bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli capsule bacteriophages. III. Fragments of bacteriophage 29
Authors: Rieger D , Freund-Mölbert E , Stirm S
Published: January 2020

A glycanase activity, catalyzing the depolymerization of host capsular polysaccharide, is associated with Escherichia coli capsule bacteriophage no. 29, a small virus with an isometric head, carrying a base plate with a set of spikes. The bacteriophage particles were disrupted by mild acid treatment (5 to 8 min at pH 3.5 and 37 C), and the enzymatically active fragments were isolated and subjected to sodium dodecyl sulfate-gel electrophoresis as well as to electron microscopy. Of the at least nine different polypeptide chains found in the complete virion, three (of 57,000 plus or minus 3,000, 29,500 plus or minus 2,000 and 13,500 plus or minus 1,000 daltons) were detected in detached base plates. They had the appearance of six-pointed stars of about 14 nm in outer diameter, with a central hole or prop, carrying six (or, possibly, a multiple thereof) spikes. Two sizes of polypeptide chains (57,000 and 29,500) were found in pure spikes, cylindrical particles of about 14.5 to 15 nm in length and 5 nm in diameter, and one (57,000) in -- still capsule depolymerizing -- spike subunits of roughly 5 nm in diameter. Phage 29 spike preparations, homogeneous in analytical ultracentrifugation and immunoelectrophoresis, were found to have a molecular weight of 245,000, as determined from the sedimentation equilibrium, and to contain equimolar amounts of the two polypeptides, probably three copies of each per organelle. The amino acid analysis of the isolated spikes revealed that aspartic acid, alanine, serine, and glycine are their dominant constituents; no amino sugars or other carbohydrates were detected in the preparations.
Heat-stable variant of human adenovirus type 5: characterization and use in three-factor crosses
Authors: Young C S
Published: January 2020

A variant of human adenovirus type 5 which is heat stable (hs) in vitro has been isolated following three rounds of heat inactivation at 52 C. The variant is genetically stable, both through vegetative viral passage and through recombination into other genetic backgrounds, which suggests that it arises from a single mutation. Three-factor crosses, using this mutant in conjunction with previously described temperature-sensitive mutants, suggest the hs mutation lies near the left-hand end of the genetic map. The mutant has been used to demonstrate the production of reciprocal recombinants in two-factor crosses. The mutational lesion is unknown, but phenotypic mixing occurs in hs times hs-+ infections, which suggests that it lies in a gene specifying a viri on structural protein. Other biological parameters examined have shown no differences from the wild-type hs-+.
Analysis of early adenovirus 2 RNA using Eco R-R1 viral DNA fragments
Authors: Craig E A
Published: January 2020

Adenovirus 2 RNA synthesized early in productive infection was analyzed by RNA-DNA hybridization. Hybridization experiments were performed with adenovirus 2 DNA and wit, the six adenovirus 2 DNA fragments generated 0y digestion with the restriction endonuclease Eco R.R1. Duplex formation between RNA and -32P-labeled viral DNA was assayed by S(1) nuclease digestion. RNA from the cytoplasm annealed 12 percent of the total viral DNA and the following percentage of each of the R.R1 fragments: 6 percent of R1-A, 24 percent of R1-B, 0 percent of R1-F, 40 percent of R1-D, 13 percent of R1-E, and 22 percent of R1-C. The early cytoplasmic RNA is composed of two sequence classes: class I, present in greatly reduced quantities at late times in infection (18 h), and class II, which remains at high concentrations at 18 h. In hybridization-inhibition experiments, hybridization of class II RNA is inhibited by late cytoplasmic RNA, whereas hybridization of class I RNA is not blocked by late cytoplasmic RNA (J. J. Lucas and H. S. Ginsberg, 1971; E.A. Craig and H. J. Raskas, 1971). To determine the location of class I and II sequences on the genome, membrane bound DNA fragments were used in hybridization-inhibition experiments. These studies demonstrated that the early cytoplasmic transcripts of R1-D belong to class II, whereas R1-C transcripts are class I sequences. The cytoplasmic RNAs transcribed from fragments A and B contain both class I and class II sequences. Analysis of cytoplasmic RNA fractionated by size demonstrated that the class I sequences include a 19 S RNA transcribed from R1-B and class II sequences include a 20S RNA derived from R1-D. Nuclear RNA purified from cultures early in infection was annealed with -32P-labeled R1 fragments. With all six fragments the nuclear RNA annealed as much or more of the DNA than did cytoplasmic RNA. Eco R1-F annealed at least 25 percent with early nuclear RNA, whereas no sequences homologous to R1-F were detected in early cytoplasmic RNA. When cultures were labeled from 2 to 6 h after infection, at least 5 percent of the -3H-labeled early nuclear viral RNA annealed to Eco R1-F. Some of these nuclear transcripts from R1-F appear to be covalently linked to sequences transcribed from a contiguous region of the genome (Eco R1-B). 8.4 percent of the RNA selected by hybridization of R1-F reannealed to R1-B, whereas no more than 1.5 percent reannealed to R1 fragments A, D, E, or C.
Antibody-induced capping of measles virus antigens on plasma membrane studied by electron microscopy
Authors: Lampert P W
Published: January 2020

