bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Deoxyribonucleic Acid Polymerase Activity in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12
Authors: Ledinko Nada
Published: January 2020

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 μg/ml); the enzyme activities had a similar broad
p
H optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-β-
d
-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.
Ribonucleic Acid Synthesis in Cells Infected with Influenza Virus
Authors: Nayak D. P. , Baluda M. A.
Published: January 2020

Virus-specific ribonucleic acid (RNA), synthesized in influenza virus-infected cells from 3.5 to 7.5 hr after infection, was studied. After velocity centrifugation in sucrose, three peaks of virus-specific RNA could be identified: 34
S
, 18
S
, and 11
S.
These RNA species are predominantly single-stranded and consist of 90% viral (plus) and 10% complementary (minus) RNA strands. Most (75%) of the complementary RNA is single-stranded, i.e., not part of RNA duplexes or replicative intermediates. The 34
S
RNA species is an aggregate of 18
S
and 14
S
RNA species. Both 18
S
and 11
S
RNA species are relatively heterogenous compared to 18
S
ribosomal RNA, and these species probably contain different RNA molecules having closely related sedimentation coefficients.
Transformation of African Green Monkey Kidney Cells by Irradiated Adenovirus 7-Simian Virus 40 Hybrid
Authors: Jensen F. , Defendi V.
Published: January 2020

The ability of adenovirus 7-simian virus 40 (SV40) hybrid (strain LL “E-46”) to replicate decreased exponentially as a function of the amount of γ-irradiation; the ability to induce SV40 and adenovirus 7 T antigen decreased at a much slower rate. Nevertheless, the virus was still able to transform African green monkey kidney cells at a radiation dosage that had completely destroyed its replication ability. All transformed colonies were positive for SV40 T antigen but were negative for adenovirus 7 T antigen. The adenovirus 7-SV40 hybrid transformed cells were superinfectible with SV40 virus. Two of the three transformed cell populations apparently did not sensitize hamsters against the appearance SV40 primary tumors, thus suggesting a deficiency in the SV40 transplantation antigen.
Enhancement of St. Louis Arbovirus Plaque Formation by Neutral Red
Authors: Marcovici Martin , Prier James E.
Published: January 2020

Experiments with St. Louis encephalitis (SLE) virus have shown that neutral red enhances the plaque size in duck embryo cell cultures. This may represent a new method for genetic studies of SLE virus population. In a mosquito pool specimen NR
+
and NR
−
particles were demonstrated. By intracerebral passage in mice, there is a selection of NR
+
particles. Similar effects were not shown for eastern, western, and California encephalitis viruses. Plaques formed by the latter virus, however, were significantly reduced in number by neutral red.
Reversible Repression of Early Enzyme Synthesis in Bacteriophage T4-infected
Escherichia coli
Authors: Cohen Paul S.
Published: January 2020

A mutant of
Escherichia coli
B, defective in its accumulation of K
+
, was found to synthesize protein at a rate proportional to the level of this cation in the growth medium. When bacteriophage T4-infected cells were incubated in growth medium containing 1 m
m
K
+
, phage deoxyribonucleic acid (DNA) was synthesized at a rate 25% that of normal, and phage protein was synthesized at a rate of 50% of normal. Deoxycytidine pyrophosphatase, a phage-directed early enzyme, shut off at a level of 55% that of normal when infected cells were incubated in medium containing 1 m
m
K
+
. However, deoxycytidine pyrophosphatase synthesis resumed in these cells when they were shifted to medium containing the normal K
+
concentration (33 m
m
). DNA synthesis also attained the rate characteristic of this K
+
concentration. These results suggest that phage DNA synthesis is not sufficient to repress early protein formation and also indicate that the inhibitor of early protein formation is an early function whose synthesis is sensitive to the same repression as that of the early proteins.
Primary Adsorption Site of Phage PBS1: the Flagellum of
Bacillus subtilis
Authors: Raimondo Linda M. , Lundh Nancy P. , Martinez Rafael J.
Published: January 2020

The adsorption of
Bacillus subtilis
phage PBS1 was studied, and it was demonstrated that the primary adsorption site for this phage is the flagellum of
B. subtilis
. The capacity of flagella to function for motility may be lost without the loss of their capacity to adsorb the phage and permit infection. Deoxyribonucleic acid injection by the phage is inhibited by cyanide, suggesting the requirement for cellular energy in the infection process.
Electron Microscopy of Herpes Simplex Virus
Authors: Nii Shiro , Morgan Councilman , Rose Harry M.
Published: January 2020

