bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells
Authors: Ozanne Brad , Vogel Arthur
Published: January 2020

Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.
Electron Microscopy Study of Reovirus Reaction Cores
Authors: Bartlett Nerida M. , Gillies Susan C. , Bullivant S. , Bellamy A. R.
Published: January 2020

A combined staining and shadowing method has been used to enhance the contrast of both reovirus cores and the RNA product that they have synthesized. After limited synthesis when all RNA strands associated with the core are nascent, many cores are observed that have more than 10 strands associated with each particle. The lengths of individual nascent mRNA molecules are not identical: many strands are extruded in the form of loops. These observations are consistent with the notion that synthesis of individual mRNA molecules occurs simultaneously at different rates at a number of different sites. The sites of extrusion are probably the 12 hollow projections located on the surface of the core.
Replication of Sindbis Virus IV. Electron Microscope Study of the Insertion of Viral Glycoproteins into the Surface of Infected Chick Cells
Authors: Birdwell Charles R. , Strauss James H.
Published: January 2020

The appearance of Sindbis virus-envelope glycoproteins in the surfaces of chicken embryo fibroblasts was studied by an indirect labeling technique. This technique involved treating infected cells sequentially with rabbit immunoglobulin G (IgG) specific for Sindbis virus followed by hemocyanin-conjugated goat (anti-rabbit IgG) IgG; surface replicas of these cells were then prepared and examined in the electron microscope. As early as 2 h after infection (and at least 1 h before mature virions were released), newly synthesized virus-envelope glycoproteins were detected at the cell surface. By 3 h after infection, cell surface membranes were extensively modified by the insertion of the Sindbis glycoproteins. When infected cells were prefixed with glutaraldehyde before labeling, the glycoproteins were distributed fairly evenly over the cell surface, although a slight clustering was observed on cells labeled early in infection. However, no evidence for large-scale clustering of virus glycoproteins corresponding to patches of budding virus was observed. Similar results were found with unfixed cells labeled at 4 C. However, when unfixed cells were labeled at 37 C, the glycoproteins were shown to be in discrete clusters, demonstrating that these glycoprotein antigens can diffuse laterally through the cell membrane at this temperature.
Transfection of Chicken Embryo Cells with DNA Extracted from Avian Virus-Producing Neoplastic Cells
Authors: Fourcade A. , Huynh T. , Lacour Fanny
Published: January 2020

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.
Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins
Authors: Yamashita Tadashi , Green Maurice
Published: January 2020

Nuclear membrane fractions were prepared by two procedures from KB cells pulse labeled with [
3
H]thymidine for 5 min late after infection with adenovirus 2: (i) the M-band technique, which yields a sharp peak containing most of the newly synthesized viral DNA, and (ii) the discontinuous sucrose gradient method, which yields three membrane fractions, one which bands at the interface between sucrose layers at density 1.18 and 1.20 g/ml and contains most of the newly synthesized viral DNA. Studies using cycloheximide to inhibit protein synthesis showed that proteins whose synthesis begins early after infection and occurs in the absence of viral DNA replication are required for viral DNA synthesis late after infection. To study the nature of these proteins, nuclear membrane fractions were isolated from cells labeled with [
3
H]leucine from 6 to 24 h postinfection in the presence of arabinosyl cytosine to block viral DNA replication, and were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Two proteins of molecular weights 75,000 and 45,000 were the major labeled polypeptides in the nuclear membrane fractions prepared from infected cells both by the M-band and the discontinuous sucrose gradient methods. These two proteins were not found in nuclear membrane fractions from uninfected cells. It is suggested that the 75,000 and 45,000 proteins may be early viral gene products that may play a role in the viral DNA replication.
Evidence for a Stable Intermediate in Leukemia Virus Activation in AKR Mouse Embryo Cells
Authors: Ihle James N. , Kenney Francis T. , Tennant Raymond W.
Published: January 2020

Analysis of the requirement for serum in the activation of the endogenous leukemia virus expression in AKR mouse embryo cells by 5-iododeoxyuridine shows that activation can be dissociated into two discrete serum-dependent events. The first involves incorporation of 5-iododeoxyuridine into DNA and results in the formation of a stable “activation intermediate” resembling the provirus formed during infection of stationary mouse embryo cells with exogenous leukemia virus. The second event, resulting in expression of the activation intermediate as synthesis of virus proteins, requires DNA replication but not 5-iododeoxyuridine.
Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus
Authors: Lothrop David , Nicholson Bruce L.
Published: January 2020