Antibodies specific for measles virus could redistribute ("cap") virus antigens on infected HeLa cells as shown by transmission and scanning electron microscopy. Using an indirect immunoperoxidase technique, infected cells showed diffuse, circumferential distribution of virus antigens over the cell surface when mixed with antibody at 4 C. At 37 C, virus-coated microvilli concentrated on one pole of the cell, leaving the remainder of the plasma membrane devoid of both viral antigens and microvillus projections. Whereas extreme polar displacement of virus-antibody complexes frequently occurred, endocytosis was rarely seen. The findings indicate that antiviral antibodies can move and cluster virus on plasma membranes and suggest that virus-antibody complexes are stripped and shed from the cell surface.
Post-transcriptional restriction of human adenovirus expression in monkey cells
Authors: Eron L
Published: January 2020

Infection of the continuous simian cell lines CV-1 and BSC-1 with human adenovirus type 2 (Ad2) is abortive. However, the restriction of Ad2 reproduction in these cells can be overcome by increasing the Ad2 infectious dose or by coinfection with simian virus 40. Vero, another established simian cell line free of detectable endogenous simian virus 40 DNA, is not restricted in its ability to promote Ad2 growth even at low input multiplicities of Ad2 and in the absence of SV40 helper. The amount of structural Ad2 proteins in total cell extracts of enhanced BSC-1 cells is at least two orders of magnitude higher than that of unenhanced cells. In contrast, comparable quantities of Ad2 mRNA specifying these proteins are found in both the enhanced and the unenhanced cell. Both sets of mRNA can be translated in a cell-free system with equal efficiency.
Structure of the DNA of the Adenovirus 7-Simian Virus 40 Hybrid, E46
+
, by Electron Microscopy
Authors: Kelly Thomas J.
Published: January 2020

The DNA molecule of the adenovirus 7-simian virus (SV) 40 hybrid, E46
+
, contains a single substitution of SV40 DNA located about 0.05 adenovirus 7 lengths from one end (arbitrarily designated the left end). The left to right direction in the SV40 DNA segment of the hybrid corresponds to the clockwise direction in the SV40 physical map. The left end point of the segment maps at about SV40 map position 0.50 ± 0.01 and the right end point at about SV40 map position 0.66 ± 0.01. The region between SV40 map positions 0.71 and 0.11 (clockwise) is deleted. Thus, the region between SV40 map position 0.50 and 0.66 (clockwise) is repeated in tandem.
Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis
Authors: Reeve J N , Cornett J B
Published: January 2020

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.
Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus
Authors: Emerson S U , Yu Y
Published: January 2020

Vesicular stomatitis virions, Indiana serotype, were solubilized with high salt solubilizer and separated by ultracentrifugation into a supernatant fraction containing L, G, NS, and M proteins and pellet fraction containing the RNA complexed with N protein. NS protein was purified from the supernatnat fluid by sequential chromatography on phosphocellulose and diethylaminoethyl cellulose columns. The purified NS protein was assayed in a standard transcription system in combination with purified L protein and purified template (pellet fraction) prepared by renografin or CsCl banding. Results of the polymerase assays indicate that both L and NS proteins are required to reconstitute transcription activity with a highly purified template composed of only RNA and N protein. The NS protein polymerase activity is destroyed by trypsin but withstands 90 C temperatures for 10 min. Cytoplasmic NS protein can substitute for virion NS protein in the in vitro transcription assay.
Inhibition of viral transcriptase by immunoglobulin directed against the nucleocapsid NS protein of vesicular stomatitis virus
Authors: Imblum R L , Wagner R R
Published: January 2020