Examination of infected cells at sequential intervals after infection revealed that the first viral forms to appear were capsids enclosing cores of low density. Not until the 6th hr were dense cores encountered, and at approximately the same time enveloped virus was seen. Envelopment occurred most frequently in close proximity to the nuclear surface, although the process was also encountered within the nuclear matrix and in the cytoplasm. There was often extensive proliferation of the nuclear membrane. Envelopment of the virus by budding from the cell surface was not observed. It was concluded that enveloped virus consitutes the infectious particle and that the unenveloped capsid is unstable outside the cell. Nevertheless, it is likely that capsids enclosing infectious nucleic acid can pass directly from one cell to another after fusion has taken place.
Destruction of L Cells by Mengo Virus: Use of Interferon to Study the Mechanism
Authors: Gauntt Charles J. , Lockart Royce Z.
Published: January 2020

Interferon, when added to L cells, inhibited the synthesis of infectious Mengo viral ribonucleic acid, hemagglutinins, and infectious virus by 85 to 95%. Serum-blocking antigens were also reduced by the action of interferon, but threefold excess amounts of these antigens accumulated in interferon-treated cultures above the amounts expected for the quantity of infectious virus that was produced in these cultures. Radioautographic analysis showed that 28 to 36% of the cells of an interferon-treated population synthesized viral ribonucleic acid and 36 to 47% produced viral antigens as determined by an immunofluorescence technique. Despite the reductions in synthesis of viral components, all cells in an interferon-treated culture underwent cytopathic effects at the same time as cells in infected cultures which had not been treated with interferon. The results are compatible with the hypothesis that the cell destruction which results from the infection of L cells with Mengo virus is due to a protein which is coded for by the virus but is not a component of the mature virion.
Analysis of Simian Virus 40-induced Transformation of Hamster Kidney Tissue In Vitro
Authors: Burns William H. , Black Paul H.
Published: January 2020

Clones of simian virus 40-transformed hamster kidney cells from which infectious virus could be recovered and clones which did not yield virus by the overlay technique were subjected to chemical (mitomycin C) induction and to co-cultivation and fusion studies with indicator cells. The results of these studies indicate that the complete viral genome is present in all clones tested but that considerable heterogeneity with respect to inducibility exists among the clones. It is suggested that differences either in the number of viral genomes per transformed cell or in the status of the viral genome in transformed cells exist among the various clones. Furthermore, the inducibility of the clones may be correlated with their malignant potential.
Herpes Simplex Virus-Host Cell Relationships in Organized Cultures of Mammalian Nerve Tissues
Authors: Feldman L. A. , Sheppard R. D. , Bornstein M. B.
Published: January 2020

Studies on the replication of herpes simplex virus in organized cultures of rat central nervous system (CNS) and peripheral nervous system (PNS) tissue demonstrated synthesis of intra- and extracellular virus, as determined by plaque assay on HEp-2 cells. Newly synthesized intracellular virus appeared 12 to 14 hr after inoculation of CNS, followed 10 hr later by the appearance of extracellular virus. In PNS cultures, where higher inputs of virus were introduced, intracellular virus appeared 6 to 8 hr after inoculation, followed by extracellular virus 12 hr later. Polykaryocyte formation was observed in CNS and PNS tissue involving neuroglial, meningeal, or Schwann cells. Neuron somas did not participate in polykaryocyte formation, but they underwent progressive morphological changes starting with increased cytoplasmic granularity followed by nucleolar distortions and disintegration, margination of nuclear chromatin, and the appearance of intranuclear inclusions. Finally, all recognizable cellular detail was lost. Immune serum globulin failed to inhibit both the progressive nature of the cytopathic effect and the synthesis of intracellular virus. These findings are discussed in relation to other in vitro systems, as well as to disease processes in man and animals.
Initiation of Vaccinia Virus Infection in Actinomycin D-pretreated Cells
Authors: Magee Wayne E. , Miller Olga V.
Published: January 2020

The early steps in vaccinia virus infection were studied in HeLa cells which had been treated with actinomycin D (1 μg/ml) and then incubated for several hours in fresh medium prior to infection. Initiation of infection occurred in such cells even though the synthesis of cellular ribonucleic acid and deoxyribonucleic acid (DNA) was severely depressed. Thymidine kinase was synthesized in amounts that exceeded those found in untreated, infected cells. The breakdown of viral “cores” to liberate viral DNA and the synthesis of viral specific DNA-polymerase also occurred but were somewhat delayed. A deoxyribonuclease resembling an exonuclease was made by the infected, pretreated cells. The time course for these events suggested that the genetic code for synthesis of thymidine kinase can be expressed before “cores” are broken down, but the DNA-polymerase can be synthesized only after liberation of the viral DNA. The amount of viral specific DNA-polymerase which was made after infection was proportional to the total number of virus synthesizing sites even beyond the point where all the cells were infected with one infectious particle. A similar relationship was observed for the amount of thymidine kinase formed and for the rate of viral DNA synthesis from
3
H-thymidine.
Effect of Cyanophage Infection on CO
2
Photoassimilation in
Plectonema boryanum
Authors: Ginzburg Dalia , Padan Etana , Shilo Moshe
Published: January 2020

