Characteristics of venezuelan equine encephalomyelitis virus ribonucleoprotein

Summary Venezuelan equine encephalomyelitis (VEE) virus grown in chick embryo fibroblasts was concentrated by differential centrifugation and purified in sucrose and caesium chloride density gradients. Treatment of the virus with Tween 80 and ether appeared to be the optimal method for obtaining biologically active subviral components. In experiments with sucrose density gradient centrifugation the sedimentation coefficient of the virus was about 380 S and that of virus ribonucleoprotein (RNP) was about 160 S. In experiments with caesium chloride equilibrium density centrifugation virions banded at the buoyant density of 1.25 g/cm³ and virus RNP banded at 1.42 g/cm³.     

Biological and physicochemical characterization of the major (1.40) and minor (1.45) component of infectious avian adeno-associated virus

Summary Two infectious components with buoyant densities of 1.40 g/cm³ and 1.45 g/cm³, designated as major (1.40) and minor (1.45) component, were detected by banding avian adeno-associated virus (AAAV) isopycnically in CsCl. In metrizamide, however, infectious AAAV banded only as a single peak at a density of 1.32 g/cm³. Biological as well as physicochemical properties of the two AAAV components recovered from CsCl density gradient were described. Concerning the minor (1.45) component, three experimental findings may suggest that the capsid structure of this AAAV population is altered in comparison with that of the major (1.40) component: (i) the sedimentation pattern characterized by an additional peak containing slower-sedimenting noninfectious material (16 S); (ii) the specific infectivity decreased by the 3.5 fold; (iii) the ready disintegration when exposed to gently denaturing conditions.     

Formation of ribonucleoprotein particles by interaction of infectious viral RNA with cytoplasmic cell fractions

Summary Virus-specific RNA's interacting with various cytoplasmic cell fractions form ribonuclein particles. The sedimentation coefficients of ribonuclein particles vary from 60S to 80S and the buoyant density is 1.30 g/cm³ to 1.42 g/cm³. With respect to infectivity the particles formed by interaction of infectious virus RNA with cellular proteins behave like virions. However, they are only partially resistant to ribonuclease treatment and are not neutralized by virus-specific immune sera. This artificially produced phenomenon may reflect some events in virus replication.     

Investigation on the biological activity of fowl plague virus ribonucleoprotein.

Fowl plague virus (FPV) ribonucleoprotein (RNP) bands in sucrose density gradient in a heterogeneous peak with sedimentation coefficients from 45 to 70 S, whereas in cesium chloride gradient it has a homogeneous density of 1.33-1.34 g/cm3. FPV RNP contains 7.4-8% RNA. Upon inoculation of chick embryo cell cultures. FPV RNP shows no infectivity, does not induce virus-specific protein synthesis and does not participate in complementation or recombination interactions with ts mutants of FPV. The biological activity of FPV RNP demonstrable under certain experimental conditions is due to admixture of undestroyed virions and is completely eliminated by treatment of the preparation with gamma-globulin fraction of antiserum to FPV haemagglutinin, but not with antiserum to RNP proteins.     

Abortive myxovirus infection of Ehrlich ascites carcinoma cells. Analysis of virus-specific structures in the ascitic fluid]

In the course of classical fowl plague virus reproduction in Ehrlich ascites carcinoma cells both hemagglutinins and S-antigen accumulate and titers of the infectious activity increase. However virus reproduction does not terminate in formation of virus, and subviral structures are found in the liquid fraction of the infected cells. Analysis of these structures has shown them to have a sedimentation coefficient of 350-370S and buovant density 1.29 g/ml. The rapidly sedimenting structure has complement-fixing hemagglutinating activity but bow infectivity.     

In vitro growth characteristics and heterogeneity of mouse hepatitis virus type 3

Summary Thein vitro virus yield of MHV3 reached 10⁷ PFU/ml in mouse DBT cells infected with a virus suspension in HEPES-buffered medium containing DEAE-dextran. The virus titer was 10⁶ PFU/ml in the presence of 10 µg actinomycin D/ml. MHV3 grown in DBT cells gave three peaks of density (1.10–1.14 g/cm³, 1.18–1.20 g/cm³, and 1.25–1.31 g/cm³) in sucrose gradients. All these peaks retained infectivity.With 2 Figures     

Isolation and characterization of a virus infecting a blue-green alga of the genus Synechococcus