In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus.
Localization of Coliphage MS2 A-Protein
Authors: Curtiss Linda K. , Krueger Robert G.
Published: January 2020

The purification of coliphage MS2 dinitrophenol (DNP) conjugates provided a system for localization of the single molecule of A-protein in the capsid of the MS2 phage particle. Three A-protein preparations isolated from unconjugated MS2, overconjugated DNP-MS2, and purified 78
S
DNP-MS2 were tested for the presence of covalently bound DNP. The binding characteristics to Dowex 1-X8 and rabbit anti-DNP bovine serum albumin (DNP-BSA) immunoglobulin G of the 78
S
DNP-MS2 and overconjugated DNP-MS2 A-protein preparations indicate that the A-protein is located on the surface of the phage particle where it can be covalently conjugated with hapten. Extensive enzymatic iodination of the A-protein of intact unconjugated MS2 substantiates this conclusion.
Requirement for Cell Division for Initiation of Transcription of Rous Sarcoma Virus RNA
Authors: Humphries Eric H. , Temin Howard M.
Published: January 2020

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) did not synthesize virus RNA until after division of the infected cells. Initiation of RNA transcription after cell division resulted in transcription of the entire viral genome and was followed shortly thereafter by appearance of progeny virus. Addition of colchicine to RSV-infected stationary chicken embryo fibroblasts resulted in inhibition of cell division and a 24-h delay in the appearance of virus RNA. RSV-infected chicken embryo fibroblasts made stationary 10 to 15 cell generations after infection continued to produce an amount of progeny virus similar to that produced in dividing RSV-infected cells. Teh virus produced from cells made stationary after virus production had begun contained RNA synthesized in stationary cells. Therefore, division of the RSV-infected chicken embryo fibroblast is required for the initiation of transcription of virus RNA, but it is not required for the maintenance of transcription of virus RNA.
Intracellular Viral RNA Species in Mouse Cells Nonproductively Transformed by the Murine Sarcoma Virus
Authors: Tsuchida Nobuo , Green Maurice
Published: January 2020

The size and quantity of virus-specific RNA in five non-virus-producing mouse cells transformed by the Moloney isolate of murine sarcoma virus (MSV) was determined. Hybridization of RNA from transformed cells with the [
3
H]DNA product of the RNA-directed DNA polymerase of the murine sarcoma-leukemia virus was used to detect and quantitate virus-specific RNA. The amount of virus-specific RNA in non-virus-producing cells was less than one-sixth of that found in virus-producing cells. A striking correlation was found between the amount of intracellular virus-specific RNA and the degree of agglutination by conconavalin A previously reported for the four non-virus-producing NIH/3T3 cell lines (Salzberg and Green, 1974). A major RNA subunit sedimenting at 26 to 28
S
was detected in all five MSV-transformed non-virus-producing cells. This could represent the RNA genome of defective MSV.
Double-Length, Circular, Single-Stranded DNA from Filamentous Phage
Authors: Wheeler Frances C. , Benzinger Rolf H. , Bujard Hermann
Published: January 2020

Wild-type and gene 3 mutant filamentous phage stocks, containing different relative amounts of multiple-length particles, were treated exhaustively with DNase and then were highly purified. The phage DNA was extracted and examined by electron microscopy. In all cases, about 0.03% of the molecules were circular dimers.
3
H-labeled phage DNA was separated as to size by sedimentation in a preformed CsCl density gradient. Individual fractions were then examined in the electron microscope, and the percentage of linear and circular monomer and dimer DNAs was determined. A peak of double-length, circular molecules (with the expected sedimentation constant of 38
S
) was found ahead of the 24
S
monomer peak. The double-length molecules had been purified 65-fold. As previously found for single-stranded DNA, the contour length of these molecules was strongly dependent upon ionic strength. Possible artifacts were ruled out, and it was shown that the double-length molecules arose from phage particles.
Morphological, Chemical, and Biological Characterization of Japanese Encephalitis Virus Virion and Its Hemagglutinin
Authors: Kitano T. , Suzuki K. , Yamaguchi T.
Published: January 2020

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.
Distribution of the Receptor Sites for Sindbis Virus on the Surface of Chicken and BHK Cells
Authors: Birdwell Charles R. , Strauss James H.
Published: January 2020