Cyanophage infection effects a rapid and complete cessation of CO
2
photoassimilation in
Plectonema
cells. From the amount of infected cells lysed, it was established that this phenomenon cannot be ascribed to lysis of the host cells either from within or from without. The possibility that the effect is due to nitrogen starvation, induced secondarily by cyanophage multiplication, was ruled out when it was found that nitrogen supplementation did not influence the inhibition. It is suggested that the arrest of CO
2
photoassimilation is an integral part of the cyanophage infection cycle in
P. boryanum
. This idea is supported by the nondependence of the cyanophage-induced inhibition on the input multiplicity, by the light requirement for the inhibition, and by the fact that infected
Plectonema
cells with inhibited CO
2
photo-assimilation support normal multiplication of the cyanophage. The pattern of light requirement for this viral inhibition further supports this suggestion.
Productive Infection of
Bacillus subtilis
168, with Bacteriophage SP-10, Dependent upon Inducing Treatments
Authors: Goldberg Ivan D. , Bryan Theodore
Published: January 2020

Strains of
Bacillus
that harbor defective phage PBSX were found to be insensitive to SP-10(C), although the phage adsorbed to these insensitive strains. Strains that did not carry the phage were sensitive to SP-10(C).
B. subtilis
168
ind
−
, which can be tranduced by SP-10(C) but is nonpermissive for the phage, was rendered phage-sensitive after treatment with ultraviolet (UV) light or mitomycin C. After induction with UV light, maximal sensitivity to SP-10(C) was obtained at a multiplicity of infection (MOI) of approximately 14; with mitomycin C induction, an MOI of approximately 1.0 was required. Phage maturation in sensitized cells was followed by plating infected streptomycin-sensitive cells in the presence of streptomycin at various stages during phase development. The latent period was estimated at 60 to 75 min. We suggest that the resistance of
B. subtilis
168 to SP-10 is controlled, at least in part, by the presence of a defective prophage.
Inactivating and Mutagenic Effects of 1-Methyl-3-Nitro-1-Nitrosoguanidine on Intracellular Bacteriophage
Authors: Zampieri Antonio , Greenberg Joseph , Warren Guylyn
Published: January 2020

1-Methyl-3-nitro-1-nitrosoguanidine (NG) was able to inactivate and mutate adsorbed but not free coliphage T2. Coliphage T4 was as sensitive to NG as T2. When treatment was delayed for 5 min or longer after adsorption of T2, the phage became increasingly refractory to NG inactivation.
Characterization of ribonucleoproteins and ribosomes isolated from lymphocytic choriomeningitis virus
Authors: Pedersen I R , Konigshofer E P
Published: January 2020

Disruption of purified lymphocytic choriomeningitis (LCM) virus with Nonidet P-40 in 0.5 M KCl followed by sucrose gradient centrifugation in 0.3 M KCl led to the isolation of two viral nucleoproteins (RNPs) as well as 40S and 60S ribosomal subunits. The largest viral RNP sedimented heterogenously at 123S to 148S and was associated with 23S and 31S viral RNA. The other viral RNP sedimented at 83S and was associated with 23S viral RNA. The buoyant density in CsCl was determined to be 1.32 g/cm3 for the viral RNP. Densities of 1.52 and 1.60 g/cm3 were determined for the 40S and 60S subunits, similar to those of the BHK-21 cells subunits dissociated by 0.5 M KCl. The viral RNPs were partly sensitive to RNase.
Size, subunit composition, and secondary structure of the Friend virus genome
Authors: Dube S , Kung H J , Bender W , Davidson N , Ostertag W
Published: January 2020

Electron microscope and gel electrophoresis studies show that the high-molecular-weight (50 to 70S) RNA extract from Friend virus (FV) is a dimer with the same basic structure previously observed for the RNAs from RD-114 virus, baboon virus, and woolly monkey virus. This observation greatly strengthens the inference that the dimer structure is a general characteristic of the RNAs of all mammalian type C viruses. The FV dimer is slightly less stable than the RNA dimer of woolly monkey virus, which is, in turn, much less stable than those of RD-114 and baboon virus. There are three FV monomer components, small (S), medium (M), and large (L), with molecular lengths of 6.7 +/- 0.6, 7.7 +/- 0.6, and 9.5 +/- 0.6 kilobases, respectively. There are approximately equal amounts of the S and M components and much less of the L component. Most of the dimers are homodimers (SS, MM, and LL). The frequency of heterodimers (SM, SL, ML) is much less than expected for a random assortment model.
Mapping of two isoleucine tRNA isoacceptor genes in bacteriophage T5 DNA
Authors: Hunt C , Hwang L T , Weiss S B
Published: January 2020

