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Results for “capsid assembly”

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PubMed1977

Changes in the capsid structure and stability of defective particles of bacteriophage R17.

Serological and chemical methods were used to compare the capsid structure and stability of R17 phage and amA31 defective particles. Immunodiffusion analysis demonstrated identity between intact R17 and amA31 capside and between dissociated subunits of both R17 and amA31 and purified coat protein. Radioimmunoassays detected an antibody in R17 antisera that binds to intact R17 but could not be absorbed from R17 antisera with amA31. The R17 antibody remaining in amA31-absorbed sera did not neutralize infectivity of R17 phage. Differences between the surface composition of R17 and amA31 capsids were also detected by iodination. Capsids of R17 bound approximately four times more 125I than amA31, which was accounted for by a decreased 125I labeling of coat protein. Finally, amA31 capsids dissociated under milder conditions of sodium dodecyl sulfate treatment than R17 capsids. The sodium dodecyl sulfate dissociation of both R17 and amA31 capsids resulted in the formation of a transient 38,000-dalton intermediate, which subsequently dissociated to coat protein monomers. Preparations of dissociated R17 capsids also contained assembly protein was also found in preparations of dissociated amA31 capsids.

Antigens, Viral↗
PubMed1979

[Contribution of two-dimensional immunoelectrophoresis technics to the study of the structure and the morphogenesis of human adenovirus].

The two-dimensional immunoelectrophoresis has been applied to human adenovirus type-2 structural analysis. This method is both qualitative and quantitative and was used in the following studies. 1) Detection of major and minor capsid antigens. 2) Identification of viral antigenic components in the infected cell material. 3) Quantification of antigens and serological characterization of ts mutants. 4) Identification of polymeric forms of structural proteins. 5) Assembly analysis of capsid subunits. 6) Stoichiometric analysis of proteins within the virion. 7) Obtaining of monospecific sera.

Adenoviruses, Human↗
PubMed1977

Kinetics of assembly of a parvovirus, minute virus of mice, in synchronized rat brain cells.

The rates of assembly of the three classes of particles of minute virus of mice were examined in synchronized rat brain cells by a combination of electron microscopy and biochemical techniques. We observed a burst of virus assembly beginning about 8 h after the end of cellular S phase. Labeled thymidine incorporated into the 1.46 g/cm3 class of full virus particles was transferred almost quantitatively to the 1.42 g/cm3 class. The 1.46 g/cm3 virus appeared to be an immediate precursor to the 1.42 g/cm3 class. Conversion of the 1.46 density virus to the 1.42 density particles was observed at the time of virus assembly. The processing was rapid and occurred primarily in the nucleus. Infected cells did not contain significant pools of viral DNA in a form that could be encapsulated in the absence of DNA synthesis. The role of the empty virus capsids in the assembly process is discussed.

Animals↗
PubMed1979

Cell-free assembly of a polyoma-like particle from empty capsids and DNA.

A polyoma-like particle (PLP) is formed when polyoma DNA and purified empty capsids are incubated in a cell-free system. The DNA of this new particle is protected against the action of pancreatic DNase. The density of the purified PLP in CsCl is 1.32 g/cm3, which is intermediate between that of polyoma virions (1.34 g/cm3) and empty capsids (1.29 g/cm3). Purified PLP sediments at 190 S in sucrose and is stable in solutions of high ionic strength. When the DNA is extracted from PLP by the use of detergent and phenol, it is found to be doublestranded with a molecular weight of approximately 1.1 x 10(6). The particles are stable in CsCl at 4 degrees for at least 5 months. Electron micrographs indicate that highly purified PLPs stained with 2% PTA have the same appearance as polyoma capsids. Neither aggregates nor complexes bound by loose ionic bonds appear reasonable to explain these results. The evidence indicates that the DNA of this new polyoma-like particle, made under cell-free conditions, is protected by the capsid.

Cell-Free System↗
PubMed1979

In vitro association of empty adenovirus capsids with double-stranded DNA.