Sindbis virus was adsorbed to chicken cells or to BHK cells, and the distribution of virus over the surface of the cell was examined by electron microscopy of surface replicas. The distribution of virus particles on the cell was used to indicate the position of virus receptors at the cell surface. When purified Sindbis virus was adsorbed at 37 C to cells prefixed with glutaraldehyde, the virus particles were evenly distributed over the surface of most cells. There was a large variability from cell to cell, however, in the number of virus particles adsorbed, and regions with different concentrations of virus particles were sometimes observed on the same cell. The concentration of virus receptors observed varied from 20 to 160/μm
2
of cell surface, and, thus, the total number of virus receptors per chicken cell is on the order of 10
5
. When virus was adsorbed to unfixed cells at 4 C, the virus particles were clustered into aggregates varying from a few particles to large crystalline arrays (the latter seen only in chicken cells). These conditions are apparently conducive to virus aggregation, and this, coupled with free lateral diffusion of the virus-receptor complex in the cell membrane at 4 C, leads to the observed clustering.
Evidence for the Presence of RNA in the Purified Virions of Vaccinia Virus
Authors: Roening G. , Holowczak J. A.
Published: January 2020

Vaccinia virus, strain WR, was propagated in HeLa cells, L mouse fibroblats, or primary chicken embryo fibroblasts in the presence of [5-
3
H]uridine. Carefully purified virions were found to contain significant amounts of labeled trichloroacetic acid-precipitable material which was rendered acid soluble when digested with pancreatic RNase or hydrolyzed in alkali. Controlled degradation of virions with Nonidet P-40 and 2-mercaptoethanol demonstrated that 65 to 80% of the [5-
3
H]uridine-labeled molecules resided in the viral core. When the total nucleic acids were extracted from viral cores prepared from virions propagated in HeLa cells, 30 to 50% of the total incorporated [5-
3
H]uridine was found in RNA; in L mouse fibroblasts, 40 to 50%; in primary chicken embryo fibroblasts, 50 to 60%. The RNA molecules do not appear to be covalently linked to the viral DNA genome but sediment in sodium dodecyl sulfate-sucrose gradients as 8 to 10
S
species relative to ribosomal RNA.
Mutants of
Bacillus subtilis
Bacteriophage φe Defective in dTTP-dUTP Nucleotidohydrolase
Authors: Dunham Linda T. , Price Alan R.
Published: January 2020

Mutants of bacteriophage φe inducing only 1 to 5% of wild-type levels of dTTP-dUTP nucleotidohydrolase give normal bursts of viable progeny phage whose DNA contains 5 to 10% thymine (but no uracil) in place of 5-hydroxymethyluracil. The relative heat lability of one phage mutant enzyme solubilized from the membrane fraction of infected cells suggests that a φe gene codes for the induced dTTPase-dUTPase.
Effect of β-Propiolactone Inactivation of Polyoma Virus on Viral Functions
Authors: Brown A. , Consigli R. A. , Zabielski J. , Weil R.
Published: January 2020

Polyoma virus was inactivated by treatment with β-propiolactone. T-antigen production, polyoma-RNA synthesis, induction of host DNA synthesis (measured by incorporation of labeled thymidine into the cell culture), and in vitro transforming ability were inactivated to a similar degree by various β-propiolactone concentrations (0.25% β-propiolactone reduced these functions approximately 96%), whereas plaque-forming ability and the ability of the virus to replicate its DNA and to synthesize capsid antigen were inactivated by a given concentration of β-propiolactone to a much greater degree (0.25% β-propiolactone led to a reduction of plaque-forming ability of over 8 logs). The significance of these data and their relationship to previously published experiments are discussed.
Location of the 5-Methylcytosine Group on the Bacteriophage φX174 Genome
Authors: Lee Amy Shiu , Sinsheimer Robert L.
Published: January 2020

Bacteriophage φX174 DNA was labeled in vivo with [
methyl
-
3
H]methionine. The methyl-labeled progeny DNA was extracted from purified bacteriophage φX174 particles and was used as template for in vitro synthesis of the complementary strand in the presence of the nucleoside triphosphates and
Escherichia coli
polymerase I. The resultant replicative form DNA was then cleaved, in separate experiments, with restriction endonucleases from
Haemophilus influenzae
and
H. aegyptius.
The DNA fragments were analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methylcytosine in the viral DNA is located in a specific region of the φX174 genome, very likely in gene
H.
Studies on the Bacteriophage 2 Receptors of
Pseudomonas aeruginosa
Authors: Castillo Francisco J. , Bartell Pasquale F.
Published: January 2020