T5 bacteriophage codes for the synthesis of more than 14 different tRNA species, which map in four separate clusters in the C segment of the T5 chromosome. In this study, two tRNAile isoacceptor species have been identified by reverse-phase chromatography and shown to be transcribed from two different tRNA loci along the T5 chromosome. The map positions of the tRNA isoacceptors were aided by the use of several T5 deletion mutants in which the position and size of the deleted DNA segments had been previously determined by heteroduplex mapping. Hybridization analysis suggests the presence of some sequence homology between the two tRNAile species.
Isolation and structural characterization of monomeric and dimeric forms of replicative intermediates of Kilham rat virus DNA
Authors: Gunther M , May P
Published: January 2020

Two virus-specific species of newly synthesized DNA were isolated from rat fibroblast cell cultures infected with the Kilham rat virus (RV). These two DNA species were purified; their behavior on hydroxyapatite chromatography and their sedimentation coefficients in sucrose gradients were determined. One of the two species corresponds to the linear double-stranded form of the RV DNA, and the other corresponds to the dimeric duplex form. After denaturation, a fraction of both species showed an intramolecular renaturation; these molecules are composed of viral strand covalently linked to complementary strand. Models for the structure of both species are posposed. Both species may be considered as double-strand replicative intermediates of the single-stranded RV DNA.
Evidence that mismatched bases in heteroduplex T4 bacteriophage are recognized in vivo
Authors: Berger H , Pardoll D
Published: January 2020

T4 heteroduplex heterozygotes are lost selectively after prolonged incubation of phage-infected Escherichia coli cells under nonreplicating conditions. The loss of heterozygosity occurs for four out of six rII sites tested and is not dependent upon T4 v gene function. The results are interpreted to indicate the existence of a base-specific system for the recognition of mismatched bases in intracellular DNA.
Nature of the single-stranded DNA in replicating adenovirus type 5 DNA
Authors: Sussenbach J S , Tolun A , Pettersson U
Published: January 2020

Single-stranded regions in replicating adenovirus type 5 DNA were isolated and hybridized in solution to the separated strands of adenovirus 2 or 5 DNA. The results showed that the two strands of adenovirus 5 DNA are exposed to almost the same extent during replication, suggesting that displacement synthesis may start from either end of the viral DNA.
Distinguishing cytomegalovirus, mycoplasma, and cellular DNA polymerases
Authors: Miller R L , Rapp F
Published: January 2020

Cytomegalovirus-induced DNA polymerase can be distinguished from infected-cell enzymes by activity in 100 mM (NH4)2SO4. Virus polymerase is stimulated to 145% of control, whereas mock-infected cell polymerase is inhibited to 12% of control without added salt. Mycoplasmas induce a DNA polymerase in cell extracts that is stimulated to 130 to 180% by 25 mM (NH4)2SO4. Mycoplasma DNA polymerase may be mistaken for a virus-induced polymerase when virus stocks are contaminated. Identification of virus, cellular, and mycoplasma DNA polymerases in total cell extracts is described using sedimentation rate and effect of inhibitors on DNA polymerase activities.
Infectious, linear, unintegrated DNA of Moloney murine leukemia virus
Authors: Smotkin D , Yoshimura F K , Weinberg R A
Published: January 2020

A closed circular, double-stranded infectious DNA of Moloney leukemia virus has been described previously. The present report characterizes a second type of infectious, unintegrated viral DNA which is linear, largely double stranded, and of mass comparable to that of the closed circular viral DNA. The linear form is of nonpermuted sequence, and SalI endonuclease cleaves at one site 45% from one end.
Interspecies determinants of Friend leukemia virus antigens involved in cytolysis of virus-pfoducing cells
Authors: Collins J J , Bolio L D , Denny T P , Bolognesi D P
Published: January 2020