In search of an anti-transcriptase, antibody was raised in rabbits to partially purified, soluble NS protein present in cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis (VSInd) virus. This antiserum gave specific reactions of identity by agar immunodiffusion with both cytoplasmic and virion NS protein. NS antiserum also preferentially precipitated NS 3-H-labeled protein from infected cytoplasmic extracts, whereas anti-whole VSInd virion serum also precipitated N 3-H-labeled protein from extracts both of infected cytoplasm and virion nucleocapsids. Transcriptase activity of VSInd cytoplasmic or virion-derived nucleocapsids was effectively inhibited by ribonuclease-free immunoglubulin prepared from homologous NSInd antiserum or from anti-whole vesicular stomatitis virus serum. Transcriptase activity of heterologous New Jersey serotype (VSNJ) nucleocapsids and virions was not appreciably affected by anti-NSInd or by anti-whole VSInd virion gamma globulin. Anti-NS gamma glubulin immediately switched off RNA synthesis by actively transcribing VSInd nucleocapsids, a finding which suggests that NS antibody inhibits RNA chain elongation.
Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization
Authors: Hayward W S , Hanafusa H
Published: January 2020

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.
Nucleic acid homology of murine xenotropic type C viruses
Authors: Callahan R , Lieber M M , Todaro G J
Published: January 2020

Two major subclasses of xenotropic (X-tropic) murine type C viruses can be distinguished by nucleic acid hybridization. The most frequently encountered subclass (MuLV-X-alpha) includes isolates from BALB/c, C57BL/6J, C58/J, AKR/J, CBA/J, and DBA/2J inbred strains and from the Asian feral mouse subspecies Mus musculus molossinus. The other subclass (MuLV-X-beta) consists of viruses isolated from the NIH Swiss and NZB/BINJ strains. Thus, significant polymorphism exists among the endogenous type C virogenes of a single species, Mus musculus. MuLV-X-alpha genes are found in strains that also have endogenous mouse-tropic viruses (either N-tropic, B-tropic, or both), whereas the MuLV-X-beta subclass is restricted to mouse strains from which mouse-tropic viruses have not yet been isolated. The results are consistent with a model which proposes that mouse-tropic endogenous viruses are derived from the MuLV-X-alpha subclass.
Sindbis virus glycoproteins form a regular icosahedral surface lattice
Authors: von Bonsdorff C H , Harrison S C
Published: January 2020

Electron micrographs of negatively stained Sindbis virus particles show that the glycoproteins are organized with trimer clustering in a T = 4 icosahedral surface lattice.
Effect of DNA delay mutations of bacteriophage T4 on genetic recombination
Authors: Leung D , Behme M T , Ebisuzaki K
Published: January 2020

Studies have been made of the effect of the DNA delay mutations of bacteriophage T4 on growth and genetic recombination in a number of Escherichia coli hosts. DNA delay mutations in genes 39, 52, 58 (61), and 60 result in abnormally high recombination frequencies. These high recombination frequencies are discussed in the context of other observations.
Effect of UV irradiation on transduction by coliphage T1
Authors: Drexler H , Kylberg K J
Published: January 2020

Lysates of the virulent coliphage T1 transduce seven markers between strains of Escherichia coli with reproducilble efficiencies which range from 10(-7) to 10(-5). The ability of a UV-irradiated lysate to transduce Arg(+), Str(R), Trp(+), Lac(+), and Pro(+) is 90% and Bio(+) is 99% inactivated by doses which inactivate plaque formation of T1 by six orders of magnitude. A dose of irradiation which causes a 1- to 2-log drop in the titer of T1 stimulated Gal(+) transduction by two- to three-fold; no other marker tested was stimulated. Irradiation causes dislinkage of some co-transducible markers but not others.
Structure, subunit composition, and molecular weight of RD-114 RNA
Authors: Kung H J , Bailey J M , Davidson N , Nicolson M O , McAllister R M
Published: January 2020

