Properties and origins of infectious rhinovirus type 14 particles of different buoyant densities.

Isopycnic centrifugation of rhinovirus type 14 (RV14), purified from infected HeLa or KB cell cultures, into CsCl gradients resolved two bands of infectious virus particles with buoyant density values of 1.409 +/- 0.007 (H virus) and 1.386 +/- 0.004 (L virus) g/ml. Only H virus was detected by incorporation of radiolabeled uridine into viral RNA, and H virus accounted for the majority of infectivity in gradients. H and L virus could not be differentiated by plaque morphology, extent of neutralization by RV14-specific antiserum, or particle size. Electron microscope studies showed that most L-virus particles were associated with an amorphous material. Treatment of L virus with proteolytic enzymes or rebanding L virus in CsCl gradients resulted in recovery of the majority of infectivity as H virus. Virus purified from cell-free fluids from infected HeLa or KB cell cultures banded only as H virus. HeLa cell cultures challenged with purified H virus and harvested at 3 h postinoculation for virus purification yielded only infectious H virus. Both H and L viruses were detected in cell cultures that had been challenged with purified H virus and harvested at 12 h postinoculation. The data suggest that H virus represents progeny virus, whereas L virus represents sequestered infectious virus particles which become associated with an amorphous material and do not enter into viral replicative processes.     

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis.To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.     

Isolation and Partial Characterization of Different Defective DNA Molecules Derived from Polyoma Virus

Supercoiled DNA molecules purified from mouse cells infected with high-multiplicity-passaged polyoma virus has a broader size distribution and sediments more slowly than DNA derived from low-multiplicity-passaged virus. The shorter DNA molecules are predominately noninfectious. Virus populations containing distinct size classes of defective virus DNA were isolated by growing virus from single cells infected by a defective and nondefective helper virus (infectious center). This technique probably results in the cloning of defective virus particles. By applying the infectious center method to DNA from various fractions of sucrose gradients it has been possible to obtain shorter circular DNA molecules ranging in size from 50 to 95% of the unit-length polyoma DNA molecule. The shorter molecules in any one preparation are homogeneous in size. This class size is retained upon repeated passage of crude viral lysates at high multiplicity. Thus far, all the purified shorter DNA molecules tested appear to be noninfectious and largely resistant to cleavage by the R(1) restriction enzyme. Some of the purified defective molecules have been found to interfere with the production of infectious virus upon co-infection with unit-length infectious polyoma DNA.     

Transformation Potentials of the Noninfectious (Defective) Component in Pools of Adenoviruses Type 12 and Simian Adenovirus 7

Pools of adenovirus 12 and simian adenovirus 7 were separated into four or five fractions by density gradient centrifugation in cesium chloride. Each fraction was analyzed for total in vitro infectivity units, total transformation activity, and for total virus particle (VP) content. Two major subpopulations were separated with mean densities of 1.30 +/- 0.02 and 1.34 +/- 0.02 g/ml, respectively. Virions in the 1.34 g/ml range were highly infectious (10(2) to 10(3) VP per infectivity unit) in contrast to virions at 1.30 g/ml density (10(4) to 10(5) VP per infectivity units). Transformation capacity was evenly distributed throughout fractions of both viruses, indicating that genetically incomplete or defective virus particles were not deficient in their ability to induce transformation. The average VP per transformation unit for adenovirus 12 (2.85 x 10(6)) and for simian adenovirus 7 (4.00 x 10(6)) did not vary significantly from fraction to fraction. These values were obtained with optimal input multiplicities of 16 to 64 VP per cell. At higher multiplicities the apparent increase in VP per transformation unit was attributable to the viral cytocidal effect on hamster cells. These studies revealed that quantitation of in vitro transformation based on VP multiplicities was more reliable than on the basis of infectious units. These estimates were independent of method of virus production, extraction, and purification.     

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.     

Persistence of herpes simplex virus type-2 genome in a human leukemic cell line

To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three K562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCl density gradients; but was readily identified by Southern blot hydridization of virus-infected intracellular DNA with purified viral DNA. Viral DNa polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.     

