bluuebunny/crossref_metadata_2025 huggingface dataset:
Function of gene 49 of bacteriophage T4. I. Isolation and biochemical characterization of very fast-sedimenting DNA
Authors: Kemper B , Janz E
Published: January 2020

Very fast-sedimenting DNA was isolated from cells after infection with gene 49 defective phage T4. This DNA appeared membrane bound throughout the time after infection and could be isolated either in the membrane-bound form (M-DNA) or free of membrane (released DNA) depending on the lysis procedure. Released DNA formed complexes of marked stability with sedimentation velocities between 1,400S and 2,100S. These complexes did not seem to contain material other than DNA. This was concluded from the results of RNA, protein, and membrane labeling experiments and density analysis. In addition, these complexes were resistant against treatment with n-butanol, phenol. chloroform-methanol, sodium dodecyl sulfate, Sarkosyl, Pronase, RNase, or lysozyme. The observation that more then 90% of the purified very fast-sedimenting DNA is retrapped by magnesium-Sarkosyl crystals (M-band) suggests that the M-band technique may not be sufficient as a test for DNA-membrane attachment.
Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40
Authors: Smith H S , Turner S , Leong J A , Rigby P W
Published: January 2020

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.
Genome structure of incomplete particles of adenovirus
Authors: Daniell E
Published: January 2020

Incomplete particles arising during productive growth of adenovirus were separated from infectious particles by density gradient centrifugation. The DNA contained in particles of low density was characterized by restriction enzyme analysis and by electron microscopy and heteroduplexing techniques. The DNA is heterogeneous in length, ranging in size from 15% of the normal genome to full length. Many individual molecules contain long, inverted terminal repetitions, which consist of the sequences extending from the normal left-hand end of the viral genome inward; the normal right end sequences appear to be missing from these molecules. The region of the genome reiterated in these molecules is that which has been implicated in transformation of rat cells by adenovirus (Gallimore, Sharp, and Sambrook, 1974; Graham, van der Eb, and Heijneker, 1974). A model for adenovirus replication is presented that accounts for the aberrant structures observed.
Differential response of type C and intracisternal type A particle markers in cells treated with iododeoxyuridine and dexamethasone
Authors: Kuff E L , Lueders K K , Orenstein J M , Wilson S H
Published: January 2020

Mouse neuroblastoma cells containing intracisternal type A particles were treated with iododeoxyuridine and dexamethasone to induce the release of type C oncornavirus particles. For 5 days after treatment, antigenic markers and DNA polymerase activities specific to particles of each of the two types were assayed in the cells and in pellets obtained by high-speed centrifugation of the culture fluid. There was a marked release of C-particle antigen (p30) and DNA polymerase activity in extracellular particulate form, reaching a maximum on day 3 after treatment and falling thereafter. In contrast, no extracellular A-particle antigen was detected, and A-particle-specific DNA polymerase activity in the medium pellets did not increase from the original very low level. Electron microscopy confirmed the presence of free type C virus particles, but not intracisternal type A particles, in the culture fluid. Although intracellular levels of C-particle antigen rose 20- to 30-fold per milligram of cell protein, intracellular A-particle antigen and DNA polymerase activity did not vary more than two-fold. The relative rate of A-particle synthesis in the treated cells, as judged by incorporation of radioactive amino acids into the major structural protein (P73), was also unchanged over the period of observation. Thus, the induction of type C virus particle formation in cultured neuroblastoma cells had no detectable effect on the quantity, synthesis rate, or location of intracisternal type A particles.
DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells
Authors: Purifoy D J , Powell K L
Published: January 2020

Affinity chromatography on single-stranded and double-stranded DNA-cellulose indicates that 12 proteins previously identified from herpes simplex virus type 2-infected cells, ranging in molecular weight from 28 X 10(3) to 186 X 10(3), bind to DNA-cellulose. The DNA-binding proteins found in infected cells differed in relative binding strengths for denatured DNA-cellulose. The virus specificity of these DNA-binding proteins was further studied by comparison with DNA-binding proteins isolated from mock-infected cells, and by immunoprecipitation of infected-cell DNA-binding proteins with antisera specific for viral antigens. The promise this technique holds for the purification and study of polypeptides involved in virus DNA replication, recombination, or repair is discussed.
Isolation of a transcriptive complex from Newcastle disease virions
Authors: Colonno R J , Stone H O
Published: January 2020

