Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl.

Although hepatitis A virus (HAV) released by infected BS-C-1 cells banded predominantly at 1.325 g/cm3 (major component) in CsCl, smaller proportions of infectious virions banded at 1.42 g/cm3 (dense HAV particles) and at 1.27 g/cm3 (previously unrecognized light HAV particles). cDNA-RNA hybridization confirmed the banding of viral RNA at each density, and immune electron microscopy demonstrated apparently complete viral particles in each peak fraction. The ratio of the infectivity (radioimmunofocus assay) titer to the antigen (radioimmunoassay) titer of the major component was approximately 15-fold greater than that of dense HAV particles and 4-fold that of light HAV particles. After extraction with chloroform, the buoyant density of light and major component HAV particles remained unchanged, indicating that the lower density of the light particles was not due to association with lipids. Light particles also banded at a lower density (1.21 g/cm3) in metrizamide than did the major component (1.31 g/cm3). Dense HAV particles, detected by subsequent centrifugation in CsCl, were indistinguishable from the major component when first banded in metrizamide (1.31 g/cm3). However, dense HAV particles recovered from CsCl subsequently banded at 1.37 g/cm3 in metrizamide. Electrophoresis of virion RNA under denaturing conditions demonstrated that dense, major-component, and light HAV particles all contained RNA of similar length. Thus, infectious HAV particles released by BS-C-1 cells in vitro consist of three distinct types which band at substantially different densities in CsC1, suggesting different capsid structures with varied permeability to cesium or different degrees of hydration.     

Isolation and Characterization of Subacute Sclerosing Panencephalitis Virus Nucleocapsids

Nucleocapsids from subacute sclerosing panencephalitis (SSPE) virus-infected CV-1 cells were concentrated by differential centrifugation employing sucrose cushion techniques and further purified by centrifugation through a linear CsCl density gradient. The bouyant density of (3)H-uridine-labeled nucleocapsids in CsCl was found to be 1.31 g/cm(3). Ultraviolet absorption spectra of the purified SSPE nucleocapsid showed an absorption maximum at 260 to 265 nm and a 280/260 ratio that corresponded to a nucleic acid content of approximately 4.3%. Negatively stained preparations of SSPE nucleocapsids were found to have a width of 18 +/- 1 nm, a periodicity of 5 to 6 nm, and a length between 1-1.4 mum, with the greatest number at 1.3 mum.     

Buoyant Density of the Hepatitis A Virus-Like Particle in Cesium Chloride

We recently visualized by immune electron microscopy a virus-like particle in the stools of patients with hepatitis A. The particle measured approximately 27 nm in diameter and morphologically resembled a picornavirus or parvovirus. To further characterize this particle, we have determined its buoyant density in cesium chloride (CsCl) by ultracentrifugation. Hepatitis A particles from three positive stool specimens were isopycnically banded in separate experiments, and the gradient fractions were examined for particles by immune electron microscopy by using hepatitis A convalescent sera. In each experiment, the particles were observed in a normal distribution about a peak fraction with a mean density of approximately 1.4 g/cm(3). The buoyant density of 1.4 g/cm(3) in CsCl together with its morphology and the reported resistance of hepatitis virus to acid, ether, and heat suggest that this particle is parvovirus-like.     

Defective bacteriophage MS2 particles containing specific RNA FRAGMENTS]

Two types of MS2 particle are revealed when phage lysates are banded in CsCl density gradient. The lower band contain normal phage particles with a density of 1.46 g/cm3. The upper band with a density of 1.44 g/cm3 containes uninfective incomplete MS2 particles. Both phage types reveal no abnormalities in the content of the coat protein and A-protein. They are nearly identical in RNA content. RNA in the normal buoyant density phage particles is native. RNA in the defective particles consists of three specific fragments with molecular weights 6.5-10(5), 5.5-10(5) and 4.4-10(5) and molar ratios 5:4:9 respectively. THE 5'-TERMINAL ANALYSIS OF RNA from defective MS2 particles reveals the presence of native pppGp. THE 3'-TERMINAL ANALYSIS OF THE INDIVIDUAL RNA fragments reveals the presence of adenosine only in the shortest fragment. RNA fragmentation in defective particles can be explained by the action of intracellular RNAses on the unprotected regions on RNA chain in structurally incomplete virions.     

