Defective Virions of Reovirus

When purified preparations of stock reovirus, type 3, were digested with chymotrypsin, the virions were converted into two different types of particle. These new particles could be separated from each other by isopycnic centrifugation in cesium chloride gradients. One particle banded at a buoyant density of 1.43 g/cm(3), the other at a density of 1.415 g/cm(3). The former particle is termed the heavy (H) particle, the latter is the light (L) particle. The ratio of H/L particles varied between 0.5 and 0.25 in various purified preparations of virus. In electron micrographs, both H and L particles had the appearance and dimensions of viral cores. H particles were infectious for L cells. When plaques formed by stock virus, or by H particles, were picked and propagated in L cells, the majority of the clones gave rise only to H particles on chymotrypsin digestion. On continued serial passage of the clones, virions containing L particles again appeared in the progeny. The simplest explanation of these results was that stock virus was comprised of two populations of virions. One type of virion which contained H particles was infectious, whereas the other, which contained L particles, was not itself infectious and could replicate only in cells coinfected with an H particle virion. Added weight was given to this hypothesis by two observations. First, a small but definite separation of H and L virions could be achieved by isopycnic centrifugation in a gradient of cesium chloride. Second, L particles and virions containing L particles were both shown to lack the largest of the ten segments of double-stranded ribonucleic acid genome. Thus, L particle virions have defective genomes.     

Irreversible conversion of the physical state of herpes simplex virus preceding inactivation by thermal or antibody treatment.

The buoyant density characteristics of infectious particles of herpes simplex virus types 1 and 2 were studied by centrifugation in sucrose and cesium chloride density gradients with a high resolution and satisfactory infectivity recovery. It was shown that two populations of infectious virions differing in buoyant density coexisted, the difference being slight but definite. The ratio of heavy (H) to light (L) viral particles varied depending upon the solute used, the strains of virus, and the cell origin. Circumstances favoring degradation of viral infectivity tended to increase the H portion. Incubation at 37 degrees C largely converted L to H, and heating at 45 degrees C converted all virions to H without infectivity. The L to H conversion was irreversible, and no populations intermediate between L and H were clearly observed. Inactivation by UV light irradiation did not affect the density pattern. That H was not an artefact due to penetration of solutes, osmotic pressure, viral aggregation, or loss of the envelope was shown experimentally. A difference in the outer shape of particles between negatively stained L and H populations was demonstrated by electron microscopy. Both cell-released and cell-bound herpes simplex virus particles gave essentially the same result with respect to the above characteristics. The effect of limiting dilutions of antiserum was similar to that of mild thermal treatment, in that denser virions increased parallel to a decrease in less dense virions. Sensitization with early immunoglobulin G, composed mainly of complement-requiring neutralizing antibody, caused the density transition, and subsequent addition of complement resulted in a further increase in the buoyant density of the sensitized virions. The DNA in virus particles neutralized with immunoglobulin G plus complement remained resistant to DNase treatment. Possible implications of the phenomena are discussed.     

Equilibrium Density Gradient Studies on Simian Virus 40-Yielding Variants of the Adenovirus Type 2-Simian Virus 40 Hybrid Population

The simian virus 40 (SV40)-yielding variants of the adenovirus type 2 (Ad.2)-SV40 hybrid (Ad.2(++)) population were studied by means of fixed-angle equilibrium density gradient centrifugation in cesium chloride. The hybrid virions of the Ad.2(++) high-efficiency yielder population banded at densities of 0.004 g/cm(3) lighter than the nonhybrid Ad.2 virions. The degree of separation of the hybrid particles was sufficient to permit greater than 100-fold relative purification by two cycles of centrifugation. Hybrid particles that produce adenovirus plaques in African green monkey kidney cells by two-hit kinetics (one-hit kinetics when assayed on lawns of nonhybrid adenovirus) were not separable from the particles that yield SV40 virus. The hybrid particle in the Ad.2(++) low-efficiency yielder population was not separable from the nonhybrid Ad.2 virions.     