An active transcriptive complex was isolated from purified virions of Newcastle disease virus. After disruption with Triton X-100 and high salt, soluble and particulate fractions were separated by density gradient centrifugation. The transcriptive complex, recovered at a density of 1.275 g/cm3, appeared as a nucleocapsid structure by electron microscopy. When analyzed by polyacryl-amide gel electrophoresis, the nucleocapsids consisted of the nucleocapsid protein, a minor protein of 53,000 molecular weight, and the large L protein. Nucleocapsids possessed less than 1% of the hemagglutinating and neuraminidase activities originally associated with virions. The active complex synthesized predominantly 11 to 20S RNA in vitro and approximately one-fourth of the RNA molecules contained polyadenylic acid segments. In the presence of S-adenosyl-L-methionine, the RNA molecules were capped and methylated at the 5' termini. The transcriptive complex was also capable of methylating exogenous Escherichia coli RNA in the absence of viral RNA synthesis.
Configurational variants of oncornavirus RNAs
Authors: Bader J P , Ray D A
Published: January 2020

Heating oncornavirus RNAs at temperatures insufficient for complete denaturation results in forms migrating between the native form (vRNA) and the completely denatured form (vRNA) after gel electrophoresis. Intermediate forms from Rous sarcoma virus or murine leukemia virus were isolated after heating of vRNA's at 58 degrees C and sedimenting in sucrose gradients, and at least four intermediates could be identified in each case. Melting of feline virus (RD-114) RNA produced one major intermediate which required a comparatively high temperature to denature, and a second intermediate occurring in conditions of low ionic strength. Although the subunit model for oncornavirus RNA is not excluded by these data, we propose that vRNA, vRNA', and intermediates may be configurational variants of the same molecule, and a monomer model for oncornavirus RNA is presented.
Herpesvirus Envelopment
Authors: Darlington Robert W. , Moss L. Howard
Published: January 2020

The growth and envelopment processes of three representative herpesviruses, equine abortion, pseudorabies, and herpes simplex, were examined in baby hamster kidney (BHK 21/13) cells by bioassay (plaque-forming units) and electron microscopy. The envelopment process was identical for all three viruses. After assembly in the nucleus, the nucleocapsid acquired an envelope by budding from the inner nuclear membrane. This membrane was reduplicated as the enveloped particle was released so that the budding process did not result in disruption of the continuity of the nuclear membrane. That portion of the nuclear membrane which comprised the viral envelope was appreciably thicker than the remainder of the membrane and exhibited numerous projections on its surface. Once enveloped, the viral particles were seen in vesicles and vacuoles in the cell cytoplasm. These appeared to open at the cytoplasmic membrane, releasing the virus from the cell. There was no detectable difference in the size or appearance of enveloped particles in intra- or extracellular locations.
Inhibition of HeLa Cell Protein Synthesis by the Vaccinia Virion
Authors: Moss Bernard
Published: January 2020

HeLa cell protein synthesis is rapidly suppressed after infection with purified vaccinia virus. This was measured in three ways. (i) In the presence of 5 μg of actinomycin D per ml, viral protein synthesis was prevented and the decline in host protein synthesis was measured directly. (ii) Virus particles irradiated with 800 ergs or more of ultraviolet (UV) light per mm
2
are defective in their ability to initiate viral protein synthesis, but they still inhibit host protein synthesis. After addition of UV-irradiated virus, the decline in host protein synthesis was measured. (iii) Polyacrylamide gel electrophoresis was used to distinguish between host- and virus-induced proteins. The following results were obtained. (i) The inhibition of HeLa cell protein synthesis begins within 20 min after infection with purified vaccinia particles. Greater than 95% inhibition occurs within 1 to 4 hr after infection, depending on the viral multiplicity used. (ii) The synthesis of viral ribonucleic acid or viral protein is not required for the inhibition of host protein synthesis. (iii) The ability of the virus particles to inhibit cell protein synthesis is lost after heat or detergent treatment. (iv) The ability of the virus particles to inhibit cell protein synthesis is retained after UV-irradiation. (v) Vaccinia viral protein synthesis in preinfected cells is resistant to the effects of superinfection with UV-irradiated vaccinia particles. (vi) Inhibition of cell protein synthesis is complete and does not involve the continued synthesis of small polypeptide fragments. (vii) A decrease in the size of host polyribosomes rapidly follows infection with vaccinia virus. The results are interpreted as a selective effect of some constituent of the vaccinia virus particle or virus-activated host enzyme on host protein synthesis at a level beyond that of transcription.
Target Volume Analysis of Vaccinia Virus: Influence of Virus Dispersion and Noninfectious Particles
Authors: Contreras Guillermo , Ohlbaum Adela
Published: January 2020

