virions by binding with VP51 总被引:1,自引:0,他引:1
The envelopment of the nucleocapsid is an important step in white spot syndrome virus (WSSV) assembly. Previous studies showed that VP26, a major envelope protein of WSSV, can interact with viral nucleocapsid. In this study, using the biotin label transfer technique, we found that the biotin label was transferred from Bio-rVP26 to the viral capsid protein VP51 or from Bio-MBP-VP51 to VP26. Far-Western analyses provided further evidence for direct interaction between VP26 and VP51. Therefore, we conclude that VP26 functions as a matrix-like linker protein between the viral envelope and nucleocapsid, which suggests that VP26 is a key factor in the envelopment of WSSV virion. 相似文献