Preparation and partial characterization of a Lactobacillus lactis bacteriophage

High-titer lysates of a bacteriophage active against Lactobacillus lactis were prepared from liquid cultures as well as from areas of confluent lysis in soft-agar overlayers. Phage concentration and purification were accomplished by means of polyethylene glycol precipitation, differential centrifugation, and cesium chloride gradient centrifugation. The buoyant density of this phagein cesium chloride was 1.4795 g/ml. Characterization of phage growth cycle by one-step growth experiments under optimal conditions showed that the latent period was about 120 min, that the rise period lasted approx. 130 min, and that the average burst-size was about 80.Abbreviations p.f.u. plaque forming units - m.o.i. multiplicity of infection - PEG polyethylene glycol - SSC standard saline citrate solution     

Comparison of JC and BK Human Papovaviruses with Simian Virus 40: Restriction Endonuclease Digestion and Gel Electrophoresis of Resultant Fragments

JC virus was found to have a buoyant density of 1.20 g/cm(3) in linear sucrose-D(2)O and 1.35 g/cm(3) in cesium chloride isopycnic gradients. DNA extracted either from JC-infected cultures or from gradient-purified virions occupied a dense position relative to linear DNA in cesium chloride/ethidium bromide gradients, and the circular configuration of the extracted DNA was confirmed by electron microscopy, with a measured molecular weight of 2.93 x 10(6). DNA from BK virus was similarly prepared and compared to JC and to an SV40 DNA standard by digestion with restriction endonuclease preparations from Haemophilus influenzae, Haemophilus parainfluenzae, and Escherichia coli. Digests were electrophoretically analyzed on gradient polyacrylamide slab gels or agarose gels, and the three viruses were found to have distinctly different cleavage patterns by this form of analysis: JC and BK viruses were almost entirely different from SV40 and significantly different from each other. Thus, JC and BK human papovaviruses appear to be discrete new members of the papovavirus group, rather than SV40 variants.     

Morphological, Chemical, and Biological Characterization of Japanese Encephalitis Virus Virion and Its Hemagglutinin

Three morphologically distinct structures, inner core, envelope, and surface projections, were observed in purified Japanese encephalitis virus virions by electron microscopy. The average diameter of each structure was 29.8 +/- 2.5, 44.8 +/- 3.2, and 53.1 +/- 4.5 nm, respectively. Double staining with uranyl acetate and phosphotungstic acid preserved these structures well. Treatment of virions with proteolytic enzymes resulted in the loss of hemagglutinating activity, surface projections, and the major polypeptide band in polyacrylamide gel electrophoresis, which corresponds to glycoprotein, one of the three virion polypeptides. Surface projections were purified by cesium chloride density gradient centrifugation after treatment of virions with Nonidet P-40. The purified materials had a density of 1.256 g/cm(3) and were composed of only glycoprotein, as revealed by polyacrylamide gel electrophoresis. Purified surface projections carried hemagglutinating activity, as well as neutralizing antibody-blocking activity, and induced neutralizing antibody in mice.     

Density of infectious virus and complement-fixing antigens of two rhinovirus strains

Dans, P. E. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), B. R. Forsyth, and R. M. Chanock. Density of infectious virus and complement-fixing antigens of two rhinovirus strains. J. Bacteriol. 91:1605-1611. 1966.-Two rhinovirus serotypes (echovirus 28 and HGP) and poliovirus type 1 were banded by isopycnic centrifugation in cesium chloride. The rhinovirus virions had a density of 1.41 g/ml, whereas that of poliovirus was 1.34. Since a number of other enteroviruses also have a density of 1.34 g/ml in cesium chloride, a basic difference in density may exist between the rhinovirus and enterovirus subgroups of the picornavirus family. Whether this difference reflects differences in ribonucleic acid content or binding of cesium ions remains to be determined. In tests with echovirus 28 two peaks of CF activity were detected: one in association with the virion (1.41 g/ml), and a larger peak of lower density (1.30 g/ml). With echovirus 28 antiserum, a heterotypically reactive complement-fixing (CF) antigen was detected in the HGP virus suspension at a density less than that of the virion (1.30 g/ml). This antigen corresponded in density to the less dense CF antigen of echovirus 28.     

Methylation of satellite deoxyribonucleic acid in mouse neoplastic and non-neoplastic cell cultures

Summary Comparisons of nucleic acid methylation between paired neoplastic and non-neoplastic mouse cell lines have shown a striking difference in the deoxyribonucleic acid (DNA) peak eluted from methylated albumin-kieselguhr columns (R. Gantt and V. J. Evans, 1969, Cancer Res. 29: 536–541). Since mouse satellite DNA is relatively highly methylated, its 5-methylcytosine content was compared with mainband DNA in these two paired cell lines to determine whether this might account for the observed differences. The cell DNA was labeled with methyl-labeled methionine and isolated from the cells by repeated neutral cesium chloride isopycnic centrifugation. The satellite DNA strands were then separated in an alkaline cesium chloride gradient. Both the 5-methylcytosine content and the relative amounts of satellite DNA were indistinguishable in the paired cell lines. Further, the results showed that both strands of satellite DNA had virtually equal amounts of 5-methylcytosine, although the heavy strand contains 1.5 times more cytosine than the light strand.     