These data indicate that 90% of monodisperse vaccinia virus (VV) has a
D
37
dose of 1.0 × 10
4
± 0.2 × 10
4
rad when exposed to the direct effect of gamma radiation from
137
Cs and
60
Co sources. This
D
37
dose yielded a targt size of approximately 10
8
daltons, close to the molecular weight of VV deoxyribonucleic acid. This target corresponds to the first component of the survival curve of VV. Noninfectious virus seemed to play a role on the slope and the proportion of the survival curve second component, suggesting that multiplicity reactivation takes place.
Structure and Development of Viruses as Observed in the Electron Microscope IX. Entry of Parainfluenza I (Sendai) Virus
Authors: Morgan Councilman , Howe Calderon
Published: January 2020

After attachment, the uncoating of Sendai virus, which was accompanied by dissolution of the plasma membrane and fusion of virus to cell, proceeded quickly. Nucleoprotein filaments were found at stages of transit from virus to cytoplasm.
Electron Microscopy of Herpes Simplex Virus III. Effect of Hydroxyurea
Authors: Nii S. , Rosenkranz H. S. , Morgan C. , Rose H. M.
Published: January 2020

The effect of hydroxyurea on the development of herpes virus is mediated through its inhibitory action on deoxyribonucleic acid (DNA) synthesis. Concentrations of the drug that suppress the production of infectious virus cause typical developmental anomalies: failure in formation of the normally dense cores or “complete” viral particles, and either faulty or no envelopment of viral capsids by membranes. The synthesis of viral capsids and virus-stimulated nuclear and cytoplasmic membranes, however, is not interrupted. Combining these results with those of time sequence experiments, the following hypotheses can be presented regarding viral development. Protein synthesis, which is characterized by capsids enclosing cores of low density, precedes DNA synthesis, which is characterized by the appearance of dense cores. Capsids with dense cores are selectively transported to the cytoplasm. Envelopment generally takes place as capsids pass from the nucleus to the cytoplasm. The process of envelopment is also selective, with the result that the majority of particles that have an envelope contain a full quota of DNA.
Deoxyribonucleic Acid Replication in λ Bacteriophage Mutants
Authors: Salzman Lois Ann , Weissbach Arthur
Published: January 2020

After ultraviolet light induction of
Escherichia coli
K-12 strain W3350(λ), several structural intermediate forms of phage deoxyribonucleic acid (DNA) are synthesized. The early defective lysogens of λ, sus O
8
, sus P
3
, and T
11
, were found to synthesize none of the DNA structural intermediates. A lysogen believed to be defective in all known phage activities, λsus N
7
, was found to be able to synthesize an early phage DNA intermediate. The lysogen λsus Q
21
, defective in late phage functions, is able to synthesize the early phage DNA intermediate and a concatenated molecule of greater molecular weight than the mature λ DNA.
Control of Lysis of T4-infected
Escherichia coli
Authors: Couse Nancy L.
Published: January 2020

The lysis of
Escherichia coli
B/5 infected with T4D
r
48 could be delayed by addition of 9-aminoacridine (9AA). Infected cells showed an early period of maximal response followed by a decline in sensitivity. The ultimate rate of lysis was also affected by the dye. Deoxyribonucleic acid (DNA), protein, and lysozyme synthesis began at the normal time in complexes inhibited by 9AA addition. The rates of synthesis of these macromolecules were lower in the presence of the dye, with DNA and lysozyme synthesis being more strongly affected than total protein synthesis. Penicillin-sensitive cell wall synthesis stopped at about 10 min after infection. Inhibition of oxidative metabolism by early potassium cyanide addition prevented lysis in the presence of intracellular lysozyme. The cyanide-sensitive event occurred at about 20 min in normal infections, and between 30 and 40 min in 9AA-inhibited infections. 9AA could alter both the time at which the cyanide-sensitive event occurred and the time of lysis. Addition of chloramphenicol did not prevent lysis once intracellular lysozyme was present. Lysis from without of infected cells consisted of three phases: an initial sensitivity, followed by a short period of resistance, and then a return to sensitivity in normal infections. The demonstration of the late return to sensitivity depended on the presence of intracellular lysozyme, and could be delayed by 9AA addition.
Association of Adenovirus Type 12 Deoxyribonucleic Acid with Host Cell Chromosomes
Authors: zur Hausen Harald
Published: January 2020