The lysogenization of
Pseudomonas aeruginosa
strain BI with phage 2 resulted in the loss of the capacity to adsorb the same phage. The absence of phage 2 receptors on the surface of the lysogenized strain BI(2)
8
was confirmed by the failure of purified slime polysaccharide (SPB) or lipopolysaccharide (LPS) to inactivate phage 2. SPB and LPS from a phage 2-resistant strain also failed to inactivate phage 2 in contrast to the phage inactivation exhibited by the SPB and LPS obtained from the wild-type strain BI. Chemically, quantitative differences were apparent when the SPB and LPS of strains BI(2)
8
and BI/2S
2
were compared with those of the wild-type strain BI. The most striking difference noted was the absence of amino sugars in the SPB of strain BI/2S
2
. The SPB of strain BI(2)
8
also contained a lower percentage of amino sugars compared with the SPB of the wild-type strain BI.
Herpes Simplex Virus and Human Cytomegalovirus Replication in WI-38 Cells II. An Ultrastructural Study of Viral Penetration
Authors: Smith Janet Duyckinck , de Harven Etienne
Published: January 2020

An electron microscope study was carried out on the early minutes of herpes simplex virus (HSV) and cytomegalovirus (CMV) penetration into WI-38 cells. Both HSV and CMV entered cells either by fusion of the viral envelope with a limiting cell membrane, or via phagocytosis. Both fusion and phagocytosis occurred within 3 min after the initiation of penetration. After fusion, the naked capsids of CMV free in the cytoplasm became coated with a fine, fibrillar material. CMV capsids thus coated retained a well-defined and easily identifiable morphology until the eclipse of visible viral particles between 1 and 1.5 days postinfection. In contrast, naked HSV capsids free in the cytoplasm were never coated. Rather, within minutes after penetration, they assumed a rounded, less regular outline, and were no longer detectable by 90 to 120 min postinfection. The free naked capsids of both viruses appeared to migrate across the cytoplasm toward the nucleus and to become located near nuclear pores. Both HSV and CMV capsids reached the nucleus as early as 5 min after the initiation of penetration. No further interaction with the nucleus could be documented. Particles were also consistently identified in the Golgi region. Phagocytosed particles generally remained within phagosomes, where they appeared to be degraded. However, stages were identified in what is believed to be the escape of enveloped viruses from phagosomes into the cytoplasm via fusion of their envelope with the phagosomal membrane.
Diffusion Constant and Dimension of Bacteriophage φX174 as Determined by Self-Beat Laser Light Spectroscopy and Electron Microscopy
Authors: Bayer M. E. , DeBlois R. W.
Published: January 2020

The diffusion constant of bacteriophage φX174 was determined by laser light self-beat spectroscopy. The method allows one to establish the diffusion constant in comparatively short time and with high accuracy. From the diffusion constant
D
37
= 1.96 ± 0.08 × 10
−7
cm
2
/s, the size of the virus particles within their aqueous milieu can be calculated. For φX174, a diffusional diameter of 31.4 ± 1.0 nm was found, in good agreement with measurements of diameters of freezeetched (32.3 ± 1.8 nm) and negatively stained particles (33.8 ± 2.1 nm), provided that the entire spikes of the virion are included. Other isometric viruses may show a complex interaction of the virion with the surrounding water and its ions.
Chromatographic Analyses of Isoaccepting tRNAs from Avian Tumor Viruses
Authors: Taylor M. W. , Wang S. , Kothari R. M. , Hung P. P.
Published: January 2020

Low-molecular-weight RNA from transforming viruses (Rous sarcoma virus-Rous-associated virus 1, Schmidt-Ruppin strain of Rous sarcoma virus, and sarcoma-B
77
), from nontransforming viruses (Rous-associated virus 1 and sarcoma-NTB
77
), and from chicken liver, chicken embryo fibroblast, and Rous sarcoma virus-Rous-associated virus 1-transformed chicken embryo fibroblast was isolated and purified. To determine if there are modified, qualitatively or quantitatively different isoaccepting species of tRNA in these avian sarcoma viruses as compared with the cell of virus origin, chicken embryo fibroblast or normal chicken liver, methionyl-, arginyl-, and lysyl-tRNA (with high amino acid acceptance activity), and aspartyl- and glutamyl-tRNA from viral-trans-formed cells (with low viral amino acid acceptance activity) were co-chromatographed on reversed phase-5 chromatography columns, and elution profiles were compared. Although in each case the elution profile between a particular viral and host cell tRNA differed quantitatively, there was no qualitative difference in the profiles of corresponding tRNAs from either transforming or nontransforming viruses examined. Minor quantitative differences in the elution profiles might be a reflection of the metabolic state of the cells, since all evidence points to acceptor activity being of host rather than viral origin. Since, with the exception of selective packaging of methionyl-tRNA (IV) species by both transforming and nontransforming viruses, no selectivity was found for isoacceptor species of other tRNAs, it seems that such preferential packaging of methionyl-tRNA (IV) species has no bearing on the event of viral transformation.
New Method for Large-Scale Preparation of Covalently Closed λ DNA Molecules
Authors: Reuben R. , Gefter M. , Enquist L. , Skalka A.
Published: January 2020