The cytolytic reactivity of a complex goat anti-feline leukemia virus (FeLV) antiserum for mouse cells (Eveline) releasing large quantities of Friend leukemia virus (FLV) was analyzed by the sensitive [14C]nicotinamide release microcytotoxicity assay. Whereas this interspecies killing reactivity could be blocked by absorption of the goat anti-FeLV serum with a preparation of disrupted FLV, absorption with purified FLV gp71, the major envelope glycoprotein, had no effect. Subsequent serum absorptions with the individual FLV structural polypeptides revealed that the lysis of the Eveline cells by the goat anti-FeLV serum is mediated by antibodies recognizing the interspecies determinant of p30, the major internal capsid protein. The expression of this internal viral component at the surface of virus-producing cells is discussed further. The results also demonstrated that removal of approximately 70% of the carbohydrate portion of gp71 with a preparation of glycosidases did not affect the integrity of its interspecies determinant; these results are in agreement with an earlier study (Bolgnesi et al., 1975) that examined primarily the group- and type-specific sites.
Induction of syncytia by Moloney murine leukemia virus in myoblasts defective in differentiation
Authors: Wong P K , Yuen P H , Kaufman S J
Published: January 2020

fu-1 cells, a nonfusing variant of the L8 line of rat myoblasts, form syncytia upon infection with murine leukemia virus (MuLV) or upon cocultivation with MuLV-infected cells; L8 cells do not form these syncytia, but do fuse into multinucleate myotubes. Syncytia of fu-1 cells form within 1 h after infection. The number of syncytia formed is proportional to the multiplicity of virus within a range of 4 to 16 and is maximum when the cell density is subconfluent. When either XC or fu-1 cells are productively infected with MuLV, they become resistant to syncytia formation by passage 3. The rapid formation of syncytia in fu-1 cells was found amenable for selection of temperature-sensitive mutants of MuLV and for screening additional variants of the L8 line.
Application of freeze-drying intact cells to studies of murine oncornavirus morphogenesis
Authors: Demsey A , Kawka D , Stackpole C W
Published: January 2020

Using a method for freeze-drying intact cells, uninfected and murine leukemia virus (MuLV)-infected JLSV9 cell surfaces, as well as murine mammary tumor virus (MuMTV)-infected cell surfaces, were examined by electron microscopy. The 10-nm knobs of MuLV and the 5-nm spikes of MuMTV were clearly revealed on the surfaces of budding viruses and were also found dispersed over the cell surface. The MuLV knobs are randomly arranged on the virus surface, whereas the MuMTV spikes are much more ordered. Because freeze-fractured budding viral envelopes are devoid of intramembranous particles, the observed surface particles do not appear to be merely accentuated intramembranous particles. This technique should permit further analysis of the morphogenesis of viral envelopes without the need for externally applied labels.
Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses
Authors: Leavitt R , Schlesinger S , Kornfeld S
Published: January 2020

Tunicamycin (TM), an antibiotic that inhibits the formation of N-acetylglucosamine-lipid intermediates, thereby preventing the glycosylation of newly synthesized glycoproteins, inhibits the growth of Sindbis virus and vesicular stomatitis virus in BHK cells. At 0.5 mug of TM per ml, the replication of both viruses is inhibited 99.9%. Noninfectious particles were not detected. All the viral proteins were synthesized in the presence of TM, but the glycoproteins were selectively altered in that they migrated faster than normal viral glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting defective glycosylation. Within 1 h after TM addition, [14C]glucosamine incorporation into glycoproteins was inhibited 20%, whereas [35S]methionine incorporation was unaffected. By 2 to 3 h after TM addition, glucosamine incorporation had fallen to 15% of control value, with methionine incorporation being 60% of normal. TM did not affect the growth of the nomenveloped encephalomyocarditis virus in BHK cells, demonstrating that TM is not a general inhibitor of protein synthesis. These data demonstrate that TM specifically inhibits the glycosylation of viral glycoproteins and that glycosylation may be essential for the normal assembly of enveloped viral particles.
Transcriptional mapping of vesicular stomatitis virus in vivo
Authors: Ball L A
Published: January 2020

Synthesis of the proteins of vesicular stomatitis virus (Indiana serotype) was studied in mouse L cells infected with virus that had been exposed to UV radiation. The UV target sizes measured during primary transcription indicated that the five genes occupy a single transcriptional unit. Thus, in infected cells, as a cell-free system, transcription of vesicular stomatitis virus RNA initiates at a single point and proceeds in the order N. NS, M, G, and L.
Selective screening procedure for the isolation of heat- and cold-sensitive, DNA replication-deficient mutants of bacteriophage SPO1 and preliminary characterization of the mutants isolated
Authors: Glassberg J , Slomiany R A , Stewart C R
Published: January 2020