The properties and subunit composition of the RNA extracted from RD-114 virions have been studied. The RNA extracted from the virion has a sedimentation coefficient of 52S in a nondenaturing aqueous electrolyte. The estimated molecular weight by sedimentation in nondenaturing and weakly denaturing media is in the range 5.7 X 10(6) to 7.0 X 10(6). By electron microscopy, under moderately denaturing conditions, the 52S molecule is seen to be an extended single strand with a contour length of about 4.0 mum corresponding to a molecular weight of 5.74 X 10(6). It contains two characteristic secondary structure features: (i) a central Y- or T-shaped structure (the rabbit ears) with a molecular weight of 0.3 X 10(6), (ii) two symmetreically disposed loops on each side of and at equal distance from the center. The 52S molecule consists of two half-size molecules, with molecular weight 2.8 X 10(6), joined together within the central rabbit ears feature. Melting of the rabbit ears with concomitant dissociation of the 52S molecule into subunits, has been caused by either one of two strongly denaturing treatments: incubation in a mixture of CH3HgOH and glyoxal at room temperature, or thermal dissociation in a urea-formamide solvent. When half-size molecules are quenched from denaturing temperatures, a new off-center secondary structure feature termed the branch-like structure is seen. The dissociation behavior of the 52S complex and the molecular weight of the subunits have been confirmed by gel electrophoresis studies. The loop structures melt at fairly low temperatures; the dissociation of the 52S molecule into its two subunits occurs at a higher temperature corresponding to a base composition of about 63% guanosine plus cytosine. Polyadenylic acid mapping by electron microscopy shows that the 52S molecule contains two polyadenylic acid segments, one at each end. It thus appears that 52S RD-114 RNA consists of two 2.8 X 10(6) dalton subunits, each with a characteristic secondary structure loop, and joined at the 5' ends to form the rabbit ears secondary structure feature. The observations are consistent with but do not require the conclusion that the two 2.8 X 10(6) dalton subunits of 52S RD-114 RNA are identical.
Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA
Authors: Brinton-Darnell M , Plagemann P G
Published: January 2020

Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.
Escherichia coli capsule bacteriophages. VII. Bacteriophage 29-host capsular polysaccharide interactions
Authors: Fehmel F , Feige U , Niemann H , Stirm S
Published: January 2020

Different interactions between particles of Escherichia coli capsule bacteriophage 29 and its receptor, the E. coli serotype 29 capsular polysaccharide have been studied. The inactivation of phage 29 (8 x 10(3) PFU/ml) by isolated host capsular glycan was found to be physiologically insignificant (50% inactivation dose equals 100 mug after 1 h at 37 C). No adsorption (less than 2 x 10(4) PFU/mug) of the viruses to K29 polysaccharide-coated erythroyctes (at 0 or 37 C) was observed either. The phage particles were, however, found to catalyze the hydrolysis of beta-D-glucosido-(1leads to 3)-D-glucuronic acid bonds (arrow) in the receptor polymer, leading, ultimately, to the formation of a mixture of K29 hexasaccharide (one repeating unit), dodecasaccharide, and octadecasaccharide: (see article). Testing derivatives of K29 polysaccharide, as well as 82 heterologous bacterial (mainly Enteriobactericeae) capsular glycans, the viral glycanase was found to be highly specific; in accordance with the host range of phage 29, only one enzymatic cross-reaction (with the Klebsiella K31 polysaccharide) was observed. These and previous results, as well as the electron optical findings of M. E. Bayer and H. Thurow (submitted for publication), are discussed in terms of a unifying mechanism of phage 29-host capsule interaction. We propose that the viruses penetrate the capsules by means of their spike-associated glycanase activity, which leads them along capsular polysaccharide strands to membrane-cell wall adhesions where ejection of the viral genomes occurs.
Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells
Authors: Flint S J , Wewerka-Lutz Y , Levine A S , Sambrook J , Sharp P A
Published: January 2020

The genomes of the two nondefective adenovirus 2/simian virus 40 (Ad2/SV 40) hybrid viruses, nondefective Ad2/SV 40 hybrid virus 1 (Ad2+ND1) and nondefective hybrid virus 3 (Ad2+ND3), WERE FORMED BY A DELETION OF ABOUT 5% OF Ad2 DNA and insertion of part of the SV40 genome. We have compared the cytoplasmic RNA synthesized during both the early and late stages of lytic infection of human cells by these hybrid viruses to that expressed in Ad2-infected and SV40-infected cells. Separated strands of the six fragments of 32P-labeled Ad2 DNA produced by cleavage with the restriction endonuclease EcoRI (isolated from Escherichia coli) and the four fragments of 32P-labeled SV40 DNA produced by cleavage with both a restriction nuclease isolated from Haemophilus parainfluenzae, Hpa1, and EcoRI were prepared by electrophoresis of denatured DNA in agarose gels. The fraction of each fragment strand expressed as cytoplasmic RNA was determined by annealing fragmented 32P-labeled strands to an excess of cellular RNA extracted from infected cells. The segment of Ad2 DNA deleted from both hybrid virus genomes is transcribed into cytoplasmic mRNA during the early phase of Ad2 infection. Hence, we suggest that Ad2 codes for at least one "early" gene product which is nonessential for virus growth in cell culture. In both early Ad2+ND1 and Ad2+ND3-infected cells, 1,000 bases of Ad2 DNA adjacent to the integrated SV40 sequences are expressed as cytoplasmic RNA but are not similarly expressed in early Ad2-infected cells. The 3' termini of this early hybrid virus RNA maps in the vicinity of 0.18 on the conventional SV40 map and probably terminates at the same position as early lytic SV40 cytoplasmic RNA. Therefore, the base sequence in this region of SV40 DNA specifies the 3' termini of early messenger RNA present in both hybrid virus and SV40-infected cells.
Study of the fine structure of adeno-associated virus DNA with bacterial restriction endonucleases
Authors: Berns K I , Kort J , Fife K H , Grogan E W , Spear I
Published: January 2020