Use of Zonal Ultracentrifuge Systems for Biophysical Studies of Rhinoviruses

This paper reports the use of zonal ultracentrifuge techniques to conduct biophysical studies of rhinoviruses grown with WI-38 cells. Good clean-out of infectivity from rhinovirus harvests was obtained with the continuous-flow B-V and B-IX rotors. Use of the B-V rotor resulted in the successful concentration of rhinovirus infectivity and antigenicity. Additional purification was achieved by the combined use of continuous-flow centrifugation and isopycnic banding procedures. Two particle sizes were found to be associated with the virus-infected cell harvests. The infectious 22-nm particle banded in density ranges of 1.38 to 1.40 g/cm(3) in CsCl and 1.26 to 1.27 g/cm(3) in potassium citrate. The 8.0 nm capsomere was composed of 2.0 nm subunits and banded with a density of protein at 1.28 g/cm(3) in CsCl. Equivalent sedimentation coefficients of 155 or 185, depending on particle density in sucrose, were calculated from rate zonal experiments by use of the B-IV zonal rotor.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

Purification of Streptomyces venezuelae phage

Kolstad, R. A. (University of Minnesota, Minneapolis), and S. G. Bradley. Purification of Streptomyces venezuelae phage. J. Bacteriol. 87:1157-1161. 1964.-Streptomyces venezuelae phage MSP8 was concentrated and purified by a combination of methods including dialysis against polyethylene glycol, partitioning between the two phases of aqueous polymer systems, gel filtration, chromatography on ECTEOLA-cellulose, and differential centrifugation. S. venezuelae phage MSP8 is 57% deoxyribonucleic acid and 43% protein. Its head is 55 by 70 mmu, and its tail is 10 by 150 mmu. Its dry weight is 250 mg per 10(15) plaqueforming units, and its density is 1.4. Phage MSP8 contains 15 mug of phosphorus and 40 mug of nitrogen per 10(12) particles. The ratio of light absorbancy at 260 to 280 mmu is 1.5. A mixture of two actinophages, MNP3 and MVP7, was separated by use of ECTEOLA-cellulose. In one fraction, 99% of the phage was MNP3; in another fraction, 99% of the phage was MVP7.     

Differences between poliovirus empty capsids formed in vivo and those formed in vitro: a role for the morphopoietic factor.

Empty capsid species formed from the self- and extract-mediated assembly of poliovirus type 1 14S particles in vitro and procapsids isolated from virus-infected cells were subjected to isoelectric focusing in charge-free agarose gels. The empty capsid formed in the self-assembly reaction had an isoelectric point (pI) of 5.0, whereas procapsids and extract-assembled empty capsids focused at pH 6.8. Unreacted 14S particles focused at pH 4.8 to 5.0. The sedimentation coefficient (s20,w) and density of the empty capsid species were also determined. Procapsids had a density in CsCl of 1.31 g/cm3, whereas empty capsids formed by self- or extract-mediated assembly had a density of 1.29 g/cm3. Both extract-assembled empty capsids and procapsids had an s20,w of 75S, whereas self-assembled empty capsids had an s20,w of 71S. Self-assembled empty capsids were not converted to pI 6.8 empty capsids by incubation with poliovirus-infected HeLa cell extracts. The dissociated polypeptides of self-assembled empty capsids (pI 5.0) and procapsids (pI 6.8) behaved identically when analyzed by isoelectric focusing in the presence of 9 M urea and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These results suggest that infected cell extracts possess a factor that influences the final conformation of the empty shell (pI 6.8, 75S) formed from 14S particles and that this influences is exerted at the initiation step or during the polymerization reaction. A small amount of this activity (less than or equal to 20% of infected extracts) was detected in uninfected cells; the significance of this remains unknown.     