A procedure is described for the selective isolation of temperature-sensitive replication-deficient mutants of Bacillus subtilis phage SPO1. A modification of the procedure permits the isolation of temperature-sensitive mutants in specific cistrons of interest. The applicability of these procedures to other viral systems is discussed. The mutations isolated were assigned to eight replication-deficient cistrons, with the cold-sensitive mutations showing a distribution strikingly different from that of the heat-sensitive mutations. As a preliminary to the identification of initiation-deficient mutants, the mutants were divided into three classes on the basis of their ability to synthesize DNA after a shift to nonpermissive temperature. We also report two incidental results: (i) the SPO1 dUMP hydroxymethylase, like the T4 dCMP hydroxymethylase, may be part of a multifunctional complex; and (ii) mutants were isolated that were replication positive but lysis deficient and failed to complement one of the replication-deficient mutants.
Gene D of bacteriophage phi X 174: absence of transcriptional and translational regulatory properties
Authors: Pollock T J
Published: January 2020

The only evidence that a gene product of phi chi 174 is involved in the regulation of phage transcription was presented by Clements and Sinsheimer (1974) for gene D. I. found, in attempting to investigate this possibility further, that the D protein had no regulatory effect. When the D protein was eliminated by various nonsense mutations, all other phage proteins were synthesized at rates nearly identical to the wild-type control. There was also no significant difference in the amount of phage-specific RNA synthesis as a proportion of the total RNA synthesis when the D mutants were compared with wild type. No evidence was detected for a polar effect of gene D nonsense mutants on the expression of genes F, G, and H. The relative molar rates of synthesis of D, F, G, and H, synthesized during a normal infection were 29, 4.9, 3.3, and 1.0, respectively, with an accuracy of +/- 20%.
Modification of simian virus 40 protein A
Authors: Tegtmeyer P , Rundell K , Collins J K
Published: January 2020

The A protein of simian virus 40 is phosphorylated in both productive and transforming infection. The phosphorylated amino acid has been identified as serine and has been localized in a single tryptic peptide of the protein. Because the A protein synthesized in infection by A mutants is phosphorylated to the same extent and in the same peptide as in infection by wild-type virus, the functional defect of the A mutants is apparently unrelated to phosphorylation. At least three distinct forms of the A protein with apparent molecular weights of 85,000, 88,000, and 100,000 can be identified in extracts of cells infected by wild-type virus. After exposure of cells to Nonidet P-40, the 85,000- and 88,000-dalton proteins were found in varying amounts in extracts of permissive cells but not in extracts of transformed cells. This finding raised the question of the possible functional importance of the smaller proteins in productive infection. However, the virtual absence of the 85,000- and 88,000-dalton proteins in some extracts of the fully permissive CV-1 cell line indicates that a conversion of the larger to the smaller forms of the A protein is not required in significant quantity for productive infection. Furthermore, a study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.
Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis
Authors: Singer I I , Rhode S L
Published: January 2020

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).
Patterns of transcription in bacteriophage P22-infected Salmonella typhimurium
Authors: Pipas J M , Reeves R H
Published: January 2020

Two peaks of RNA synthesis (early and late) are directed by bacteriophage P22 in lytic infections of Salmonella typhimurium. Late RNA synthesis is not seen in P22 23- infections; neither early nor late RNA synthesis occurs in P22 24- infections. Genes 23 and 24 of P22 appear to be analogous to genes Q and N of lambda, respectively.
Cell-free translation of RNA synthesized in vitro by a transcribing nucleoprotein complex prepared from purified vesicular stomatitis virus
Authors: Preston C M , Szilagyi J F
Published: January 2020

The RNA species synthesized in vitro by a transcribing nucleoprotein (TNP) complex of vesicular stomatitis virus (VSV) were translated with high efficiency in a fractionated cell-free system derived from reticulocytes. The use of TNP complexes isolated from VSV Indiana, VSV New Jersey, and Chandipura viruses showed that in each case the predominant polypeptides synthesized had electrophoretic mobilities identical to their virion N, NS, and M polypeptides in proportions reflecting those found in infected cells rather than purified virions. A minor polypeptide corresponding to unglycosylated polypeptide G was also observed, but the in vitro synthesis of polypeptide L was not detected. The addition of RNase inhibitor to transcription mixtures markedly increased the rate of RNA synthesis. Furthermore, the messenger activity of the RNA was significantly enhanced. The inclusion of S-adenosyl L-methionine during transcription substantially increased the messenger activity of the product RNA, suggesting a requirement for methylation. Fractionation by oligodeoxythymidylic acid-cellulose chromatography revealed that the RNA required a polyadnylic acid tract for messenger activity.
Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses
Authors: Deppert W , Walter G , Linke H
Published: January 2020

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.
Identification of the defective genes in three mutant groups of influenza virus
Authors: Ritchey M B , Palese P
Published: January 2020