A combination of mutations in bacteriophage λ and its host
Escherichia coli
K-12 provides a convenient system for the isolation of large quantities of covalently closed circular DNA molecules. We describe two procedures for the large scale preparation of λ DNA in the duplex circular form.
Cold-Sensitive Mutants of Bacteriophage φX174. II. Comparison of Two Cold-Sensitive Mutants
Authors: Segal David J. , Dowell Clifton E.
Published: January 2020

Cold-sensitive bacteriophage φX174 mutants, another class of conditional lethals, were examined with regard to growth parameters, DNA synthesis, and particle properties. Two mutants,
cs
70 and
cs
82, were examined. Mutant
cs
70 was eclipse defective, showing altered eclipse kinetics at permissive temperature (40 C) and failing entirely to eclipse at restrictive temperature (25 C). Mutant
cs
70 replicated well at 25 C if allowed prior eclipse at 40 C. Mutant
cs
82 had wild-type eclipse at both temperatures but was defective in single-strand synthesis at 25 C, which led to delayed progeny phage appearance, decreased progeny phage synthesis rate, and greatly reduced burst size. The
cs
82 block could not be bypassed by temperature shift. Since complementation analysis of
cs
70 and
cs
82 was not feasible due to the unique properties of these mutants, those φX174 properties affected by the virus coat were examined as an index of a mutation in a coat protein gene. Mutant
cs
70 had aberrant attachment kinetics at both 25 C and 40 C, evidence of a coat protein alteration. Mutant
cs
70 also exhibited significantly decreased thermal stability, further evidence of an altered virus structure. Mutant
cs
82 had increased thermal stability, but the difference was not sufficient to allow unequivocal assignment of this mutant to a coat protein gene. Both mutants had wild-type antiserum inactivation and host range, although
cs
70 was subject to less of (low-level) plating restriction by endogenous F
+
factors.
Fragment Maps of φX-174 Replicative DNA Produced by Restriction Enzymes from
Haemophilus aphirophilus
and
Haemophilus influenzae
H-I
Authors: Hayashi Marie N. , Hayashi Masaki
Published: January 2020

φX-174 replicative form (RF) DNA was cleaved by restriction enzymes from
Haemophilus aphirophilus
(Hap) and
H. influenzae
(H-I strain Osaka) (HinH). Five fragments were produced by Hap and eight by HinH. The positions of all of the Hap fragments and six of the HinH fragments were mapped on the genome by heteroduplex transfection and DNA-DNA hybridization methods. In addition, fragment sizes of φX-174 RF DNA and S13 RF DNA by Hap and HinH were compared. Considerable differences of the size of the fragments produced from these closely related phage genomes were observed.
Intermediates of Bacteriophage MS2 Assembly In Vivo
Authors: Bonner Philip H.
Published: January 2020