Preparations of purified
3
H-labeled adenovirus type 12 (
3
H-Ad. 12) were analyzed for radioactive impurities by Millipore filtration and ultracentrifugation. It was found that only about 1% of the isotope activity was separable from the virions. Exposure of hamster cell lines to
3
H-Ad. 12 resulted in nonlytic infections, and autoradiography indicated that viral deoxyribonucleic acid (DNA), or parts thereof, became associated with host cell chromosomes. Usually, the label was observed in the form of small clusters of grains, as described previously for lytic adenovirus infections of human embryonic kidney cells. The uptake of labeled virus by the PT-K1 line of ratkangaroo cells was close to background level. These cells did not adsorb the virus to any significant extent. Ultraviolet irradiation of the virus for as long as 8 min did not affect viral adsorption onto susceptible cells, nor did it alter the association of viral DNA with host cell chromosomes. The capacity of the virus to induce chromosomal aberrations decreased linearly with an increase in the dose of irradiation, but the decrease occurred at a rate which was four- to fivefold slower than the rate of inactivation of viral infectivity. These results suggest that the capacity to induce chromosomal aberrations is controlled by viral genes.
Mechanism of Enhancement of Virus Plaques by Cationic Polymers
Authors: Wallis Craig , Melnick Joseph L.
Published: January 2020

It has been assumed that plaque enhancement by cationic polymers is due to their binding of sulfated polysaccharides in agar. However, viruses that are enhanced by cationic polymers, diethylaminoethyl-dextran, and protamine were found
not
to be inhibited by polyanions in agar under the usual overlay conditions. In the case of adenovirus, enhancement by protamine seems to be due to the protamine serving as a source of arginine; enzymes released from the cultured cells digest the protamine and provide a reservoir of arginine for the cells. Other viruses (herpes and echovirus types 3, 4, 5, and 6) known to be susceptible to agar inhibitors were found to be enhanced by cationic polymers even under starch gel and methylcellulose overlays, which are free of polyanions. Since cationic polymers enhance the diffusion of virus through agar or starch gel, plaque enhancement seems to be the result of the gel becoming positively charged so that viruses can move effectively through them. The observation that starch gel and methylcellulose enhance plaque formation with viruses known to be inhibited under agar was also reinvestigated. When the consistency of the agar gel was reduced to the same viscosity of starch gel and methylcellulose overlays, the same plaque counts and sizes were observed under all three overlays.
Physiological and Genetic Aspects of Abortive Infection of a
Shigella sonnei
Strain by Coliphage T7
Authors: Hausmann R. , Gomez Beatriz , Moody Beverly
Published: January 2020

Phage T7 adsorbed to and lysed cells of
Shigella sonnei
D
2
371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (
ss
−
mutants) have been isolated which show normal growth in strain D
2
371-48. Upon mixed infection of this strain with T7 wild type and an
ss
−
mutant, infection was abortive; no complementation occurred. The DNA of the
ss
−
mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D
2
371-48 were isolated from
ss
−
mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of
ss
−
mutants did not produce
ss
−
recombinants when they were crossed among themselves. When independently isolated
ss
−
mutants were crossed with each other, wild-type recombinants were found;
ss
−
mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [
ss
+
function (for suicide in
Shigella
)] which leads to the breakdown of phage DNA in the
Shigella
host.
Adenovirus Transformation of Hamster Embryo Cells
Authors: Casto Bruce C.
Published: January 2020

Inoculation of hamster embryo cell cultures with human adenovirus type 12 (Ad12) or simian adenovirus (SA7) resulted in the formation of foci of morphologically transformed cells within 12 days. The rapid appearance of well-defined foci was dependent upon the transfer of cells into new plates, with sufficient dilution after virus adsorption, and was independent of virus dose. Dose-response studies showed linearity of focus formation with dilution of Ad12 or SA7. Results averaged from several experiments show plaque-forming unit to focus-forming unit ratios of approximately 1.8 × 10
6
for Ad12 and 2.6 × 10
5
for SA7. Other experiments showed that most of the adenovirus involved in transformation was adsorbed by 3 hr. Cell lines derived from SA7 transformed cells produced tumors within 19 days when inoculated intradermally into young adult hamsters. Such cell-induced tumors histologically resembled SA7 virus-induced hamster tumors. Formation of tumors with SA7 transformed cells was inhibited by prior immunization of test animals with SA7 or Ad12 virus.
Isolation and Characterization of Viruses from the Kidneys of
Rana pipiens
with Renal Adenocarcinoma Before and After Passage in the Red Eft (
Triturus viridescens
)
Authors: Clark H. Fred , Brennan James C. , Zeigel Robert F. , Karzon David T.
Published: January 2020