Properties of "diplophage": a lipid-containing bacteriophage.

We describe the purification and properties of Dp-1, a bacteriophage isolated from Diplococcus pneumoniae. The phage was sensitive to the organic solvents deoxycholate and Sarkosyl, and its infectivity was reduced by treatment with phospholipase C. Electron microscopy indicated the presence of a double-layered coat around the phage particles. Purified phage preparations contained lipid amounting to about 8.5% of the dry weight of the phage, and thin-layer chromatography resolved the lipids into four components. The phage had a buoyant density in CsCl of 1.47 g/cm3, and a sedimentation constant in 0.1 M NaCl of 313S. Analysis in acrylamide gel electrophoresis indicated the presence of three major proteins. Dp-1 DNA shows a density of 1.681 g/cm3. Neutralizing antisera against the phage have a low potency (K less than 120/min).     

Properties of Intracytoplasmic A Particles Isolated from Oncornavirus-Producing Human Cells

A particles with the diameter of 70 to 80 nm were isolated from the cytoplasm of HEp-2, HeLa, and AO cells producing oncornavirus of Mason-Pfizer-like type. Most of the A particles banded at 1.23 to 1.24 g/ml, whereas 3 to 10% banded at 1.29 g/ml in equilibrium sucrose gradients. They banded at 1.30 g/ml in CsCl gradients suggesting that they contained 8% RNA. Individual A particles sedimented at 200 to 250S in velocity sucrose gradients, but their significant part was found aggregated and sedimented at more than 300S. They were resistant to RNase digestion. A particles possessed polymerase activity which was preferentially activated by Mn(2+) rather than by Mg(2+), the RNA template being 60S RNA. Cross-hybridization with two DNA products and immunoassay showed that A particles and Mason-Pfizer-like oncornavirus produced by the same cells contained neither homological RNA sequences nor common antigens, suggesting that A particles are not intracellular precursors of Mason-Pfizer-like oncornavirus but represent an independent oncornavirus. Hybridization of A particle RNA with excess of cellular DNA revealed about 20 proviral copies per HEp-2 cell genome and no proviral copies in human embryo and placenta cell genomes.     

Molecular structure of adeno-associated virus variant DNA

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.     

Mechanical Transmission, Purification and Properties of an Isolate of Maize Stripe Virus from Venezuela

A Venezuelan isolate of maize stripe virus (MStpV) was successfully transmitted mechanically and by the leafhopper Peregrinus maidis from field infected plants to sweet cv. Iochief. After purification of maize infected with MStpV, fine spiral filamentous particles about 4 nm in diameter and with variable lengths were consistently associated with a nucleoprotein band present in CsCl or Cs₂SO₄ isopycnic gradients. Purified preparations exhibited a typical nucleoprotein absorption spectrum with a maximum at 260–263 nm and a minimum at 240–243 nm and a 260–280 ratio of 1.38. The density of the nucleoprotein in CsCl gradients was estimated at 1.29 g/ml. The sedimentation coefficient was calculated at 62 S. The nucleoprotein consisted of 5 % single stranded RNA and a capsid protein of molecular weight 33.500 daltons. Large quantities of non-capsid proteins were isolated from infected tissue with a molecular weight of 17.500 and 16.500 daltons. Peregrinus maidis, injected with purified MStpV preparation failed to transmit the disease to healthy plants. However, they were infectious when injected with clarified infected plant sap. Antisera against capsid and non-capsid proteins from MStpV-Florida strain reacted positively with the Venezuelan antigens.     