A new virus infecting the unicellular blue-green alga, Synechococcus strain NRC-1, has been isolated. The virus, named S-1, possesses a head 500 Å in diameter and a narrow, noncontraotile tail 1400 Å in length. The sedimentation coefficient of whole virus particles is 353 S and the buoyant density is 1.501 g/cm³. The major structural proteins have molecular weights of 10,000, 11,000, and 39,000 daltons. S-1 DNA has a contour length of 13.3 ± 0.3 μm and a S_{20, w} of 30.8 ± 1.5 S. These values imply a molecular weight of 23–26 × 10⁶ daltons. The buoyant density in CsCl of S-1 DNA is 1.729 g/cm³, suggesting a GC content of 70%. A slightly higher value, 74%, is derived from the thermal denaturation temperature of S-1 DNA.     

Characterisation of rubella virus hemagglutinin rosettes

Purified rubella virus treated with Triton X-100 was subjected to centrifugation in a sucrose density gradient containing nonionic detergent β-D-octylglucoside. The result of this treatment was the formation of hemagglutinating rosettes containing viral glycoproteins VP₂ (50,000 mol. wt.) and VP₃ (63,000 mol. wt.). The rosettes have a 26 S sedimentation coefficient and a density of 1.2 g/cm³ in sucrose. Electron microscopy revealed 15 nm rosettes with a hollow center. The molecular weight of the rosettes was extrapolated at 850,00     

Physicochemical properties of two distinct types of virus-like particles from Helminthosporium victoriae

Two serologically distinct types of virus-like particles (VLPs) were isolated from Helminthosporium victoriae. The first, which was isolated from three normal and two diseased isolates, sedimented at a rate of 190 S when centrifuged in linear-log sucrose density gradients. The second, which was found only in the two diseased isolates, had a sedimentation coefficient of 145 S. No VLPs were detected in two other normal isolates of the fungus. The 190 and 145 S VLPs both were polyhedral and 35 to 40 nm in diameter and had typical nucleoprotein absorbancy spectra. The 190 S type of VLP was electrophoretically distinct from the 145 S VLP when electrophoresed on either polyacrylamide gels or cellulose acetate strips. Equilibrium density gradient centrifugation in cesium chloride revealed a single density component for the 190 S VLPs with a buoyant density of 1.4325 g/cm³ The 145 S VLPs could be resolved into two to four components with buoyant densities of 1.3813 to 1.4138 g/cm³. SDS polyacrylamide gel electrophoresis of the 190 S VLPs revealed two major proteins of molecular weight 83,000 and 88,000 present in equimolar amounts. The 145 S type of VLP had three major proteins with molecular weights of 92,000, 97,000, and 106,000; these were also present in equimolar amounts. Both the 145 and 190 S VLPs contained double-stranded RNA (dsRNA). The 190 S type of VLP had a single species of dsRNA with a molecular weight of 3.0 × 10⁶. There were four classes of dsRNA associated with the 145 S VLPs, and their molecular weights were 2.4, 2.2, 2.1, and 2.0 × 10⁶.     

Identification of point mutations in the genome of the poliovirus Sabin vaccine LSc 2ab, and catalogue of RNase T1- and RNase A-resistant oligonucleotides of poliovirus type 1 (Mahoney) RNA

In harvests of a feline calicivirus (FCV) grown in feline embryo cells two populations of viral components were observed after centrifugation in sucrose and CsCl gradients. Particles in the first (PI) were mature virions since they had a buoyant density of 1.39 g · cm^?3, a sedimentation coefficient of 170 S, were infectious and showed characteristic calicivirus morphology by electron microscopy. The second particle (P2), which in terms of protein was 10 times as abundant as P1, had a buoyant density of 1.31 g · cm^?3, a sedimentation coefficient of 15 S, and contained little or no infectivity. Evidence that P2 was virus specific was that P2 as well as P1 contained a polypeptide with a molecular weight of 65,000 which is the weight of the viral capsid protein, and P1 and P2 shared an antigenic determinant which was responsible for inducing neutralizing antibodies. The 15 S component was not generated from the virion by conditions encountered during viral growth and purification. It is considered likely that the 15 S particle is a stable product of FCV infection and may be a subunit in the assembly of the viral capsid.     