Viruses were isolated from kidneys of normal and renal tumor-bearing Vermont
Rana pipiens
after subinoculation into red eft newts (
Triturus viridescens
). Organs of efts inoculated with viable cell suspensions from four of seven tumor-bearing kidneys yielded virus (LT-1, -2, -3, -4) when inoculated into TH-1 (
Terrapene
heart) cell culture. One tumor-bearing kidney also yielded virus (L-4) by direct inoculation into TH-1 cells. An additional isolate (L-5) was obtained from 1 of 52 normal Vermont frog kidneys inoculated directly into TH-1 cells. LT-1 was propagated with cytopathic effect (CPE) in each of 38 cell types tested, of fish, amphibian, reptilian, avian, and mammalian origin, at 23 or 30 C. LT-1 through LT-4, L-4 and L-5, and FV-1 through FV-3 each induced similar CPE in all cells tested. LT-2, however, induced CPE that progressed at a slower rate than that caused by the other isolates and produced smaller plaques (<0.8 mm) under starch gel overlay. Each of the viruses replicated to high titer in embryonated eggs incubated at 30 C. The viruses also grew in efts and adult newts, but not in bullfrog (
Rana catesbeiana
) tadpoles or adult leopard frogs. Tumor induction in adult leopard frogs inoculated with LT-1 was not demonstrated. Electron microscopic observations of LT-1 and LT-2 viruses revealed cytoplasmic particles, hexagonal in cross section, approximately 120 to 140 mμ in diameter, containing a dense nucleoid. LT-1 and LT-2 viruses were indistinguishable from FV-1 and
Tipula
iridescent virus. LT-1 was presumed to be a deoxyribonucleic acid virus on the basis of 5-bromodeoxyuridine inhibition. The isolates were ether-sensitive. On the basis of biological, physicochemical, and antigenic similarities, LT-1 through LT-4, L-4, L-5, FV-1 through FV-3, and isolates recently recovered from the bullfrog and the newt may represent strains of the same amphibian cytoplasmic virus.
Isolation and Characterization of Transducing Bacteriophage BP1 for
Bacterium anitratum (Achromobacter
sp.)
Authors: Twarog R. , Blouse L. E.
Published: January 2020

A small transducing phage has been isolated against a strain of
Bacterium anitratum
. The particle has a head dimension of 450 A and a tail approximately 200 A long. The latent period is 16 min and the average burst size is 98. The intact particle has an absorption maximum and minimum at 260 and 237 mμ, respectively. The sedimentation coefficient (
S
20
) is 460. The phage contains double-stranded DNA with an S°
20,w
of 32.8. Molecular weight estimates of the deoxyribonucleic acid ranged from 2.33 × 10
7
to 2.66 × 10
7
based on sedimentation velocity studies. The percentage guanine plus cytosine compositions of the deoxyribonucleic acid, determined by melting temperature and cesium chloride equilibrium centrifugation, were 40.7 and 42.0, respectively.
Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA
Authors: Wovcha M G , Chiu C S , Tomich P K , Greenberg G R
Published: January 2020

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.
Sequence arrangement in herpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease digests and evidence for inversions in redundant and unique sequences
Authors: Wilkie N M , Cortini R
Published: January 2020

It has been proposed by Sheldrick and Berthelot (1974) that the terminal sequences of herpes simplex virus type 1 (HSV-1) DNA are repeated in an internal inverted form and that the inverted redundant sequences delimit and separate two unique sequences, S and L. In this study the sequence arrangement in HSV-1 DNA has been investigated with restriction endonuclease cleavage, end-labeling studies, and molecular hybridization experiments. The terminal fragments in digests with restriction endonucleases Hind III, Hpa-1, EcoRI and Bum were identified and shown to be consistent with the Sheldrick and Berthelot model. Inverted fragments which contain unique sequences as well as redundant sequences, and which the model predicts, were identified by DNA-DNA hybridization studies. Further cleavage of Bum fragments with Hpa-1 also revealed inversions of the terminal sequences that contained unique sequences. The results obtained showed that the unique sequences S and L are relatively inverted in different DNA molecules in the population, resulting in the presence of four related genomes with rearranged sequences in apparently equal amounts. The redundant sequences bounding S do not share complete sequence homology with those bounding L, but hybridization studies are presented which show that the terminal 0.3% of the genome is repeated in every redundant sequence.
Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX
Authors: Buxton R S
Published: January 2020