Isolation and characterization of a Pseudomonas aeruginosa bacteriophage with a very limited host range

A Pseudomonas aeruginosa bacteriophage, phi PLS743, with extremely limited host range has been isolated. It belongs to the virus family Podoviridae, morphological type C1, and possesses a head diameter of 45 nm. The phage has a buoyant density in CsCl of 1.516 g/cm3, and its mass is 45 x 10(6) daltons. The phage particles are composed of double-stranded DNA (49.9 mol% G + C; 42.4 kilobase pairs) and 11 structural proteins (66% by weight). The major head protein, P5, has a Mr of 34,500. The DNA is not cut by SalI or XhoI restriction endonucleases, but is cut by PvuII (1 site), KpnI and BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), and HindIII (12 sites). A restriction endonuclease map is presented.     

Characterization and classification of virus particles associated with hepatitis A. I. Size, density, and sedimentation.

Virus-like particles were purified from stools of patients in an epidemic of hepatitis A in Germany. When reference MS-1 chimpanzee pre-inoculation and convalescent sera were used, the close serological relationship of the purified particles to well-known isolates of hepatitis A could be established. On the other hand, the physicochemical characteristics of the particles were determined in parallel to the characteristics of a marker parvovirus (LuIII) and a marker picornavirus (poliovirus type 2). It could be shown that the majority of the hepatitis A-associated particles band at 1.34 g/ml in CsCl and, like poliovirus, sediment at about 160S. In addition, a distinct hepatitis A antigen was observed, which banded at 1.305 g/ml and sedimented between 50 and 90S. A further component accumulated in the density range of between 1.38 and 1.44 g/ml. However, it seemed to be rather labile. Upon reisolation from CsCl and sedimentation in sucrose, it resolved into a 160S, a 90 to 100S, and a 50S form. The size of the 160S particles (27 to 29 nm) could be readily distinguished from that of the parvovirus (22 to 24 nm). It is concluded, therefore, that hepatitis A-associated virus particles are more likely to be classified with the picornaviruses than with the parvoviruses.     

Purification and some particle properties of anthriscus yellows virus, a phloem-limited semi-persistent aphid-borne virus

Anthriscus yellows virus (AYV), a phloem-limited virus transmitted in the semi-persistent manner by the aphid Cavariella aegopodii, was purified by treatment of leaf extracts with cellulasc, followed by differential and sucrose density gradient centrifugation. ‘The preparations contained isometric particles c. 29 nm in diameter which were unstable unless stored in buffer at pH 8.0 containing 1 mM CaCl₂,. The particles sedimented as two components, ’full‘ nucleoprotein particles with A₂₆₀/A₂₈₀= 1.83 containing about 42% nucleic acid, and ’empty‘ protein shells with A₂₆₀,/A₂₈₀= 0.73; their buoyant densities in CsCl solutions were 1.52 and 1.27 g/cm³. Respectively. Yields of ihe nircleoprotein particles were c. 1.75 mg/kg leaf tissue. The particles contained a single species of RNA, of mol. wt 3.6 × 10 “(10 000 nucleotides). Particle protein preparations contained four electrophoretic species, of mol. wt (× 10³) 35.0, 28.3, 23.3 and 22.3.C. aegopodii did not transmit AYV from purified preparations. A rabbit injected with AYV preparations produced antibodies that coated AYV particles in electron microscope tests, but gave variable reactions in immunosorbent electron microscopy (ISEM), depending on the composition of the medium. No reactions were obtained in enzyme-linked inimunosorbent asjay (ELISA). No serological relationship was detected in ISEM between AYV and any of 10 viruses that resembled it in one or more properties.     

Adenovirus type 2 assembly analyzed by reversible cross-linking of labile intermediates.