Large scale production and purification of human retrovirus-like particles related to the mouse mammary tumor virus

Abstract Human retrovirus-like particles related to mouse mammary tumor virus (MMTV) are secreted in a steroid-dependent manner by the breast cancer cell line T47D. We report the successful large scale production and purification of these particles from culture supernatants of T47D cells and describe the experimental conditions established for this purpose. Thus, mg amounts of particles were produced by large scale culturing of T47D cells in an autoharvesting roller bottle system and purified by differential centrifugation and continuous flow ultracentrifugation on density gradients with a 50% recovery and a 350-fold enrichment.     

Effect of the Purification of Virus Antigens on the Production of Specific Complement-Fixing Antibodies

The effect of viral purification procedures on the antibody response of guinea pigs to immunization with reovirus type 2 and echovirus type 19 was investigated. Three grades of antigens were employed: (i) infectious monkey kidney tissue culture fluid (TCF), (ii) virus sedimented in the ultracentrifuge and suspended in phosphate-buffered saline, and (iii) virus purified by centrifugation in CsCl density gradients. The antibody response of the guinea pigs was studied by the hemagglutination inhibition, complement fixation, and serum neutralization tests. Only sera produced from virus purified by CsCl density gradients reacted specifically with homologous antigen in the complement fixation test. Sera from animals receiving tissue culture fluid virus or sedimented virus cross-reacted with heterologous antigens such as tissue culture fluid from uninfected monkey kidney cells. All sera, however, reacted specifically in hemagglutination inhibition and serum neutralization tests. Sera from intranasally infected animals (reovirus type 2), even though reacting specifically in the complement fixation test, had much lower titers than sera from animals inoculated intramuscularly.     

Purification of the Moloney and Rauscher Murine Leukemia Viruses by Use of Zonal Ultracentrifuge Systems

The B-IV and B-IX zonal ultracentrifuge rotors were applied to the concentration and purification of the Moloney and Rauscher murine leukemia viruses from large volumes of infected tissue culture fluids and animal materials. Potassium tartrate, potassium citrate and sucrose gradients were used to obtain viral concentrates from the density 1.16 to 1.18 zone. Proteolytic enzyme digestion of tissue culture preparations prior to zonal ultracentrifuge processing was effective in releasing virus from cell debris and producing highly purified, though nonleukemogenic, viral concentrates. Infected Rauscher mouse plasma was processed to give highly purified infectious virus fractions. A single centrifugation of crude Rauscher mouse spleen homogenates resulted in partially purified infectious concentrates with high virus particle counts.     

Purification of Rabies Virus Grown in Tissue Culture

Extracellular rabies virus, grown in monolayer cultures of BHK21 cells in the presence of medium supplemented with bovine serum albumin, was purified by the following procedure. Virus was precipitated from infectious tissue culture fluid by zinc acetate and was resuspended in a solution of ethylenediaminetetraacetate. The suspension was filtered through a Sephadex column and was treated with ribonuclease and deoxyribonuclease. The virions were then pelleted by centrifugation at high speed and were resuspended in buffer solution. Banding of the virus by centrifugation in a sucrose density gradient was the final step in the purification procedure. Purified preparations contained bullet-shaped virus particles of variable length and little (up to 5%) contaminating host-cell material. Most of the virions were "complete", i.e., 180 nm long, but some virus particles were shorter. The length distribution of the virions was nonrandom. Shorter virions seemed to be noninfectious and showed markedly decreased hemagglutinating activity. The complement-fixing activity and the ribonucleic acid to protein ratio of the virions were not related to the length of the virus particles. Although the properties of extracellular and intracellular viruses were similar, the procedure was not suitable for purification of intracellular rabies virus.     

Composition and Size of Shope Fibroma Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified virions of Shope fibroma virus (SFV) (by using DNA from Microccocus lysodeikticus as marker) had a buoyant density of 1.6996 +/- 0.0003 g/ml), hence a guanine plus cytosine (G + C) content of 40.4 +/- 0.3%, which is close to the G + C content of the DNA of susceptible rabbit cells (40.9 +/- 0.4%) and different from that of vaccinia virus DNA (35.5 +/- 0.4%). For the determination of the molecular weight of DNA, SFV and vaccinia purified virions, treated with Pronase and detergent, were cosedimented in sucrose density gradients. Results showed that SFV-DNA has a molecular weight of about 153 x 10(6) daltons. By electron microscopy, only one molecule corresponding to this value was observed (its length was 80.3 mum). The others had a median size of 49.8 mum +/- 0.9.     