Seven complementation-recombination groups of temperature-sensitive (ts) influenza WSN virus mutants have been previously isolated. Recently two of these groups (IV and VI) were shown to possess defects in the neuraminidase and the hemagglutinin gene, respectively, and two groups (I and III) were reported to have defects in the P3 and P1 proteins which are required for complementary RNA synthesis. In this communication we report on the defects in the remaining three mutant groups. Wild-type (ts+) recombinants derived from ts mutants and different non-ts influenza viruses were analyzed on RNA polyacrylamide gels. This technique permitted the identification of the P2 protein, the nucleoprotein, and the M protein as the defective gene products in mutant groups II, V, and VII, respectively. Based on the physiological behavior of mutants in groups II and V, it appears that P2 protein and nucleoprotein are required for virion RNA synthesis during influenza virus replication.
Temperature-sensitive defect of vesicular stomatitis virus in complementation group II
Authors: Combard A , Printz-Ane C , Martinet C , Printz P
Published: January 2020

The prototype member of the complementation group II temperature-sensitive (ts) mutants of vesicular stomatitis virus, ts II 052, has been investigated. In ts II 052-infected HeLa cells at the restrictive temperature (39.5 degrees C), reduced viral RNA synthesis was observed by comparison with infections conducted at the permissive temperature (30 degrees C). It was found that for an infection conducted at 39.5 degrees C, no 38S RNA or intracytoplasmic nucleocapsids were present. For nucleocapsids isolated from ts II 052 purified virions or from ts II 052-infected cells at 30 degrees C, the RNA was sensitive to pancreatic RNase after an exposure at 39.5 degrees C in contrast to the resistance observed for wild-type virus. The nucleocapsid stability of wild-type virus when heated to 63 degrees C or submitted to varying pH was not found in nucleocapsids extracted from ts II 052 purified virions. The data suggest that for ts II 052 there is an altered relationship between the viral 38S RNA and the nucleocapsid protein(s) by comparison with wild-type virus. Such results argue for the complementation group II gene product being N protein, so that the ts defect in ts II 052 represents an altered N protein.
Lipids of rabies virus and BHK-21 cell membranes
Authors: Blough H A , Tiffany J M , Aaslestad H G
Published: January 2020

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.
Receptor interaction between eastern equine encephalitis virus and chicken embryo fibroblasts
Authors: Marker S C , Connelly D , Jahrling P B
Published: January 2020

The attachment of eastern equine encephalitis virus to chicken embryo fibroblasts was studied at 0 degrees C. The binding specifically responsible for initiating infection was studied in the initial experiments by employing plaque-forming ability as the measured response. Results from these initial studies were closely paralleled in studies of binding of radiolabeled virus under the same conditions. Binding that had occurred at the pH optimum, pH 6.5, could be reversed only at higher pH. The observed pH dependence of virus attachment suggested the interaction of at least two ionizable species in the initial binding of virus to cell, and that one to three attachments must occur between virus and cell prior to infection.
New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA
Authors: Darlix J L , Bromley P A , Spahr P F
Published: January 2020

Based on the observation that in vitro transcription of Rous sarcoma virus (RSV) RNA by avian myeloblastosis virus DNA polymerase renders the RNA PROGRESSIVELY MORE SENSITIVE TO Escherichia coli RNase H digestion, a new procedure for the in situ analysis of this process has been developed. In vitro transcription products of 32P-labeled RSV RNA are first treated with RNase H, the resistant fraction is then digested to completion with RNase T1, and the oligonucleotides are analyzed by a fingerprint technique. By using the established order of these oligonucleotides along the RNA molecule, a comparison of the yields of each oligonucleotide, before and after transcription, allows qualitative and quantitative in situ analyses of the transcription process. Using this new procedure, we find that upon transcription of purified RSV RNA, DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA molecule, and that most of these RNA molecules are competent templates for limited transcription at these specific sites. We also show that purified RSV 70S RNA contains a low-molecular-weight DNA hybridized to a nucleotide sequence near the center of the subunit molecule. Furthermore , we find that the low-molecular-weight nucleic acid fraction extracted from purified RSV virions contains DNA that can hybridize to RSV 70S RNA and that the virion DNA in such hybrids can function as a primer for an extensive in vitro reverse transcription.
RNA-dependent DNA polymerase associated with equine infectious anemia virus
Authors: Archer B G , Crawford T B , McGuire T C , Frazier M E
Published: January 2020