The in vivo process of virion assembly was studied in rifampin-treated, MS2-infected
Escherichia coli
during late times of infection—after 18 min postinfection. Differential sucrose gradient sedimentation of infected-cell lysates taken at various times after radioactive labeling indicated a definite temporal order of appearance of phage-specific protein in assembly-related structures. Labeled MS2 protein appears first as a low-molecular-weight peak at the tops of gradients, then as a peak at 40
S
and as a large number of almost unseparable structures between 40 and 80
S
, and finally as 80
S
mature phage particles. During the chase of a short labeling period, radioactive phage protein was found to disappear from gradients in the same temporal order as it appeared; the soluble peak disappears first, followed by the 40 to 70
S
region. The chased label appears quantitatively in the 80
S
phage peak. Labeled phage RNA was found to appear first in the 40
S
peak, then in the structures between 40 and 70
S
, and finally in 80
S
phage particles. The order of disappearance of labeled phage RNA during a chase is the same as its appearance. Resedimentation of the 40 to 70
S
region indicated the presence of distinct structures at 60 and 70
S
and many indistinct ones between 40 and 60
S
. The smaller intermediates exhibit separable maturation protein-rich and coat protein-rich segments, indicating nonrandom binding of the two proteins during the initial steps of assembly. Larger, discrete intermediates appear at 60 and 70
S
. Treatment of the various structures with pancreatic RNase results in destruction of those from 40 through 60
S;
treatment of the 70
S
structure results in the conversion of some of it to a 45
S
peak, presumably the complete capsid. A small fraction of the 80
S
phage peak is also sensitive to RNase, resulting in a similar 45
S
peak. Pulse-chase experiments indicate that structures from 40 through 60
S
as well as the RNase-sensitive 70
S
structure are assembly intermediates, but that the RNase-insensitive 70
S
and the RNase-sensitive 80
S
structures are not.
Prophage Induction of Noninducible Coliphage 186
Authors: Woods Walter H. , Egan J. Barry
Published: January 2020

Coliphage 186 has been regarded as a member of the noninducible group of coliphages. Evidence that prophage 186 is induced by ultraviolet irradiation or by treatment with nalidixic acid or mitomycin C is now presented. The phage yields were similar to those from lysogens of the inducible phage lambda, and the induction required a
recA
+
host. A noninducible mutant of 186 was isolated from its heat-inducible derivative, 186
cIts
, that was no longer inducible by ultraviolet irradiation but remained heat inducible. That zygotic induction of 186 after transfer from a lysogenic male to a non-lysogenic recipient did not occur is indicated by the following findings: (i) there was only a slight increase in phage titer; (ii) similar levels of recombinants were obtained for markers adjacent or distal to the phage integration site, whether the recipient was lysogenic or not, and there was no effect on the gradient of marker transfer; (iii) lysogenic recombinants were readily found and the co-transfer of 186 with adjacent markers was the same to lysogenic or non-lysogenic recipients. Thus, 186 formed an inducible prophage that did not display zygotic induction. Nevertheless, it shared many properties with the noninducible phage P2 as outlined in the discussion.
Nucleic Acid Homology of Murine Type-C Viral Genes
Authors: Callahan R. , Benveniste R. E. , Lieber M. M. , Todaro G. J.
Published: January 2020

The nucleic acid sequence homology between various murine, endogenous type-C viruses (three host range classes of BALB/c virus, the AT-124 virus, and the CCL 52 virus) and two laboratory strains of murine leukemia virus (Rauscher and Kirsten) was determined by DNA:RNA hybridization. The viral sequences exhibit varying degrees of partial homology. DNA:DNA hybridizations were performed between [
3
H]DNA probes prepared from N- and X-tropic BALB/c endogenous viruses and cellular DNAs from BALB/c, NIH Swiss, and AKR inbred mouse strains as well as from California feral mice and the Asian mouse subspecies
Mus musculus molossinus
and
M. musculus castaneus.
All of these strains of mice are shown to possess multiple (six to seven per haploid genome), partially related copies of type-C virogenes in their DNAs. Thermal melting profiles of the DNA:RNA and DNA:DNA hybrids suggest that the partial homology of the viral nucleic acid sequences is the result of base alterations throughout the viral genomes, rather than the loss of discrete segments of viral sequences.
Avian Tumor Virus Proteins and RNA in Uninfected Chicken Embryo Cells
Authors: Chen J. H. , Hayward W. S. , Hanafusa H.
Published: January 2020

The content of proteins P19 and P15 (mol wt 19,000 and 15,000, respectively) of avian leukovirus in various types of uninfected chicken embryos has been determined by radioimmunoassay. All chicken embryos examined, including embryos which have thus far been classified as group specific (gs) antigen negative by complement fixation tests, contained these viral proteins as well as P27 as previously reported. The embryos known as “gs antigen-positive” type contained about five times as much of these viral proteins as did the “gs antigen-negative” type. The ratio of the three viral proteins was similar for all types of embryos, suggesting that the genes for these proteins are coordinately controlled. In contrast to the relatively high levels of viral internal proteins in gs antigen-negative cells, the amounts of virus-specific RNA detectable by molecular hybridization were extremely low. The levels of helper activity, which presumably reflect the level of viral envelope glycoprotein, were also generally low or undetectable in these cells. Thus, the expression of the gene for envelope glycoprotein does not appear to be controlled coordinately with the genes for viral internal proteins.
Antigenic characterization of type C RNA virus isolates of gibbon apes
Authors: Tronick S R , Stephenson J R , Aaronson S A , Kawakami T G
Published: January 2020