Morphogenesis of the Nucleoprotein of Vesicular Stomatitis Virus

Accumulation of the nucleoprotein of vesicular stomatitis virus (VSV) in the cytoplasm of BHK-21 cells and in two of four human cell lines was demonstrated. Appearance and progression of the nucleoprotein inclusions paralleled development of virus-specific immunofluorescence and production of virus progeny. The inclusions appeared early as discrete foci of filamentous material which eventually increased in size to form large masses which replaced normal cytoplasmic constituents. The filamentous strands were found in close proximity to budding virions. The inclusion material was extracted from infected cells and purified in cesium chloride gradients. The isolated filaments resembled the ribonucleoprotein isolated from purified virions. They incorporated (3)H-uridine, exhibited virus-specific complement-fixing activity, had a buoyant density of 1.32 g/cm(3), and appeared as single wavy strands the width of which varied from 2.5 to 8.5 nm, depending on the angle of viewing.     

Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA.

The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication.     

Propagation and Purification of High-Titer Human Cytomegalovirus

High-titered yields of human cytomegalovirus (CMV), strain AD 169, were produced in WI-38 cells in large roller bottles. Maximum plaque titers were observed by the 4th day after infection at which time infectivity in the medium was 200 times greater than that associated with the cells. Virus released into the medium was recovered by sedimentation in a sucrose gradient in a continuous-flow centrifuge rotor. Maximal viral infectivity was found at a sucrose concentration of 42%, equivalent to a density of 1.18 g/cm(3). Deoxyribonucleic acid extracted from these preparations was about 80% viral and 20% cellular as judged by equilibrium centrifugation in cesium chloride density gradients.     

Generation and neutralization of pseudovirions of human papillomavirus type 33.

Since human papillomaviruses (HPV) cannot be propagated in cell culture, the generation of infectious virions in vitro is a highly desirable goal. Here we report that pseudovirions can be generated by the assembly of virus-like particles (VLPs) in COS-7 cells containing multiple copies of a marker plasmid. Using recombinant vaccinia viruses, we have obtained spherical VLPs of HPV type 33 (HPV-33) which fractionate into heavy and light VLPs in cesium chloride density gradients. VLPs in the heavy fraction (1.31 g/cm3) carry the plasmid in DNase-resistant form and are capable of transferring the genetic marker located on the plasmid to COS-7 cells in a DNase-resistant way (pseudoinfection). The minor capsid protein L2 is not required for encapsidation but is essential for efficient pseudoinfection. Antiserum to HPV-33 VLPs inhibits VLP-mediated DNA transfer with high efficiency. Antisera to VLPs of HPV-18 and HPV-16 are not neutralizing, although the HPV-16 antiserum exhibited some cross-reactivity with HPV-33 VLPs in an enzyme-linked immunosorbent assay. In a cell binding assay, the titer of the HPV-33 VLP antiserum was 1:200 compared to the neutralization titer of 1:10(5). This indicates that neutralization is essentially due to the inhibition of cellular processes after VLP binding to cells. The encapsidation of marker plasmids into VLPs provides a sensitive and fast assay for the evaluation of neutralizing potentials of antisera against papillomavirus infections.     

On the Nature of Recombinants Formed during Transformation in Hemophilus influenzae

During the process of transformation in Hemophilus influenzae integration of donor DNA, i.e. the formation of recombinant DNA, involves the incorporation of single-stranded DNA. Evidence was obtained from cesium chloride density gradient centrifugation of DNA from donor-recipient complexes that integration was accompanied by the formation of hybrid DNA with a density intermediate with respect to heavy, ²H, ¹⁵N, donor and light, ¹H, ⁴N recipient DNA. On denaturation the position of the heavy donor DNA moved closer to, but not all the way toward, the density position of the original donor DNA. In addition to supporting the idea of single-stranded incorporation, this evidence suggested that the integrated donor DNA was covalently linked to light recipient DNA. The DNA was taken up in the double-stranded form and no detectable amounts of denatured DNA could be found during the transformation process. However, during the process of integration an amount of donor atoms, equivalent to the amount of hybrid DNA formed, appeared in recipient DNA, and indicated that while one strand of DNA was integrated the other was broken down and resynthesized. The density of the hybrid DNA, as well as rebanding of denatured hybrid, indicated that the size of the integrated piece of DNA was large, approximately 6 x 10⁶ daltons.     

Structure and Composition of a Small Particle Prepared from a Simian Adenovirus.