A physical map of the adeno-associated virus type 2 genome has been constructed on the basis of the five fragments produced by the restriction endonucleases HindII + III from Hemophilus influenzae. There are three endo R-HindII cleavage sites and one endo R-HindIII site. Evidence has been obtained to support the existence of two nucleotide sequence permutations in adeno-associated virus DNA, the start points of which have been estimated to be separated by 1% of the genome. The three cleavage fragments produced by endo R-Eco RI have been ordered and oriented with respect to the endo R-HindII + III cleavage map.
Mapping simian virus 40 mutants by construction of partial heterozygotes
Authors: Mantel N , Boyer H W , Goodman H M
Published: January 2020

Simian virus 40 temperature-sensitive mutants ts A28, A30, B1, B11, and D101 are associated with the region of the genome defined by the restriction endonuclease fragments Hind-I, H, F, G, and E, respectively.
Dengue virus-induced modifications of host cell membranes
Authors: Stohlman S A , Wisseman C L , Eylar O R , Silverman D J
Published: January 2020

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.
Purification and properties of spleen necrosis virus DNA polymerase
Authors: Mizutani A , Temin H M
Published: January 2020

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.
Size of virus-specific RNA in B-34, a hamster tumor cell producing nucleic acids of type C viruses from three species
Authors: Tsuchida N , Gilden R V , Hatanaka M
Published: January 2020

B-34 is the designation of a hamster tumor-derived cell line induced by the Harvey sarcoma virus. This cell line produces virions which contain structural proteins common to edogenous hamster viruses and nucleic acid sequences of hamster, mouse, and rat origin. The sedimentation characteristics of the intracellular virus-specific RNA was determined in sucrose gradients after treatment with dimethylsulfoxide by molecular hybridization using complementary DNA of strict virus specificity. Hamster virus-specific RNA sedimented at 35S (major peak) as is characteristic of productive infection by type C leukemia viruses of other species. Rat virus-specific RNA sedimented at 30S which is characteristic of the sarcoma virus-related genome found in nonproducer cells transformed by Kirsten sarcoma virus. Both Harvey and Kirsten sarcoma viruses contain a related but not necessarily identical 30S rat-specific component which is also found in normal cultured rat cells. Mouse cells producing Harvey sarcoma virus also contain a rat-specific 30S RNA. Mouse virus-derived sequences also sedimented at 30S in B-34 cells and in a similar size range in Harvey virus-infected mouse cells. The possibility that the mouse and rat-derived sequences are present on a single 30S RNA species which would then be related to sarcomagenic potential is one attractive hypothesis suggested by these data.
Variants of N-tropic leukemia virus derived from BALB/c mice
Authors: Hopkins N , Jolicoeur P
Published: January 2020

Clonal lines derived from cultures of NIH/3T3 cells infected with N-tropic leukemia virus from BALB/c mice differ in the amount and type of N-tropic virus they produce. Three biologically distinguishable N-tropic viruses were found: the large XC plaque-forming virus of hartley et al. (1969) (LP-N), A SMALL XC plaque-forming virus (sp-n), and a non-plaque-forming virus (NP-N). SP-N and NP-N are less infectious than LP-N. Upon prolonged passage in NIH/3T3 cells NP-N gives rise to highly infectious LP-N.
DNA polymerases in polyoma virus-infected mouse kidney cells
Authors: Wintersberger U , Wintersberger E
Published: January 2020