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.     

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.     

Purification and partial characterization of virus-like particles from Schneider line 2 Drosophila cells

A procedure has been developed for the purification of virus-like particles (VLPs) from Schneider line 2 Drosophila cells. The VLPs were precipitated with polyethylene glycol from the cytoplasmic fraction of lysed cells and further purified by equilibrium centrifugation in CsCl density gradients, in which they band at a density of 1.366 g/ml. Electron micrographs of these preparations revealed polyhedral particles with a diameter of 310–330 Å. We have also found particles of this size in thin sections of the intact cells. Sedimentation of the VLPs through 10–70% sucrose gradients yields a sedimentation coefficient of 235 S. Preliminary studies show that the VLPs contain double-stranded RNA species of 10 S, 14.5 S, 16 S, and 18 S.     

Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy.

We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.     

Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer

Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency. Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates. We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity. Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles. We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells. We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus. We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells.     

Proteins of Vasicular Stomatitis Virus: I. Polyacrylamide Gel Analysis of Viral Antigens

Infection of L cells with vesicular stomatitis virus results in the release, into the cell-free fluid, of four antigenic components separable by rate zonal centrifugation on sucrose gradients. The largest antigens are the infectious (B) particle and a shorter noninfectious, autointerfering (T) particle. The two small antigens are characterized by sedimentation coefficients of approximately 20S and 6S. Treatment of purified B or T particles with sodium deoxycholate results in the release from the particle of a nucleoprotein core which can be purified on sucrose gradient and which has a sedimentation coefficient characteristic of the virus from which it arose. Utilizing purified antigens labeled with (14)C-amino acids during growth, we examined the protein constituents of each antigen by acrylamide-gel electrophoresis. The proteins of B and T particles are identical, each containing one minor (virus protein 1) and three major (virus proteins 2, 3, and 4) proteins, numbered in order of increasing mobility. Virus protein 3 originates from the nucleoprotein core, whereas proteins 2 and 4 come from the coat. The origin of virus protein 1 is not known. The 20S antigen contains a single protein equivalent to virus protein 3, whereas the 6S antigen shows a single protein which is similar to, but probably distinct from, virus protein 2.     

Large-Scale Purification of Rubella Virus and Preparation of an Experimental Split Rubella Virus Vaccine

Large-scale purification of rubella virus from tissue culture fluid and preparation of an experimental rubella subunit vaccine are described. The virus, purified isopycnically in sucrose gradients, was Tween 80-ether treated, and the product was filtered sterile. The vaccines were of two types, formalinized and nonformalinized. They were not infectious, had only a slight pyrogenicity, and caused no harmful symptoms in animals. The immune response in guinea pigs was better when the nonformalinized vaccine was used and the dosage was 2,500 hemagglutinating units per injection (0.5 ml).     

The purification and quantitation of myosin from cultured cells.

Myosin has been purified from the following cultured cell lines: normal rat kidney fibroblast (NRK), HeLa-Rhino (HeLa), human choriocarcinoma, human acute lymphoblastic leukemia, rat hepatoma (HTC), monkey kidney (VERO), pigmented mouse melanoma, Y-1 rat adrenal cortex, and growth hormone-secreting GH-1. Myosin constitutes 0.5-5.4% of the protein of these cells. It was not detected in washed human erythrocytes or in two types of mouse plasmacytoma cells. Two methods for the purification of myosin from cultured cells have been employed. With Method I highly purified myosin was prepared by Sepharose 4B and DEAE-cellulose chromatography from 10(10) L-929 cells as well as from mouse uterus. Those myosins have similar molecular and subunit weights as well as ATPase activity but are immunologically distinct. Method II involving ultracentrifugation and Sepharose 4B chromatography, is suitable for the production of moderately pure myosin in good yield from as few as 5-10(7) cells (five 100-mm Petrie dishes).