A mutant of Bacillus subtilis 168 has been isolated in which the defective phage PBSX was heat inducible, whereas another phage, phi105, was not so induced. A culture of the mutant grown at 30 degrees C, when shifted to 45 degrees C, began to lyse after 45 min; cell viability began to decrease after 10 min. Heat-induced lysis of the mutant was prevented by chloramphenicol. DNA, RNA, protein, and peptidoglycan synthesis were normal at the nonpermissive temperature up to the time of lysis. The site of xhi-1479 mutation causing this phenotype was linked (50%) in phage PBS1-mediated transduction to the host marker metC and to another PBSX marker xtl and was thus thought to map within the PBSX prophage. The order of markers was argC-thiB-metA-xhi-metC. The xhi mutation was thus distinct from another mutation, tsi-23, causing a similar heat inducibility of PBSX (Siegel and Marmur, 1969), which was unlinked to the metC marker. tsi-23 is therefore thought to be a host mutation, and the available evidence for a scattered phage genome being the cause of the defective nature of PBSX is thus less tenable. It was shown that the mutant, besides carrying the xhi mutation, also carried another closely linked mutation, xki-1479, which caused the PBSX produced to have no killing activity on the sensitive strain W23. The xki mutation was separated from xhi by recombination.
Physical maps for Herpes simplex virus type 1 DNA for restriction endonucleases Hind III, Hpa-1, and X. bad
Authors: Wilkie N M
Published: January 2020

It has been proposed that the genome of herpes simplex virus type 1 (HSV-1) consists of two internal unique sequences, S and L, bounded by two sets of redundant sequences (P. Sheldrick and N. Berthelot, 1974). In this arrangement, terminal sequences (TRs and TRl) are repeated in an internal inverted form (IRs and IRl) and delimit S and L. Furthermore, a body of evidence has accumulated that suggests that S and L themselves are inverted, giving rise to four related forms of the HSV genome. In this study the ordering of restruction endonuclease fragments of HSV-1 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by Hind III, Hpa-1, and X. bad have been constructed for the four related forms of the HSV-1 genome. TRs and IRs were found to be between 3.5 x 10(6) and 4.5 x 10(6) daltons, TRl and IRl about 6 x 10(6) daltons, S about 8 x 10(6) to 9 x 10(6) daltons, and L about 6.8 x 10(6) daltons.
Generation of defective virus after infection of newborn rats with reovirus
Authors: Spandidos D A , Graham A F
Published: January 2020

When 2-day-old rats were inoculated subcutaneously with the R2 strain of reovirus type 3 or with a class B (352) or class C (447) temperature-sensitive (ts) mutant, 5 to 10% of the animals died from acute encephalitis within 12 days. Approximately half of the survivors recovered rapidly and grew normally, but the remainder became runted. Two phases of infection are distinguished in the animals: an acute phase during which infectious virus reaches a maximum titer in brain and other tissues by 10 days p.i. and thes runting of the rats and the slow disappearance of virus from their brains over a period of 2 months or so. Virus isolated from chronically infected brains generally retained the genetic character (ts or wild type) of the inoculated virus, but two exceptions to this are described. Defective virions lacking the L1 segment of the viral genome (L1 defectives) were generated in rat brains during the acute phase of infection. Defective virus was also generated during the chronic phase, but during this period defectives were found with multiple segments deleted from the genome in addition to L1 defectives. In another type of experiment defective virus exerted a marked protective effect when inoculated intracerebrally with R2 virus. In the absence of defectives all animals died, but in their presence 17 of 23 animals survived and 15 of 23 became runted and chronically infected. The formation and evolution of defective particles in the brains of these rats were similar to those found in rats chronically infected after subcutaneous inoculation of reovirus. We conclude that the formation of defective virus particles may play a role in the initiation and maintenance of chronic neutropic infections with reovirus.
Spontaneous mutations affecting the host range of the B77 strain of avian sarcoma virus involve type-specific changes in the virion envelope antigen
Authors: Zarling D A , Mosser A G , Temin H M
Published: January 2020

Previously it was shown that the host-range gene of the Bratislava strain of avain sarcoma virus (B77 virus) spontaneously mutates with a very high rate. The wild-type B77 virus called B77 virus-II, mutates either to virus that efficiently infects duck cells (B77 virus-III) or to virus that does not mutate to the ability to infect duck cells (B77 virus-I) (Zarling and Temin, 1976). No significant differences in either the virion envelope glycoproteins or other major virion proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, pseudotypes of B77 virus-I with proteins of a transformation-defective mutant of B77 virus-III formed foci efficiently in duck cells. An alteration in the envelope protein of B77 virus-I was demonstrated by experiments in which B77 firus-I was fused into duck cells with UV-irradiated Sendai virus and formed foci. Neutralization experiments further demonstrated that B77 virus host-range mutants have altered type-specific envelope antigens. Thus, the spontaneous mutations in the host-range gene of B77 virus involve changes in the type-specific virion envelope antigen.
Complexity and polyadenylic acid content of visna virus 60-70S RNA
Authors: Vigne R , Brahic M , Filippi P , Tamalet J
Published: January 2020