Infection of arrested mouse kidney cells by polyoma virus results in the induction of the cellular 6-8S DNA polymerase activity. Levels of this enzyme increase two- to threefold in the cytoplasm but seven- to tenfold in nuclei and nuclear extract, suggesting an accumulation of the enzyme in the nucleus. Experiments using the inhibitor of DNA synthesis, fluordeoxyuridine, indicate that this accumulation is linked to active DNA synthesis. The activity and cellular distribution of the small 3.4S DNA polymerase remains unchanged.
Differential expression of helper viral structural polypeptides in cells transformed by clonal isolates of woolly monkey sarcoma virus
Authors: Aaronson S A , Stephenson J R , Hino S , Tronick S R
Published: January 2020

Cell lines transformed by woolly monkey sarcoma virus (WSV) in the absence of infectious virus production were analyzed for the expression of woolly monkey helper viral p30, p12, and gp70 antigens. Several lines produced high levels of both p30 and p12, whereas gp70 was not detectable. One transformed clone expressed only p12, and in another cell line, none of the helper viral antigens were detected. The properties of each sarcoma virus bred true upon transmission, indicating that each variant represents a distinct genotype. The different cell lines were examined with respect to properties characteristic of the transformed state. The in vitro growth properties and oncogenicity of each WSV-transformed clone were indistinguishable, indicating that transformation by WSV occurs independently of the expression of at least three helper viral polypeptides.
Qualitative Complementation Test for Temperature-Sensitive Mutants of Herpes Simplex Virus
Authors: Chu Chien-Ts , Schaffer Priscilla A.
Published: January 2020

A simple, rapid, qualitative method for classifying temperature-sensitive mutants of herpes simplex virus into functional complementation groups has been developed. The positive reaction observed in this test reflects the ability of mutant pairs to interact by both complementation and recombination.
Multiplication of Coxsackie B
1
Virus in Synchronized HeLa Cells
Authors: Suarez Monica , Contreras Guillermo , Fridlender Bertold
Published: January 2020

A higher yield of Coxsackie B
1
virus was obtained when HeLa cells were infected late during S phase as compared to the amount produced by random cultures.
Purification of influenza viral complementary RNA: its genetic content and activity in wheat germ cell-free extracts
Authors: Etkind P R , Krug R M
Published: January 2020

Influenza viral complementary RNA (cRNA) was purified free from any detectable virion-type RNA (vRNA), and its genetic content and activity in wheat germ cell-free extracts were examined. After phenol-chloroform extraction of cytoplasmic fractions from infected cells, poly(A)-containing viral cRNA is found in two forms: in single-stranded RNA and associated with vRNA in partially and fully double-stranded RNA. To purify single-stranded cRNA free of these double-stranded forms, it was necessary to employ, as starting material, RNA fractions in which cRNA was predominantly single stranded. Two RNA fractions were successfully employed as starting material: polyribosomal RNA and the total cytoplasmic RNA from infected cells treated with 100 mug of cycloheximide (CM) per ml at 3 h after infection. In WSN virus-infected canine kidney (MDCK) cells, the addition of CM at 3 h after infection stimulates the production of cRNA threefold and causes a very large increase in the proportion of the cytoplasmic cRNA which is single stranded; double-stranded RNA forms are greatly reduced in amount. Total cRNA was obtained by oligo(dT)-cellulose chromatography, and single-stranded cRNA was separated from double-stranded forms by Sepharose 4B chromatography. The cRNA preparation purified from polyribosomes consists of 95% single-stranded cRNA, with the remaining 5% apparently being double-stranded RNA forms. The cRNA preparation purified from CM-treated cells (CM cRNA) is even more pure: 100% of the radiolabeled RNA is single-stranded cRNA. Annealing experiments, in which a limited amount of 32P-labeled genome RNA was annealed to the cRNA, indicate that the purified cRNA contains at least 84 to 90% of the genetic information in the vRNA genome. Purified viral cRNA (CM cRNA) is very active in directing the synthesis of virus-specific proteins in wheat germ cell-free extracts.
Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid
Authors: Huang E S
Published: January 2020

Phosphonoacetic acid specifically inhibited human cytomegalovirus DNA synthesis in virus-infected human fibroblasts as detected by virus-specific nucleic acid hybridization. Inhibition was reversible; viral DNA synthesis resumed upon the removal of the drug. The compound partially inhibited DNA synthesis of host cells in the log phase of growth but had little effect on confluent cells. Studies of partially purified enzymes indicated that phosphonoacetic acid specifically inhibited virus-induced DNA polymerase and had only a slight effect on normal host cell enzymes. The drug was shown to interact directly with virus-induced enzyme but not with the template-primers.
Chemical stability of bacteriophage T7 early mRNA
Authors: Yamada Y , Whitaker P A , Nakada D
Published: January 2020