Equine infectious anemia (EIAV) is shown to have an associated RNA-instructed DNA polymerase similar in its cofactor requirements and reaction conditions to the RNA tumor virus DNA polymerases. Demonstrating this DNA polymerase activity requires a critical concentration of a nonionic detergent, all four deoxyribonucleoside triphosphates, and a divalent metal ion. The reaction is sensitive to RNase, and a substantial fraction of the FNA synthesized is complementary to viral RNA. The detection of a complex of tritium-labeled polymerase product DNA-template RNA, which sedimented at 60S to 70S, provided evidence that EIAV contains high-molecular-weight RNA. These results, obtained with both virus propagated in cell culture and virus from the serum of an experimentally infected horse, indicate that EIAV may properly be considered a member of the family Retroviridae. They may also be pertinent to the mechanism(s) of viral persistence and periodic recrudescence of disease in chronically infected horses.
Inhibition of viral RNA methylation in herpes simplex virus type 1-infected cells by 5' S-isobutyl-adenosine
Authors: Jacquemont B , Huppert J
Published: January 2020

5' S-isobutyl-adenosine (SIBA), a structural analogue of S-adenosylhomocysteine, reversibly blocks the multiplication of herpes simplex type 1 virus. In the presence of SIBA, viral protein synthesis is inhibited. After removing SIBA the synthesis of proteins starts rapidly again. The new polypeptides are mainly alpha proteins (Honess and Roizman, J. Virol. 14:8-19, 1974,), normally the first to be synthesized after infection. The rapid synthesis of proteins after release of inhibition seems to be directed by mRNA formed in the presence of SIBA as indicated by experiments using actinomycin D but which was undermethylated as shown by analysis of methyl groups on RNA. SIBA inhibits the methylation of mRNA and especially that of the 5' cap. Capping of mRNA thus seems to be essential for efficient translation. The analogue affected various methylations to different extents.
Bacteriophage conversion of spore-negative mutants to spore-positive in Bacillus pumilus
Authors: Bramucci M G , Keggins K M , Lovett P S
Published: January 2020

A pseudolysogenic phage, PMB1, was isolated from soil on the basis of its ability to increase the sporulation frequency of the oligosporogenic Bacillus pumilus strain NRS 576 (sporulation frequency, less than 1%). Several spore-negative mutants (sporulation frequency, less than 10-8) derived from strain NRS 576, which were converted to spore positive by infection with PMB1, were subsequently identified. PMB1 repeatedly grown on a given spore-negative mutant (e.g., GW2) converted GW2 cells to spore positive. Each plaque-forming unit initiated the conversion of a spore-positive clone in semisolid agar overlays. GW2 cells remained spore positive as long as they maintained PMB1. Return of PMB1-converted cells to the orginal spore-negative phenotype correlated with loss of PMB1. In liquid media, PMB1 infection increased the sporulation frequency of mutant GW2 over 106-fold. More than half of the spore-negative mutants we isolated from strain NRS 576 were converted to spore positive by PMB1 infection. PMB1-induced spores of the spore-negative mutant GW2 were somewhat more heat sensitive than uninfected or PMB1-infected spores of the spore positive parent of GW2. PMB1-induced spores of GW2 do not differ from wild-type spores in morphology by phase-contrast microscopy, dipicolinic acid content, or rate of sedimentation through Renografin gradients.
Purification of hepatitis A virus from chimpanzee stools
Authors: Bradley D W , Hornbeck C L , Cook E H , Maynard J E
Published: January 2020

Hepatitis A virus antigen was purified from early acute-phase chimpanzee stools by a rapid three-step procedure using 7% polyethylene glycol precipitation, CsCl banding, and Sepharose 2B column chromatography. Electron microscopic examination of the hepatitis A virus entigen preparation revealed highly purified hepatitis A virus particles.
Effects of x irradiation on a temperate bacteriophage of Haemophilus influenzae
Authors: Boling M E , Randolph M L
Published: January 2020

The inactivation of bacteriophage HP1c1 by X rays in a complex medium was found to be exponential, with a D0 (the X-ray exposure necessary to reduce the survival of the phage to 37%) of approximately 90 kR. Analysis of results of sucrose sedimentation of DNA from X-irradiated whole phage showed that the D0 for intactness of single strands was about 105kR, and for intactness of double strands, it was much higher. The D0 for attachment of X-irradiated phage to the host was roughly estimated as about 1,100 kR. Loss of DNA from the phage occurred and was probably due to lysis of the phage by X irradiation, but the significance of the damage is not clear. The production of single-strand breaks approaches the rate of survival loss after X irradiation. However, single-strand breaks produced by UV irradiation, in the presence of H2O2, equivalent to 215 kR of X rays, showed no lethal effect on the phage. Although UV-sensitive mutants of the host cell, Haemophilus influenzae, have been shown to reactivate UV-irradiated phage less than does the wild-type host cell, X-irradiated phage survive equally well on the mutants as on the wild type, a fact suggesting that other repair systems are involved in X-ray repair.