The genomic complexity of visna virus was measured by quantitative analysis of 18 RNase T1-resistant oligonucleotides from 60-70S RNA. T1-resistant oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. Visna virus had a genomic complexity of 3.6 X 10(6) daltons, very close to the size of a single 30-40S RNA subunit. It was therefore concluded that the visna virus genome is largely polyploid. Visna virus 60-70S RNA polyadenylic acid segment was purified by T1 RNase digestion followed by oligodeoxythymidylic acid-cellulose column chromatography. It contained over 99% AMP and had a size of about 200 nucleotides. The binding capacities on oligodeoxythymidylic acid-cellulose of native 60-70S RNA and purified 30-40S RNA subunits were examined. It was concluded that two out of three intact subunits contain a polyadenylic acid segment.
Possible in vitro repair of viral RNA by ligase-like enzyme(s) in poliovirus-infected cells
Authors: Yin F H
Published: January 2020

A soluble polymerase-template complex prepared from poliovirus-infected cells was found to incorporate radioactive UTP into trichloroacetic acid-insoluble RNA linearly for 8 h in the presence of ATP and Mg2+. Radioactive CTP or GTP was not incorporated under identical conditions. Nearest-neighbor analysis of the in vitro product demonstrated that ATP was added to the viral RNA in the form of polyadenylic acid; UTP was added internally to the 3'-OH group of all four nucelotides. The data can best be explained by the addition of the UTP to the 3'-OH groups of single-stranded breaks in the double-stranded viral RNA and ligation to the adjacent 5'-phosphate groups. The enzymatic activity was also found in encephalomyocarditis virus- and rhinovirus type 1A-infected cells but not in uninfected cells.
Effect of spermidine on the RNA-A protein complex isolated from the RNA bacteriophage MS2
Authors: Leipold B
Published: January 2020

The polyamine spermidine has recently been reported to be a substantial component of the RNA phage particle. Its effect on the isolated RNA-A protein complex of the phage MS2 is investigated here. This complex infects intact Escherichia coli cells via F-pili, as does the whole phage. It is shown that the infectivity of the complex on intact E. coli cells was enhanced by incubation with spermidine. Optimal stimulation (20-fold) of the complex infectivity was achieved by incubation with 3 x 10(-4) M spermidine for 20 to 30 min at 37 degrees C. This gave a more compact structure to the complex, as could be seen by its faster sedimentation in sucrose gradients. Although spermidine and Mg2+ are known to partially replace one another in several systems, no enhancement of the infectivity of the complex, but only its considerably faster sedimentation in sucrose gradients, occurred after incubation with 3 x 10(-4) M Mg2+. Only if the Mg2+ concentration was raised by more than one order of magnitude could increased infectivity of the complex be observed. At concentrations of spermidine and Mg2+ that maximally stimulated the infectivity of the complex on intact E. coli cells, no increase in infectivity of phenol-extracted RNA to E. coli spheroplasts was detected. From these in vitro results, the role of the polyamine spermidine in the RNA phage particle for the infecting, RNA-A protein complex molecules in phage infection is discussed.
Synthesis of mRNA guanylyltransferase and mRNA methyltransferases in cells infected with vaccinia virus
Authors: Boone R F , Ensinger M J , Moss B
Published: January 2020

Guanylyltransferase and methyltransferases that modify the 5'-terminals of viral mRNA's to form the structures m7G(5')pppAm- and m7G(5')pppGm- appear to be synthesized afte- vaccinia virus infection of HeLa cells. Elevations in these enzyme activities were detected within 1 h after virus inoculation and increased 15- to 30-fold by 4 to 10 h. Increases in the guanylyl- and methyltransferase activities were prevented by cycloheximide, an inhibitor of protein synthesis, but not by cytosine arabinoside, an inhibitor of DNA synthesis. The latter results suggest that the mRNA guanylyl- and methyltransferases are "early" or prereplicative viral gene products. The guanylyltransferase and two methyltransferases, a guanine-7-methyltransferase and nucleoside-2'-methyltransferase, were isolated by column chromatography from infected cell extracts and found to have properties similar or identical to those of the corresponding enzyme previously isolated from vaccinia virus cores. In contrast, enzymes with these properties could not be isolated from uninfected cells.
Induction and biological properties of defective interfering particles of rabies virus
Authors: Wiktor T J , Dietzschold B , Leamnson R N , Koprowski H
Published: January 2020

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.
Structure of the inverted terminal repetition of adenovirus type 2 DNA
Authors: Wu M , Roberts R J , Davidson N
Published: January 2020

