Purification and structure of Semliki Forest virus isolated from mouse brain

1968 ◽  
Vol 14 (2) ◽  
pp. 153-159 ◽  
Author(s):  
P. Faulkner ◽  
S. M. McGee-Russell
Keyword(s):  
Semliki Forest Virus ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Cesium Chloride ◽  
Equilibrium Density ◽  
Thin Sections ◽  
Virus Particles ◽  
Fundamental Symmetry ◽  
The Core ◽  
Intact Virus

Semliki Forest virus from brains of infected suckling mouse was separated by cesium chloride equilibrium density gradient centrifugation into two fractions, both able to agglutinate red blood cells of goose. The heavy fraction (HF density 1.24) had more infectivity, had a sedimentation coefficient of 285 S, and contained intact virus particles, diameter about 75 mμ. The light fraction (LF density 1.205) of equivalent HA (hemagglutinating activity), had less than 1% of the infectivity of the HF and did not contain intact virus particles. It consisted of "empty" virus particles and fragments of the fragile envelope material. The complete virus particle consists of an envelope of complex character surrounding a well-defined core. Shadow-cast preparations of HF show pointed and truncated shadows which suggest cubical symmetry, and the core of full particles, in thin sections, shows an angular, often pentagonal or hexagonal configuration. This suggests that the cubical symmetry of the full particle depends upon the fundamental symmetry of the core, which appears to be icosahedral.

Isolation and preliminary characterization of herpes Channel Catfish virus DNA

1980 ◽  
Vol 26 (2) ◽  
pp. 130-134 ◽  
Author(s):  
Jean Robin ◽  
Alice Rodrigue
Keyword(s):  
Channel Catfish ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Sodium Deoxycholate ◽  
Cesium Chloride ◽  
Equilibrium Density ◽  
Channel Catfish Virus ◽  
Phenol Extraction ◽  
Infected Cells

The DNA of Channel Catfish virus (CCV) was selectively extracted from infected cells with a 5% solution of sodium deoxycholate, deproteinized using sodium sarcosinate and pronase, and purified by phenol extraction followed by equilibrium density gradient centrifugation in a cesium chloride solution. CCV DNA displays a buoyant density of 1.715 g/cm3 in such a solution, as would be expected from a duplex DNA containing 56.1% of guanine plus cytosine. As estimated from both its sedimentation coefficient and length in the electron microscope, CCV DNA is a linear duplex molecule of approximately 85 × 106 daltons.

Morphology of the California Encephalitis Virus (CEV)

1972 ◽  
Vol 30 ◽  
pp. 314-315
Author(s):  
Z. Vaituzis ◽  
P.S. Reichelderfer ◽  
F.M. Hetrick
Keyword(s):  
Gradient Centrifugation ◽  
Cesium Chloride ◽  
Staining Technique ◽  
Prototype Strain ◽  
Uranyl Acetate ◽  
Encephalitis Virus ◽  
Virus Concentration ◽  
Virus Propagation ◽  
Thin Sections ◽  
Intact Virus

Negative staining technique with potassium phosphotungstate (pH 7.2) and thin sections stained with uranyl acetate (pH 4.2) and lead citrate (pH 11.0) were used to study the fine structure of the prototype strain, BFS-283, of the California encephalitis virus group. The BHK-21 cell line was employed in this study for virus propagation. Monolayers were infected using a multiplicity of infection of 0.1. Following virus adsorption the cells were incubated at 37 C for 48 or 72 hrs. at which times samples were harvested for electron microscopy.Conventional methods for virus concentration and purification (e.g. centrifugation, cesium chloride gradient centrifugation, dialysis) failed to yield intact virus particles. These preparations contained disintegrating virions averaging 75 nm in diameter (Fig. 1). Treatment of such purified preparations with a 1.0% solution of sodium desoxycholate removed the lipid envelope and associated material revealing a nucleocapsid core of approximately 43 nm (Fig. 2).

Über die Spaltung physikalisch intakter Poliovirus-Teilchen in Nucleinsäure und leere Proteinhüllen durch Wärmebehandlung

1965 ◽  
Vol 20 (9) ◽  
pp. 870-878 ◽  
Author(s):  
O. Drees ◽  
Ch. Borna
Keyword(s):  
Protein Concentration ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Virus Protein ◽  
Analytical Ultracentrifuge ◽  
Sucrose Density Gradient Centrifugation ◽  
Virus Particles ◽  
Phosphate Buffered Saline

Purified preparations of poliovirus devoid of contaminating nucleic acid and so-called “C antigen” released their particle-bound ribonucleic acid (RNA) almost quantitatively on heating at 40°C for 48 hours in phosphate-buffered saline (pH 7.6). This process occurred without disruption of the virus protein and left, in addition to the free RNA and traces of undegraded virus particles, empty protein shells in the reaction mixture.The liberated RNA sedimented in the analytical ultracentrifuge as a very diffuse boundary with sedimentation coefficients (s20, w) in the range from about 8 S to less than 1 S. The shell material which could be isolated by means of sucrose density-gradient centrifugation proved to be homogeneous in the analytical ultracentrifuge. Its sedimentation coefficient (s20,w) extrapolated to zero protein concentration was found to be 78 S.