Type C RNA viruses initially isolated from a lymphosarcoma of a gibbon ape and from a fibrosarcoma of a woolly monkey are very closely related immunologically. However, recent studies have shown that these viruses are distinguishable in a radioimmunoassay for the 12,000-molecular-weight polypeptide (p12) of the woolly monkey virus. In the present report, an immunoassay has been developed for the p12 polypeptide of the gibbon ape type C virus. This assay is shown to further distinguish the woolly monkey and gibbon ape viruses. In type-specific assays for the p12 polypeptides of these viruses, two new type C viruses isolated from gibbons in a second colony, characterized by high incidence of hemopoietic neoplasia, are immunologically distinguishable from the original gibbon ape virus. The p12 type-specific immunoassays described in the present report may be of importance in studying the natural history of these viruses and their relationship to tumors of primates.
Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy
Authors: Jacobson A B , Bromley P A
Published: January 2020

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10-6 plus or minus 0.25 times 10-6.
In Situ Lysis of φ29- and SPO1-Infected
Bacillus subtilis
Authors: Hagen Elaine W. , Anderson Dwight L.
Published: January 2020

An improved method of in situ lysis of bacteriophage-infected
Bacillus subtilis
was developed and used to study φ29 and SPO1 phage structures produced by individual cells.
Analysis of uridine incorporation in chicken embryo cells infected by vesicular stomatitis virus and its temperature-sensitive mutants: uridine transport
Authors: Genty N
Published: January 2020

The shut-off of RNA synthesis in chicken embryo cells, after infection with vesicular stomatitis virus, is partially due to a reduced capacity of the infected cells to transport uridine. Permeability to uridine decreases exponentially after infection. This loss of ability to transport uridine may be caused either by structural components of the input virions or may result from the expression of the viral gene products. In the latter case, only minor levels of viral transcription is sufficient to modify cellular permeability, since, even at low multiplicities, RNA minus temperature-sensitive (ts) mutants of vesicular stomatitis virus bring about a significant diminution of uridine incorporation in cells infected under nonpermissive conditions. Experiments with mutants of group III suggest that the M protein of the viral envelope may play a role in the sequence of events that modifies uridine transport. In addition to this cause of the diminution of incorporation of uridine by infected cells, another mechanism is noted which requires protein synthesis.
Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine
Authors: Jerkofsky M , Rapp F
Published: January 2020

Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.
RNA polymerase associated with virions of pike fry rhabdovirus
Authors: Roy P , Clark H F , Madore H P , Bishop D H
Published: January 2020

The association of an RNA-dependent RNA polymerase activity with virions of pike fry rhabdovirus has been demonstrated by both in vitro and in vivo studies. The temperature optimum for the in vitro assay is around 20 C, although enzyme activity can be observed at 4 C. Preparations of pike fry virus possess a glycoprotein, a membrane protein, a nucleoprotein, an L protein, and a phosphoprotein, as well as an RNA of about 3.8 times 10-6 mol wt. A protein kinase activity has been found associated with virus preparations. In vitro RNA product analyses indicate that the virus-associated enzyme functions principally as a transcriptase synthesizing viral-complementary, heteropolymeric RNA.
RNA-dependent DNA polymerase activity of RNA tumor viruses. V. Rous sarcoma virus single-stranded RNA-DNA covalent hybrids in infected chicken embryo fibroblast cells
Authors: Leis J , Schincariol A , Ishizaki R , Hurwitz J
Published: January 2020

RNA-DNA covalent hybrids containing viral RNA have been isolated from nuclear fractions of Rous sarcoma virus-infected chicken embryo fibroblast cells shortly after virus infection. The formation of covalent hybrid structures depends upon a functional reverse transcriptase in vivo, since its appearance in cells is temperature dependent when infected with Rous sarcoma virus mutant LA335, which contains a temperature-sensitive reverse transcriptase.
Role of simian virus 40 gene A function in maintenance of transformation
Authors: Brugge J S , Butel J S
Published: January 2020