Dimethyl-4,4'-dithiobisbutyrimidate dihydrochloride was used as a cleavable cross-linking reagent to maintain the structure of labile intermediates in adenovirus type 2 assembly. Analysis on sucrose gradients of nuclear adenovirus particles revealed two size classes, with sedimentation rates of 750 and 600S. After reversible fixation with diimido ester, the different classes were further separated on CsCl gradients and characterized with regard to their buoyant density, DNA content, and polypeptide composition. The 750S particles banded at 1.345 g/cm3 in CsCl, contained a DNA with a sedimentation coefficient of 34S in alkaline sucrose gradients, and had a polypeptide composition similar to that of young virions. The 600S population consisted of two types of particles with buoyant densities of 1.315 and 1.37 g/cm3. The 1.315-g/cm3 particles contained a DNA fragment of 7--11S and lacked the core proteins V and VII. In their place were found precursors P VI and P VIII and two nonvirion proteins with molecular weights of 50,000 (50K) and 39,000 (39K). 34S DNA was present in the 1.37-g/cm3 particles, which also lacked core proteins V and VII, as well as the 50K and 39K. Pulse-chase labeling kinetics suggested that the 1.315-g/cm3 particles were anterior to the 1.37-g/cm3 particles, themselves preceding the 1.345-g/cm3 young virions, and that the release of both 50K and 39K, possible scaffolding proteins, was required for entry of viral DNA.     

Virus-like particles from killer, neutral, and sensitive strains of Saccharomyces cerevisiae.

Procedures were developed for purification of virus-like particles (VLPs) from killer, neutral, and sensitive strains of Saccharomyces cerevisiae. Morphologically similar spherical VLPs measuring 40 nm in diameter were extracted from all three phenotypes. The particles were partially purified by high-speed centrifugation through a layer of CsCl (1.26 g/cm3) and sucrose density gradient centrifugation. Gradient-purified preparations contained three centrifugal species that sedimented at approximately 43, 102, and 162S. The 43S component is considered to be an artifact. Preparations from killer strains contained three double-stranded RNA (ds-RNA) components with molecular weights of 1.19 x 10(6), 1.29 x 10(6) and 2.54 x 10(6). VLPs from neutral and sensitive strains contained only the largest ds-RNA species. VLP preparations were subsequently separated into two major density components by CsCl equilibrium gradient centrifugation. The light component banding at 1.28 to 1.30 g/cm3 was void of nucleic acid, and the heavy component banding at 1.40 g/cm3 contained only the largest ds-RNA species.     

Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CsCl

When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm³. Infectious EEE virus banded in two positions; most of the virus banded at 1.20 g/cm³ and a lesser amount banded at 1.22 to 1.23 g/cm³. Analysis of radioactive profiles of CsCl-fractionated EEE virus labeled with either ³²PO₄ or ³H-uridine suggested that the hemagglutinin was stripped from the intact EEE virion. The viral origin of the hemagglutinin was verified by inhibition with specific antiserum. Attempts to differentiate between infectious EEE virus of the different buoyant densities showed that the denser particle was neither a virus contaminant nor a density mutant. No evidence was obtained to indicate that the denser particle was an immature form of EEE virus. The two infectious EEE species obtained after CsCl fractionation were indistinguishable antigenically. Furthermore, unfractionated as well as CsCl-fractionated EEE virus sedimented at about 260S in sucrose gradients. These results together with the results of rebanding experiments suggested that the denser EEE species (1.23 g/cm³) results from a salt (CsCl)-induced alteration or breakdown of the EEE virion (1.20 g/cm³), and that it arises as the hemagglutinin is stripped from the surface of the EEE virion.     

Proteinaceous Virus-like Particles from an Isolate of Aspergillus flavus

Virus-like particles were purified from a single nonaflatoxin-producing isolate of Aspergillus flavus. The virus-like particles were spherical, measuring 27 to 30 nm in diameter, were electrophoretically homogeneous, and sedimented at approximately 49S. The particles had a buoyant density of 1.28 g/cm(3) in CsCl and contained no detectable nucleic acid.     

I-DNA suppressed by Ethidium Bromide and accumulated with Dactinomycin

THE I-DNA in cytoplasmic particles^1–4 of embryonic chick cells has a 260 nm/280 nm ratio of 1.95 (ref. 5) and a buoyant density of CsCl of 1.700–1.703 g/ml.; mitochondrial DNA has a buoyant density of 1.708 g/ml.^6,7. Other reports of non-mitochondrial cytoplasmic DNA different from nuclear DNA have appeared recently^8–10.