An improved procedure for the purification of apple chlorotic leaf spot virus

1992 ◽  
Vol 72 (3) ◽  
pp. 937-941
Author(s):  
Delano James ◽  
Paul L. Monette
Keyword(s):  
Density Gradient ◽  
Leaf Spot ◽  
Gradient Centrifugation ◽  
Chenopodium Quinoa ◽  
Sucrose Density Gradient ◽  
High Yield ◽  
Spot Virus ◽  
Virus Particles ◽  
Intact Virus ◽  
Chlorotic Leaf

A reproducible procedure is described for the purification of apple chlorotic leaf spot virus (CLSV) from Chenopodium quinoa. Two cycles of sucrose density-gradient centrifugation in a magnesium-containing buffer were used. A relatively high yield of virus particles was obtained with little degradation observed. The average yield was 2.3 mg 100 g−1 fresh leaf tissue. The modal lengths of purified virus particles and particles from leaf sap were 692 nm and 704 nm, respectively.Key words: Chenopodium quinoa, sucrose density gradient, magnesium-containing buffer, intact virus

Semliki forest virus core protein fragmentation: Its possible role in nucleocapsid disassembly

1993 ◽  
Vol 13 (6) ◽  
pp. 333-347 ◽  
Author(s):  
Andreas Schlegel ◽  
Johann Schaller ◽  
Pia Jentsch ◽  
Christoph Kempf
Keyword(s):  
Capsid Protein ◽  
Semliki Forest Virus ◽  
Core Protein ◽  
Gradient Centrifugation ◽  
Biological Significance ◽  
Acidic Ph ◽  
Virus Particles ◽  
Virus Core ◽  
Core Proteins ◽  
Protein Fragmentation

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243–255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.

Nucleotide sequence and genome organization of Apple latent spherical virus: a new virus classified into the family Comoviridae

Microbiology ◽  
2000 ◽  
Vol 81 (2) ◽  
pp. 541-547 ◽  
Author(s):  
Chunjiang Li ◽  
Nobu Yoshikawa ◽  
Tsuyoshi Takahashi ◽  
Tsutae Ito ◽  
Kouji Yoshida ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Direct Sequencing ◽  
Gradient Centrifugation ◽  
Chenopodium Quinoa ◽  
Capsid Proteins ◽  
Gene Arrangement ◽  
Equilibrium Density ◽  
Apple Latent Spherical Virus ◽  
Virus Particles ◽  
The Family

A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1·41 and 1·43 g/cm3 in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3′ poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa (‘23K’; ORF1) and 235 kDa (‘235K’; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa (‘108K’). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the Comoviridae. The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.

scholarly journals Proof-of-concept for the yadokari nature: a capsidless replicase-encoding but replication-dependent (+)ssRNA virus hosted by an unrelated dsRNA virus

2021 ◽  
Author(s):  
Subha Das ◽  
Md Mahfuz Alam ◽  
Rui Zhang ◽  
Sakae Hisano ◽  
Nobuhiro Suzuki
Keyword(s):  
Density Gradient ◽  
Reverse Genetics ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Cesium Chloride ◽  
Dsrna Virus ◽  
Equilibrium Density ◽  
Proof Of Concept ◽  
Ssrna Virus

We have previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense ssRNA virus, yadokari virus 1 (YkV1), and an unrelated dsRNA virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix . We have proposed that YkV1 diverts the YnV1 capsid to trans-encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp, and whether YnV1 capsid co-packages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs on the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein co-translationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsid solely packaging YkV1 dsRNA and RdRp. These results provide proof-of-concept that a capsidless (+)ssRNA virus is hosted by an unrelated dsRNA virus. Importance Viruses typically encode their own capsids that encase their genomes. However, a capsidless (+)ssRNA virus, YkV1, depends on an unrelated dsRNA virus, YnV1, for encapsidation and replication. We have previously shown that YkV1 highjacks the capsid of YnV1 for trans-encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the hetero-capsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains on the YkV1 polyprotein are important for its replication. The active RdRp must be cleaved by a 2A-like peptide from the C-proximal protein. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with YnV1 capsid solely packaging YkV1 dsRNA and RdRp. This study provides proof-of-concept of a virus neo-lifestyle where a (+)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

Intramitochondrial Viruses of Reptilian Cell Origin

1972 ◽  
Vol 30 ◽  
pp. 318-319
Author(s):  
Philip D. Lunger ◽  
H. Fred Clark
Keyword(s):  
Particle Production ◽  
Outer Zone ◽  
Density Variation ◽  
Core Region ◽  
Mitochondrial Membranes ◽  
Virus Particles ◽  
The Core ◽  
Vipera Russelli ◽  
Maturation Stages ◽  
Type Particle

In the course of fine structure studies of spontaneous “C-type” particle production in a viper (Vipera russelli) spleen cell line, designated VSW, virus particles were frequently observed within mitochondria. The latter were usually enlarged or swollen, compared to virus-free mitochondria, and displayed a considerable degree of cristae disorganization.Intramitochondrial viruses measure 90 to 100 mμ in diameter, and consist of a nucleoid or core region of varying density and measuring approximately 45 mμ in diameter. Nucleoid density variation is presumed to reflect varying degrees of condensation, and hence maturation stages. The core region is surrounded by a less-dense outer zone presumably representing viral capsid.Particles are usually situated in peripheral regions of the mitochondrion. In most instances they appear to be lodged between loosely apposed inner and outer mitochondrial membranes.

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