Mouse, hamster, and human cells were transformed at the permissive temperature by mutants from simian virus 40 (SV40) complementation group A in order to ascertain the role of the gene A function in transformation. The following parameters of transformation were monitored with the transformed cells under permissive and nonpermissive conditions: morphology; saturation density; colony formation on plastic, on cell monolayers, and in soft agar; uptake of hexose; and the expression of SV40 tumor (T) and surface (S) antigens. Cells transformed by the temperature-sensitive (ts) mutants exhibited the phenotype of transformed cells at the nonrestrictive temperature for all of the parameters studied. However, when grown at the restrictive temperature, they were phenotypically similar to normal, untransformed cells. Growth curves showed that the (ts) A mutant-transformed cells exhibited the growth characteristics of wild-type virus-transformed cells at the permissive temperature and resembled normal cells when placed under restrictive conditions. There were 3-to 51-fold reductions in the levels of saturation density, colony formation, and uptake of hexose when the mutant-transformed cells were the elevated temperature as compared to when they were grown at the permissive temperature. Mutant-transformed cells from the nonpermissive temperature were able to produce transformed foci when shifted down to permissive conditions, indicating that the phenotypically reverted cells were still viable and that the reversion was a reversible event. SV40 T antigen was present in the cells at both temperatures, but S antigen was not detected in cells maintained at the nonpremissive temperature. All of the wild-type virus-transformed cells exhbited a transformed cells exhibited a transformed phenotype when grown under either restrictive or nonrestrictive conditions. Thers results indicate that the SV40 group A mutant-transformed cells are temperature sensitive for the maintenance of growth properties characteristics of transformation. Virus rescued from the mutant-transformed cells by the transfection method was ts, suggesting that the SV40 gene A function, rather than a cellular one, is responsible for the ts behavior of the cells.
Elution and uncoating of Coxsackievirus B3 by isolated HeLa cell plasma membranes
Authors: Roesing T G , Toselli P A , Crowell R L
Published: January 2020

Plasma membranes isolated from HeLa cells on discontinuous sucrose gradients were assayed for their capacity to elute and uncoat coxsackievirus B3 at 37 C. Because the viral receptors are limited to the surface of HeLa cells, the addition of radioactively labeled virus to the cells prior to cell homogenization provided a useful marker for locating the plasma membranes during the fractionation procedure. Four bands were formed on the discontinuous sucrose gradients with approximately 70% or more of the membrane-associated viral label being recovered in the most dense bands, designated as bands 3 and 4. Bands 3 and 4 also possessed the plasma membrane marker enzymes, Na+, K+ adenosine triphosphatase and 5'-nucleotidase and revealed typical structures characteristic of plasma membranes as revealed by electron microscopy. Pelleted and washed membranes from band 3 both eluted and uncoated B3 32P-labeled virus, whereas membranes from band 4 eluted virus but failed to uncoat it. The membranes from band 4 were shown to inhibit the viral uncoating activity when mixed with membranes of band 3. Characteristically, unfractionated homogenates of cell membranes eluted but did not uncoat virus. The finding of a naturally occurring inhibitor of virus uncoating provides for the first time a way to distinguish between the membrane activities of virus elution and virus uncoating. The inhibitor remains to be characterized.
Nongenetic complementation of group V temperature-sensitive mutants of vesicular stomatitis virus by UV-irradiated virus
Authors: Deutsch V
Published: January 2020

Cells infected with temperature-sensitive (ts) mutants of complementation group V of vesicular stomatitis virus (VSV) give an enhanced yield at nonpermissive temperature when co-infected or superinfected with UV-irradiated virus. Virions produced in these mixed infections are temperature sensitive and do not complement ts V45. Rescue of group V mutants is multiplicity dependent. It can occur in the presence of cycloheximide; kinetics of rescue are similar in the absence or in the presence of the drug. Rescue is due to nongenetic complementation and is interpreted as a trigger effect on maturation of a small quantity of biologically active protein V molecules provided by UV-irradiated virus. These results are comfirmed by rescue of ts V45 by UV-irradiated, defective, interferring T particles.
Temperature-sensitive host range mutants of herpes simplex virus type 2
Authors: Koment R W , Rapp F
Published: January 2020

Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblasts cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties.
Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus
Authors: Wright P J , Di Mayorca G
Published: January 2020

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.
Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII
Authors: Summers J
Published: January 2020

Digestion of polyoma viral DNA with a restriction enzyme from Haemophilus aegyptius generates at least 22 unique fragments. The fragments have been characterized with respect to size and physical order on the polyoma genome, and the 5' to 3' orientation of the (+) and (-) strands has been determined. A method for specific radiolabeling of adjacent fragments was employed to establish the fragment order. This technique may be useful for ordering the fragments produced by digestion of complex DNAs.