Mayor, Heather D. (Baylor University College of Medicine, Houston, Tex.), Richard M. Jamison, Liane E. Jordan, and Joseph L. Melnick. Structure and composition of a small particle prepared from a simian adenovirus. J. Bacteriol. 90:235-242. 1965.-When tissue-culture fluids infected with simian adenovirus SV15 are examined in an electron microscope, either as fresh harvests or after treatment with Genetron, typical mature adenovirus particles are found. These are 65 to 70 mmu in diameter, with an icosahedral capsid built from 252 capsomeres. Also present is a population of small polyhedral particles approximately 20 mmu in diameter. These small particles can be separated from the mature virions by ultrafiltration or density gradient centrifugation. The small particles have a density of 1.43 in cesium chloride. They contain protein and double-stranded deoxyribonucleic acid. They appear to possess cubic symmetry of the icosahedral type, with a coat composed of 12 subunits each at the vertex of an icosahedron.     

Some properties of a new virus of Pieris rapae

A new insect virus of Pieris rapae was purified using a chloroform-butanol treatment followed by two differential and sucrose gradient centrifugations. The sedimentation coefficient of the purified virion was approximately 132 S, and it banded at a density of 1.39 g/cm³ in cesium chloride. The virion has a nonenveloped capsid with icosahedral symmetry. Several virions were shown to have a regular hexagonal contour about 25 nm in diameter and to be composed of many capsomeres. Full and empty viral particles, with 12 capsomeres around the periphery of the capsid, were noted. In some particles a small core has been observed which is spherical, about 15 nm in diameter. Both purified virus and partially purified virus preparations from dead, infected larvae gave only one precipitin band with a reaction of identity when tested against the antiserum to partially purified virus. When crude extracts of uninfected larvae and purified virus were tested against antiserum to partially purified virus, the pure virus produced a precipitin band. The band was formed independently and did not join to the band of the uninfected insect producing a typical reaction of nonidentity.     

Purification and characterization of a simple ribonucleoprotein particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells.

A ribonucleoprotein complex whose RNA complement consists exclusively of small nuclear RNA species (snRNA) has been purified from particles containing heterogenous nuclear RNA (hnRNP) from HeLa cells. This was accomplished by taking advantage of their ability to band at a density of about 1.43 g/cm3 in plain cesium chloride as well as in cesium chloride gradients containing 0.5% sarkosyl without prior aldehyde fixation. After these two steps of equilibrium density centrifugation, these snRNPs were still largely contaminated by free proteins (and especially phosphoproteins). A final step of purification by velocity sedimentation in a sucrose gradient containing 0.5 M cesium chloride and 0.5% sarkosyl was efficient in completely eliminating all free proteins. U1, U2, U4, U5 and U6 species according to the nomenclature of Lerner et al. (Nature, (1980) 283, 220-224) were found in these purified snRNPs, while a significant part of U6 and a small amount of U2 were found in the bottom fraction. 5S species behaved entirely as free RNA and is presumably a contaminant of cytoplasmic origin. Electrophoresis of proteins from snRNP labeled in vivo with (35S) methionine, revealed four bands with migrations corresponding to molecular weights ranging between 10,000 and 14,000 daltons.     

Properties of the Deoxyribonucleic Acid Contained in the Defective Particle Coliphage 15

Escherichia coli strain 15 TAU, which requires thymine, arginine, and uracil for growth and harbors an apparently defective prophage, was induced by exposure to ultraviolet light (580 ergs/mm(2)) or to mitomycin C (5 mug/ml). Phage particles (coliphage 15) were recovered from the resulting lysate by treatment with deoxyribonuclease, filtration, and several cycles of differential centrifugation. Analysis of the phage particles obtained by using cesium chloride density gradient centrifugation in a preparative ultracentrifuge resulted in the resolution of three components. The major component had a peak density of 1.52 to 1.53 g/cm(3) followed by components with densities of 1.5 and 1.49 g/cm(3). The guanine plus cytosine content of coliphage 15 deoxyribonucleic acid (DNA) was determined by both analytical ultracentrifugation in cesium chloride and by thermal denaturation in standard saline citrate buffer. Respective values of 46.4 +/- 1% and 46.6 +/- 1% guanine plus cytosine content were obtained. Coliphage 15 DNA formed molecular hybrids with messenger ribonucleic acid (RNA) from both uninduced and ultraviolet-induced cultures of E. coli 15 TAU, but did not hybridize with E. coli ribosomal RNA. The molecular weight of coliphage 15 DNA was determined by constant velocity sucrose density gradient centrifugation to be about 33 x 10(6) daltons.     

RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.

An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.     

Separation of Treponema pallidum from Tissue Substances by Continuous-Flow Zonal Centrifugation

The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 10(9) (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.