Several lines of evidence suggest that empty adenovirus capsids are preassembled intermediates in the pathway of virion assembly. We have observed that purified empty capsids of subgroup B adenoviruses have a remarkable affinity for DNA in vitro. The products of capsid-DNA association are sufficiently stable, once formed in low-salt solution, to permit purification and characterization in CsCl density gradients. Neither virions nor the DNA-containing incomplete particles of subgroup B adenoviruses can give rise to such in vitro reaction products. The average molecular weight of the empty adenovirus capsids is about 123 X 10(6), consistent with the absence of viral core peptides and a small deficiency of exterior shell polypeptides. Electron microscopy of negatively stained capsids and the capsids bound to DNA reveals a typical adenovirus size and architecture. The particles appear with a surface discontinuity that is presumed to expose the DNA binding site(s). The DNA molecules associated with the empty capsids are susceptible to the actions of DNase and restriction endonucleases. The dependence of rate of capsid-DNA association on DNA length suggests randomly distributed binding sites on the DNA molecules. Although the DNA molecules can successively acquire additional empty capsids, the empty particles themselves are restricted to interactionwith only one DNA molecule. Electron microscopy of the capsid-DNA complexes spread in cytochrome c films shows that the particles are bo-nd along the contour of extended duplex DNA. The amount of DNA within each bound particle appears to be less than 300 base pairs, as estimated by the length of the DNA molecules visible outside of the bound particle. The empty capsid-DNA association product described in this report provides an interesting substrate for further investigation of the DNA packaging process in a defined in vitro system, with extracts or purified components from infected cells.

Adenoviridae↗
PubMed1976

Capsid transformation during packaging of bacteriophage lambdaDNA.

Assembly pathways of complex viruses might not be simple additions of one protein after another with rigid tertiary structure. It might in fact involve shifts in subunit structure, movement of subunits relative to each other to form new arrangements, transient action of proteins and protein segments, involvement of structure forming 'microenvironments' of the host. Thus morphogenesis of the bacteriophage lambda head starts with the formation of a core-containing DNA-free petit lambda particle. In a first transition, and dependent on a host function, the core is released, minor protein components of the capsid are processed and the particle's structure is altered, as shown by a change of its hydrodynamic properties. The resulting 'prehead' undergoes a second transition triggered by a complex of DNA and recognition protein (A-protein). This transition is more drastic than the first one. The particle doubles its volume without increasing in protein mass, the shell becomes thinner, and the surface structure is changed. Concomitantly with this process, the DNA becomes packaged and the particle becomes able to bind the small 'D-protein' in amounts equimolar to the capsid protein, which it could not do before. The D-protein addition probably causes another shift of the capsid structure. DNA packaging is completed, and the DNA is cut from concatemeric precursors to unit length molecules. Binding sites are created for the tail connector molecules which in turn allow the independently assembled tail to attach. Research on these processes proceeds along several lines: comparison of physical and chemical properties of particles accumulating in mutants; pulse-chase experiments on assembly precursors; morphogenesis in vitro; and model transitions of aberrant lambda polyheads.

Capsid↗
PubMed1975

Electron microscopic studies on assembly of herpes simplex virus upon removal of hydroxyurea block.

The release of hydroxyurea-treated, herpes simplex virus-infected cells from the drug-induced block resulted in the prompt assembly of infectious virus. Electron microscope observations at sequential intervals following removal of the drug revealed considerable synchrony of replication. This synchrony permitted stages in the complex process of core assembly to be examined in detail. The data suggest that after partial or complete assembly the nucleoprotein enters the differentiated capsid to become enfolded.

Cell Nucleus↗
PubMed1976

A study of the states of aggregation of alfalfa mosaic virus protein.

The states of aggregation of alfalfa mosaic virus (AMV) protein have been characterized by sedimentation velocity experiments and electron microscopy. The main association product is a spherical particle with an s value of about 30S. It is highly likely that the assembly of this particle starts with dimers of the 25000 molecular mass unit resulting in an icosahedral particle made of 30 dimers. No intermediate aggregation products have been detected. The clustering pattern of the protein in the cylindrical part of the AMV capsid favours the concept of dimers as the active assembling units.

Capsid↗
PubMed1979

Electron microscopy of the morphogenesis of Bacillus subtilis bacteriophage SP3.

The capsid of Bacillus subtilis bacteriophage SP3 is assembled via a prohead intermediate which subsequently encapsulates DNA and attaches a tail. The prohead contains a ring-like core structure. The spokes which extend from the core to the inner prohead surface are thought to form a scaffold for the polymerization of the prohead. Ninety percent of the proheads are assembled prior to the onset of DNA encapsulation. The first mature phage particles are observed at 45 min after infection; titres of intracellular phage demonstrate their infectivity. The core is visible in phage ghosts.

Bacillus subtilis↗
PubMed2024

A RNA Dodecahedral Cage Inside a Human Virus Plays a Dual Biological Role in Virion Assembly and Genome Release Control.