T7 early mRNA produced by a gene 1 amber mutant phage, T7 am27, is chemically stable interms of acid insolubility and T7 DNA hybridizability. However, the messenger activity of individual T7 early mRNA species, transcripts of gene 1, gene 0.7, and gene 1.3, decay with a half-life of about 6.5 min at 30 C. An extensive secondary structure is present in all T7 early mRNA species and is probably responsible for the chemical stability of the RNAs after the loss of functional activity. It is unlikely that ribosomes protect T7 early mRNA from nucleolytic degradation.
Origin of the Polyoma Virus-Associated Endonuclease
Authors: McMillen Janis , Center Melvin S. , Consigli Richard A.
Published: January 2020

Endonuclease activity can be found associated with highly purified preparations of polyoma virus. Evidence has been obtained that this enzyme is not an integral part of the virus but is contributed by the fetal calf serum used in maintenance of polyoma-infected cells. This finding is based on: (i) the lack of virion-associated endonuclease activity when virus is produced using serum-free media and (ii) the production of polyoma antibody which neutralizes fetal calf serum endonuclease activity.
Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D
Authors: Shah D. B.
Published: January 2020

After infection of
Escherichia coli
B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200
S
replicative intermediate into 63
S
DNA molecules even in the absence of capsid assembly. Although these 63
S
molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.
Strand-Specific Transcription of Polyoma Virus DNA Early in Productive Infection and in Transformed Cells
Authors: Beard Peter , Acheson Nicholas H. , Maxwell Ian H.
Published: January 2020

The DNA strand origin of nuclear and cytoplasmic polyoma-specific RNA in productively infected mouse cells and in a line of polyoma-transformed hamster cells was determined by hybridization of unlabeled RNA with radioactively labeled separated strands of polyoma DNA. Early in the productive cycle (10 h postinfection) nuclear viral RNA is complementary to only about 40% of the E strand of viral DNA. No RNA complementary to the L strand was detected even when the RNA was first self-annealed to enrich for possible minor species. Early cytoplasmic RNA is complementary to the same 40% of the E strand. Thus, only that part of the polyoma genome which codes for early viral messenger RNA appears to be transcribed. Late in infection, nuclear viral RNA is complementary to most or all of the L strand and to at least 60% of the E strand. Late cytoplasmic viral RNA hybridizes to 40 to 45% of the E strand and 50 to 55% of the L strand. The transformed cell nuclear viral RNA is complementary to 60% of the E strand, whereas cytoplasmic RNA is complementary to 40% of the E strand and comprises the same polyoma-specific sequences as are found in RNA early in productive infection. No L strand transcripts could be detected. Thus, in the transformed cells and late in productive infection, viral RNA sequences in the cytoplasm are a specific subset of those in the nucleus.
Orientation of the Complementary Strands of Polyoma Virus DNA with Respect to the DNA Physical Map
Authors: Kamen Robert , Sedat John , Ziff Edward
Published: January 2020

The chemical polarities of the two strands of polyoma virus DNA with respect to the DNA physical map have been determined by hybridization of restriction endonuclease fragments specifically labeled with [
125
I]dCMP at their 3′ termini to asymmetric polyoma complementary RNA (the product of in vitro transcription of viral DNA by
Escherichia coli
RNA polymerase). The orientations of the polyoma-specific stable RNA transcripts present in the cytoplasm of productively infected mouse cells have been deduced from this result: the 5′ ends of the early and late viral transcripts map very near the origin of viral DNA replication.
Growth and genetic stability of the ts-1 mutant of respiratory syncytial virus at restrictive temperatures
Authors: Schnitzer T J , Richardson L S , Chanock R M
Published: January 2020

An in vitro study was performed to define in greater detail those factors which favored the growth of the ts-1 mutant of respiratory syncytial virus under restrictive conditions and the emergence of genetically altered virus with decreased temperature sensitivity. Replication of ts-1 occurred at each of the restrictive temperatures of 37, 38, and 39 C, even through plaque formation was not observed. The level of virus growth under restrictive conditions was inversely related to the incubation temperature and directly related to the multiplicity of infection. These relationships appeared to reflect the effect of restrictive temperature in reducing the quantity of virus produced and released from an infected cell. Under restrictive conditions the production of genetically altered virus which exhibited reduced temperature sensitivity was directly related to the multiplicity of infection and inversely related to temperature. Production of genetically altered virus was not observed under permissive conditions.
Simian virus 40 DNA replication: characterization of gaps in the termination region
Authors: Chen M C , Birkenmeier E , Salzman N P
Published: January 2020