Several secondary structure features involving the ends of single strands of adenovirus type 2 DNA have been studied by electron microscopy by both the gene 32-ethidium bromide technique and a modification of the standard formamide-cytochrome c technique. A duplex stem of length 115 +/- 10 nucleotide pairs due to pairing between the two members of the inverted terminal repetition is observed in the single-stranded circles that form upon annealing single-stranded linear molecules. This duplex stem is shown to lie at the ends of the DNA by using several reference markers: (i) a newly discovered secondary structure feature (a loop of length ca. 500 nucleotides with a 20-nucleotide pair duplex stem) that maps 73% of the full length from the left end of the molecule and (ii) a duplex region due to a hybridized restriction fragment. There is also some secondary structure within each end of linear single strands. There is some variation in the morphology of the end strucures, and we propose that these involve base pairing, as in a tRNA clover leaf, rather than an exact single hairpin-type inverted repeat. These observations are consistent with the hypothesis that there is a foldback structure at the 3' ends of the DNA that functions as a primer for the initiation of replication.
Interaction of Sindbis virus glycoproteins during morphogenesis
Authors: Jones K J , Scupham R K , Pfeil J A , Wan K , Sagik B P , Bose H R
Published: January 2020

In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell.
Envelope antigens of Sindbis virus in cells infected with temperature-sensitive mutants
Authors: Bell J W , Waite M R
Published: January 2020

Indirect fluorescent-antibody studies of living and fixed chick cells infected with temperature-sensitive mutants of Sindbis virus suggest that functional envelope glycoprotein E1 must be inserted through the plasma membrane before E2. PE2 and E2 do not affect the insertion of E1. The experiments also suggest that normal PE2, a glycosylated precursor to E2, reacts with anti-E2 serum; the abnormal PE2 made by a temperature-sensitive PE2 cleavage-defective mutant did not. Abnormal E1 proteins made by E1-defective mutants also failed to react with anti-E1 serum.
Macromolecular synthesis in cells infected by frog virus 3. VI. Frog virus 3 replication is dependent on the cell nucleus
Authors: Goorha R , Willis D B , Granoff A
Published: January 2020

Previous evidence indicated that frog virus 3 (FV3), an icosahedral DNA virus, replicates exclusively in the cytoplasm. However, data presented here demonstrate that FV3 does not replicate in UV-irradiated or enuleated chicken embryo or BSC-1 cells and that virus-specific DNA synthesis is not initiated in such cells. Primary transcription was not detected in infected enucleated cells. These results demonstrate that a functional nucleus is essential for FV3 replication.
Evidence That a Precursor Glycoprotein Is Cleaved to Yield the Major Glycoprotein of Avian Tumor Virus
Authors: England James M. , Bolognesi Dani P. , Dietzschold Bernhard , Halpern Michael S.
Published: January 2020

A glycoprotein designated pr90, which is recognized by anti-gp85 serum, is present in lysates of pulse-labeled transformed cells. Under chase conditions, a reduction in the level of labeled pr90 is observed concomitant with the appearance of labeled, cell-associated viral glycoprotein.
Circular forms of Uukuniemi virion RNA: an electron microscopic study
Authors: Hewlett M J , Pettersson R F , Baltimore D
Published: January 2020

Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3' and 5' ends of linear molecules.
Shutoff of Host RNA Synthesis in Bacteriophage T4-Infected
Escherichia coli
in the Absence of Host DNA Degradation and Nuclear Disruption
Authors: Snustad D. Peter , Bursch Claudia J. H.
Published: January 2020

In contrast to its effect on host DNA synthesis, nuclear disruption in phage T4-infected
Escherichia coli
B/5 cells has no effect on the shutoff of host RNA synthesis. Host RNA synthesis is shut off normally after infection with T4 multiple mutants that fail to induce both nuclear disruption and host DNA degradation.
Control of protein synthesis in Semliki forest virus-infected cells
Authors: Lachmi B , Kääriäinen L
Published: January 2020

Protein synthesis in Semliki forest virus-infected chicken embryo cells was studied by labeling them with [35S]methionine for short periods at different times after infection, with or without synchronization of protein synthesis by the hypertonic block technique. The rate of host-cell protein synthesis declined almost linearly in inverse correlation to the increase in the amount of virus specific RNA. At 5.5 h postinfection, the host-cell protein synthesis was reduced by about 70%. The viral structural proteins were detectable with certainty at 3.5 h postinfection, and their rate of synthesis increased linearly parallel to the amount of their messenger, the 26S RNA. This suggests that the rate of synthesis of the structural proteins is controlled at the level of transcription. The rate of synthesis of the virus-specific nonstructural proteins attained its maximum between 3 and 4 h postinfection and declined thereafter, wheras the amount of their messenger, the 42S RNA, continued to increase linearly in the cells. Thus, the messenger activity of the 42S RNA is reduced in the late phase of infection compared with its activity in the early phase.