Human rhinoviruses (RV) are among the most frequent human pathogens. As major causative agents of common colds they originate serious socioeconomic problems and huge expenditure every year, and they also exacerbate severe respiratory diseases. No anti-rhinoviral drugs or vaccines are available so far. Antiviral drug design may benefit from an understanding of the role during the infectious cycle of the interactions in the virion between the capsid and the viral nucleic acid. The genomic RNA inside the human RV virion forms a dodecahedral cage made of 30 double-stranded RNA elements that interact with equivalent sites at the capsid inner wall. RNA dodecahedral cages also occur in distantly related insect and plant viruses. However, the functional role(s) of the interactions between any dodecahedral cage and the capsid remained to be established. Here we describe an extensive structure-function mutational analysis of the capsid-RNA dodecahedral cage interface in the RV virion, to dissect the role of the interactions between the capsid and the cage-forming RNA duplexes in: (i) infection by RV; (ii) virus biological fitness; (iii) virion assembly; (iv) virion stability; and (v) viral RNA uncoating. The results reveal that the capsid-bound dsRNA dodecahedral cage in the human RV virion is a multifunctional structural element. Two structurally overlapping subsets of RNA duplex-capsid interactions promote virus infectivity and biological fitness by respectively facilitating virion assembly or restraining the untimely, unproductive uncoating of the viral RNA genome. These results provide new insights into virion morphogenesis and genome uncoating, and have implications for antiviral drug design.

RNA, Viral↗
PubMed1977

DNA packaging and the pathway of bacteriophage T4 head assembly.

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction.

Coliphages↗
PubMed1978

Studies of virus structure by laser-Raman spectroscopy. Turnip yellow mosaic virus and capsids.

Laser-Raman spectroscopy of the turnip yellow mosaic virus (TYMV) and its capsid indicate the following features of the structure and assembly of the virion. The secondary structure of coat-protein molecules in TYMV is comprised of 9 +/- 5% alpha-helix, 43 +/- 6% beta-sheet, and 48 +/- 6% irregular conformation and is not altered by the removal of the RNA from the capsid. Introduction of as many as 200 chain scissions per RNA molecule also does not affect the overall secondary structure of the encapsulated RNA, which is 77 +/- 5% in the A-helix form. Tryptophan and cysteine residues of the coat protein appear to be in contact with the solvent, while only one of three tyrosines per coat protein is available for hydrogen bonding of its p-hydroxyl group with H2O molecules. Both cytosine and adenine residues of TYMV RNA are protonated in substantial numbers near pH 4.5, suggesting elevation of their respective pKa values within the virion. The Raman data are consistent with chemical evidence favoring interaction between protonated bases of RNA and amino acid side chains of coat protein in TYMV.

Capsid↗
PubMed1977

Transient association of Semliki Forest virus capsid protein with ribosomes.

HeLa cells infected with Semliki Forest virus were exposed to [35S]methionine for 1 min and chased for various periods. The analysis of labeled ribonucleoproteins showed that the viral capsid protein associated first with the large ribosomal subunit in polysomes, from which it was chased to assembling nucleocapsids and to free monosomes.

Capsid↗
PubMed2026

RNA-repelling Anionic Clusters in Human Rhinovirus Cooperate with Cationic Residues to Promote Virion Assembly and Restrain RNA Release.

Research on virus nucleic acid-protein interactions is important to understand infection and guide antiviral drug design. In previous studies we showed that the human rhinovirus (RV) genomic RNA is organized as a dodecahedral cage formed by 30 RNA duplex elements anchored to capsid concavities. We showed also that capsid-RNA duplex interactions include conserved tryptophans, neutral polar residues, and many positively charged residues that promote virion assembly and restrain RNA release by stabilizing the negatively charged RNA duplex structure. The present study expands our understanding of the capsid-RNA duplex interface in RV by addressing the structural and functional roles of conserved patches of negatively charged capsid residues interposed between each RNA duplex and its binding site at the capsid inner surface. The initial hypothesis was that electrostatic repulsion between anionic residues and RNA phosphates would lead to functional effects opposite to those previously found for cationic residues that can electrostatically attract RNA phosphates. In fact, those anionic residues do not oppose, but act together with cationic residues at the RNA duplex binding sites to promote virion assembly and restrain RNA release. Cryogenic electron microscopy analysis showed that negatively charged residues at the capsid-RNA duplex interfaces have a different structural role than positively charged residues, even though they all play similar functional roles. A tentative model is discussed to explain the functional effects of the complex distribution of negative and positive electrostatic potential found at capsid-RNA duplex interfaces in RV.

capsid↗