A class of precursor DNA (pDNA) II molecules has been identified as the immediate precursor of simian virus 40 DNA I. A pDNA II molecule contains a strand of newly synthesized DNA with an interruption located in the region where DNA synthesis terminates (4). These pDNA II molecules have been isolated and further characterized. They are converted to covalently closed structures (simian virus 40 DNA I) only when they are treated in vitro with both T4 DNA polymerase and Escherichia coli ligase. After in vitro repair of pDNA II with T4 DNA polymerase and nucleoside triphosphates, approximately 7 mol of alpha-[32P]dATP is incorporated per mol of DNA II. Alkaline sucrose analysis of these gap-filled molecules, after they have been cleaved with Eco RI restriction endonuclease, has demonstrated that gaps are specifically located in the termination region. alpha-[32P]dATP is incorporated equally into the two labeled products that are generated by RI cleavage of these molecules. This indicates the presence of gaps in both the newly synthesized plus the minus strands. Electrophoretic analysis of the gap-filled molecules, after they have been cleaved with endonuclease Hind, has shown that gaps are localized in Hind fragments G and B and to a minor degree in fragment J. pDNA II molecules have the following properties. There is a gap in the newly synthesized linear DNA strand contained in the pDNA II molecule. Nicked pDNA II molecules cannot be detected. The two molecules that arise by segregation contain gaps in both of the complementary strands. Based on the amount of alpha-[32P]dATP incorporated and the rate of exonuclease III digestion of gap-filled molecules, it is estimated that the size of the gaps is between 22 and 73 nucleotides. Models for termination of DNA synthesis are proposed based on these findings.
Immunocytochemical localization of simian virus 40 T antigen with peroxidase-labeled antibody fragments
Authors: Tabuchi K , Lehman J M , Kirsch W M
Published: January 2020

T antigen was localized in simian virus 40 lytically infected and transformed cells by Fab' antibody conjugates at the ultrastructural level. This virus-specific protein appeared in the cytoplasm of permissive cells as early as 3 h after infection. At later time intervals, the T antigen was localized in both the cytoplasm and nucleus and finally (24 h) in the nucleus. These results suggest a synthesis of T antigen on cytoplasmic ribosomes, with subsequent transfer to the nucleus.
Recombinational-type transfer of viral DNA during bacteriophage 2C replication in Bacillus subtilis
Authors: Hoet P , Fraselle G , Cocito C
Published: January 2020

The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 10(6) daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities. Parental DNA, labeled with either [3H]uracil of [32P]phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands. Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close. These data exclude a degradation of the infecting DNA and reutilization of nucleotides. Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage. Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained. Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules. Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments. These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegatative pool, thus mimicking a dispersive transfer of the infecting viral genome. The pieces of transferred parental DNA have an average size of 10 x 10(6) daltons.
Purification and serological characterization of the major envelope glycoprotein from AKR murine leukemia virus and its reactivity with autogenous immune sera from mice
Authors: Ihle J N , Denny T P , Bolognesi D P
Published: January 2020

The major envelope glycoprotein (gp71) from AKR murine leukemia virus (MuLV) was purified and its serological reactivity with heterologous and autogenous immune mouse sera was examined. Homologous and interspecies competition radioimmunoassays using antisera to Rauscher-MulV gp69/71 or Friend-MuLV gp71 or antisera to feline leukemia virus to precipitate 125I-labeled gp71 from various MuLV showed that distinct differences exist between Rauscher- or Friend-MuLV and AKR-MuLV glycoproteins. Characteristically the AKR-MuLV gp71, in contrast to FLV or RLV gp71, does not compete fully in homologous or interspecies radioimmunoassays with iodinated Friend of Rauscher glycoproteins. Purified 125I-labeled AKR-MuLV gp71, in contrast to the Rauscher- or Friend-MuLV glycoproteins, reacts with normal (autogenous immune) mouse sera in direct radioimmune precipitation assays. Competition experiments further demonstrate that this is a predominant immunological reactivity of normal mouse sera which had previously been detected by radioimmune precipitation assay against intact virions.