Characterization of virus-like particles produced by an influenza A virus

Summary The influenza strain 413 1,1 segregated as a stable recombinant during passage of the isolate 19/N which was obtained after double infection of chick embryo fibroblasts by virus N and the fowl plague virus (FPV) mutantsts 19. Its gene constellation was determined by molecular hybridization. Upon infection of chick embryo cells by this recombinant strain, two particle populations of high (H) and low (L) buoyant densities were produced. By biological and biochemical parameters, the H-population (=1.22 g/cm³) cannot be distinguished from standard infectious influenza virus. In contrast, the noninfectious L-particles (=1.14 g/cm³) lack all virus-specific glycoproteins (HA, NA) as well as the matrix protein M and are visualized by electron microscopy as spikeless particles. Significant changes in the quantitative composition of the phospholipid bilayer are evident as compared to the H-particles. In addition to the previously characterized eight genes both populations contain a variety of smaller RNA fragments which hybridize with complementary RNA and presumably represent degradation products of full-length genes.With 6 FiguresParts of the results were presented at the Symposium on Negative Strand Viruses and the Host Cell, Cambridge, U.K., 1977.     

Proteins specified by African swine fever virus

Summary At least 28 polypeptides have been identified in intracellular virus, with molecular weights ranging from 11,500 to 243,000 daltons. By treatment with Nonidet P-40 and 2-mercaptoethanol it is possible to obtain subviral particles that have lost some proteins and have a density in CsCl of 1.31 g/cm³ which is higher than that of the complete virus (1.23 g/cm³). After addition of NaCl the virus loses its major protein VP73 which indicates that it is localized in the viral envelope. Cores obtained after this treatment are made up of at least 14 proteins. Incorporation of³H-fucose and³H-glucosamine in intracellular virus occurs in three minor components. The protein VP42 is possibly the cell actin and appears to be strongly associated with the virus. It is not possible to eliminate it under conditions where the viral envelopes desappear morphologically. At least the proteins VP172, VP162, VP146 and VP73 act as antigens in the natural infection.With 5 Figures     

Subfractionation of CsCl-purified H-1 parvovirus on metrizamide gradients.

The different density classes of H-1 parvovirus, collected within 30 hr of parection of parasynchronous cultures, following the standard CsCl purification step, have been shown to be heterogeneous. Rebanding of the denser form (HF, ? = 1.46 g/cm³) and the less dense form (LF, ? = 1.42 g/cm³) of infectious virus in the nonionic density generating solute, metrizamide, showed that both HF and LF virus bands were heterogeneous in density. The infectivity banded with isotopically labeled virus protein and DNA at 1.32 g/cm³ for both HF and LF virus. Amounts of protein and DNA which varied from preparation to preparation, but which were greater from the HF virus band, were distributed throughout the rest of the gradient, but predominated in a peak at a density of 1.2 g/cm³. The protein in this peak was without hemagglutinating activity but had the molecular weights and proportions of the H-1 virion proteins (VP1, VP2′, and VP2). The DNA was of the same size as H-1 DNA monomers and its proportion to the protein was similar to that of the infectious peak. The DNA was susceptible to micrococcal nuclease digestion. The nature of this noninfectious viral material thus seemed to be incompletely assembled virus. Radio-labeled H-1 virus collected after 72 hr of infection formed a discrete single peak in both CsCl (? = 1.42 g/cm³), and metrizamide gradients (? = 1.32 g/cm³). There was no significant amount of the 1.20 g/cm³ viral protein-DNA complex in these mature preparations.     

Properties of virions and ribonucleoproteins of Venezuelan equine encephalomyelitis virus.

The physicochemical properties of Venezuelan equine encephalomyelitis (VEE) virus and of its ribonucleoprotein (RNP) were studied. Upon purification in a discontinuous or linear sucrose gradient, the losses of infectivity were small (25%), and 96% of cellular proteins were removed. The purified virus was homogeneous with respect of sedimentation rate (s20, w = 265 S). The CsCl gradient was unsuitable for purification of infectious virus because the latter was destroyed at high CsCl concentrations. Buoyant density of the virus in CsCl after formaldehyde fixation was 1.21--1.22 g/cm3. Treatment of the virus with 1% Nonidet P-40 proved to be the most effective method for isolation of the RNP. The structures thus obtained contained practically all of the viral RNA and about 20% of viral proteins, and were homogeneous with respect of sedimentation rate (153 S) and buoyant density (1.40--1.42 g/cm3 in CsCl after formaldehyde fixation). The RNPs were sensitive to ribonuclease.     

Sendai virus nucleocapsids in the nucleoli of infected cells

Summary Uncoiled parental Sendai virus nucleocapsids were revealed in the nucleoli of Sendai virus infected Ehrlich ascites tumor cells early in infection. Their sedimentation coefficient and buoyant density were similar to that of parental nucleocapsids isolated from the cytoplasm (150–180S in a sucrose gradient and 1.35–1.37 g/ml in a CsCl gradient). Later in infection the nucleocapsids were involved into complexes with a sedimentation coefficient about 500S. Similar results were obtained with nucleoli of chicken embryo lung cells. The complex in Ehrlich tumor cells contained 23–26S nascent RNA, its synthesis being inhibited by cycloheximide.     

Effect of physical and chemical treatment on the buoyant density of reovirus

Summary Reovirus type 2 was treated with genetron or chloroform, heat (52° to 80°C), trypsin, and combinations of heat and trypsin. The treated virions were analyzed in a CsCl density gradient. Genetron and chloroform had no effect on the buoyant density of the virion, heat treatment resulted in a virus particle with a lowered density (1.35 g/cm³), and trypsin treatment rendered virus cores with a density of 1.42 g/cm³. When heat-treated virus was treated with trypsin, the particles also had a density of 1.42 g/cm³; however, trypsinized particles became more dense (1.46 g/cm³) when heated after the enzyme treatment.     

Different patterns of replication in influenza virus-infected KB cells

Summary When KB cells were infected either with the fowl plague (FPV) Rostock strain (Hav1N1) or the WSN (H0N1) strain of influenza A virus the yield of cell-associated haemagglutinin and neuraminidase polypeptides was essentially comparable, but virus particles were not produced in the FPV-KB system. WSN virus-infected KB cells synthesized normal amounts of mature virus particles and had all the characteristics of a permissive replication cycle. Biosynthesis and transport of RNP antigen from nucleus to cytoplasm of infected cells were traced by immunofluorescent staining at 4 and 8 hours after the beginning of infection. While the fluorescent-stained material was totally confined to the nuclei in FPV-infected KB cells, RNP antigen migrated out of the nucleus during the replicative cycle of WSN virus in the same host cell. Patterns of virus-specific protein synthesis were studied by pulse-labelling with³⁵S-methionine. The most significant feature concerned the amplification of synthesis of virus-induced matrix (M) protein which did not occur in FPV-infected cells but occurred normally during WSN infection. The different patterns of replication in the same host cell when infected by different influenza A viruses is discussed.With 3 Figures     

Purification studies of rubella virus

Summary Equilibrium centrifugation of rubella virus in potassium citrate density gradients revealed a heterogeneous population of virus particles, with 90% of the infectivity being recovered at a density of 1.20g/cm³ and the remaining 10% at 1.10 g/cm³. Pretreatment of the virus material with 1% Tween 80 gave a homogeneous virus peak at 1.20 g/cm³, presumably by splitting aggregates of virus and cell debris.Chromatography through DEAE Sephadex A25 revealed two distinctly different virus fractions, with 10% appearing in the void volume and 90% being eluted with 0.3–0.5M MgSO₄. These fractions were shown not to be influenced by pretreatment with Tween.This work was supported by grant Nr. 65-77 from the Swedish Foundation for Cancer Research.     

The polypeptide composition of avian infectious bronchitis virus particles

Summary Egg grown avian infectious bronchitis virus (IBV) centrifuged on sucrose density gradients was found to consist of a major virus peak of density 1.17 to 1.18 g/cm³ and occasionally two minor virus peaks of density 1.21 to 1.22 g/cm³ and 1.13 g/cm³. Three different IBV strains were examined and no morphological differences were detected between virus particles of different densities or from different strains. The polypeptides of the different density virus particles from the three IBV strains were analysed on polyacrylamide gels. In all cases 7 polypeptides were observed, although there were differences in the proportions of these polypeptides in particles of different densities and those from the different strains. The polypeptides have been called VP1 (molecular weight 130,000), VP2 (105,000), VP3 (97,000), VP4 (81,000), VP5 (74,000), VP6 (51,000) and VP7 (33,000). Additional polypeptides were produced if slightly harsher treatments were used.With 4 Figures