bluuebunny/crossref_metadata_2025 huggingface dataset:
Origin of Polysaccharide Depolymerase Associated with Bacteriophage Infection
Authors: Bartell Pasquale F. , Orr Thomas E.
Published: January 2020

Analyses, by construction of phage growth curves, indicated that the polysaccharide depolymerase was synthesized by
Pseudomonas aeruginosa
strains B and BI after infection with phage 2. The kinetics of biosynthesis of the depolymerase were found to parallel closely the rate of formation of phage-directed virions, and alterations in the experimental conditions of infection were reflected by alterations in the production of enzyme. Infection with other
Pseudomonas
phages, 84 and 1197, did not result in the synthesis of depolymerase. The enzyme was not detectable in uninfected cultures, and no evidence was obtained for the existence of inhibitors or activators of enzyme activity in extracts of uninfected or infected cells. The results of experiments employing chloramphenicol or an auxotorphic mutant (BI
arg
−
) suggested that protein synthesis de novo was essential for production of the enzyme. Various mutants of phage 2 (
pdp
1
,
pdp
2
), which alter the synthesis of the polysaccharide depolymerase, have been isolated. These experimental results strongly support the role of the phage genome in the synthesis of this enzyme.
Specific Effect of Guanidine in the Programming of Poliovirus Inhibition of Deoxyribonucleic Acid Synthesis
Authors: Powers C. D. , Miller B. A. , Kurtz H. , Ackermann W. W.
Published: January 2020

Inhibition of HeLa cell deoxyribonucleic acid (DNA) synthesis, which occurred by the 4th to 5th hr after infection with poliovirus, could be blocked completely by guanidine only when it was present before the 2nd hr. At the 2nd hr, there was no significant ribonucleic acid (RNA)-replicase activity, and addition of guanidine inhibited all production of virus but allowed 57% of maximal DNA inhibition to develop. Maximum DNA inhibition developed in cells infected for 4 hr in the presence of guanidine when the guanidine was removed for a 10-min interval. RNA-replicase activity was not enzymatically detectable and viral multiplication did not develop in these cells unless the interval without guanidine was extended to 60 min. The interpretation of the data was that the effect of guanidine on viral-induced inhibition of DNA synthesis was distinct and not a consequence of the inhibition of RNA-replicase.
Isolation of Bacteriophage for
Clostridium tetani
Authors: Roseman Diane , Richardson Robert L.
Published: January 2020

The purpose of this report is to note the isolation of a
Clostridium tetani
phage from soil and to specify the procedure used to obtain suitable lawns for plaque formation.
Separation of Replicative Intermediate from Single-stranded Ribonucleic Acid by Sedimentation at Low Ionic Strength
Authors: Fenwick M. L.
Published: January 2020

By sedimentation in 2.5 × 10
−4
m
sodium ethylenediaminetetraacetate at 25 C, replicative intermediate could be separated from single-stranded ribonucleic acid, both viral and ribosomal. All of the label in the replicative intermediate after a brief pulse of titrated uridine was resistant to ribonuclease.
Changes in Nuclear Basic Proteins During Pseudorabies Virus Infection
Authors: Stevens J. G. , Kado-Boll G. J. , Haven C. B.
Published: January 2020

As a preliminary study to investigation of the possible role played by basic proteins in the genetic regulation of virus-infected cells, acid-extractable proteins synthesized during pseudorabies virus infection were investigated. The synthesis of histones was found to decrease in a gradual manner, and arrest was complete by 6 hr after infection. Five virus-induced acid-extractable proteins appeared in nuclei of infected cells after 4 hr of infection. Four of these proteins were virus structural proteins; one was not. All these proteins contained tryptophan and, therefore, were not “classic” histones.
Characteristics of the Deoxyribonucleic Acid of Tφ3, a Bacteriophage for
Bacillus stearothermophilus
Authors: Egbert Larre N.
Published: January 2020

It is shown that the individual strands of bacteriophage Tφ3 DNA are intact and that heat-denatured Tφ3 DNA forms a bimodal distribution in a neutral CsCl density gradient.
Cell Fusion by Various Strains of Newcastle Disease Virus and Their Virulence
Authors: Kohn Alexander , Fuchs Pinhas
Published: January 2020

Some properties of eight strains of Newcastle disease virus (cell-fusing ability, hemolysin, heat stability of hemagglutinin or of hemolysin) do not correlate with virulence of these strains.
Bacteriophage-induced Inhibition of Host Functions
Authors: Bose Subir K. , Warren Richard J.
Published: January 2020

Degradation of bacterial deoxyribonucleic acid (DNA) after infection with T4 bacteriophage was studied in an endonuclease I-deficient host. The kinetics of degradation were similar to those seen in other hosts with a normal level of this enzyme. Irradiation of extracellular phage with ultraviolet (UV) destroyed the capacity of the infecting virus to induce extensive breakdown of host DNA, which was, however, converted to high-molecular-weight material. Addition of chloramphenicol to T4-infected cells provided data which can be interpreted to indicate the involvement of at least two endodeoxyribonucleases and one exodeoxyribonuclease having a high degree of specificity. A model is proposed showing the sequential action of two endodeoxyribonucleases followed by an exodeoxyribonuclease in the degradation of host DNA. The appearance of these hydrolytic enzymes requires protein synthesis. Infections leading to partial degradation only (UV-irradiated phages, gene 46 mutants) effectively inhibited the synthesis of bacterial messenger ribonucleic acid and of β-galactosidase.
Colorado Tick Fever Virus: Growth in a Mosquito Cell Line
Authors: Yunker C. E. , Cory J.
Published: January 2020

Two strains of Colorado tick fever virus grew in Singh's
Aedes albopictus
cells. In one of three experiments, virus growth continued for 7 weeks.
Differences in pathogenicity among cloned sublines of a murine leukemia virus
Authors: Manly K F , Buffett R F
Published: January 2020

Five clones of the lymphatic leukemia virus 334C were isolated by a procedure designed to maintain homogeneity of the clones. Three of these induced leukemia in mice with the time course of the uncloned parental virus, one induced leukemia with a delayed time course, and one seemed to be biologically inactive. When the clone inducing leukemia most rapidly and the clone inducing leukemia least rapidly were subcloned, the subclones retained the leukemogenicity of the parental clones. The electrophoretic patterns of purified virion proteins and hybridization of viral RNAs with virus-specific DNA suggest that these clones are two closely related variants, not unrelated viruses. Furthermore, in mice infected with these two clones, viral RNA appears in thymuses and spleens at the same time after infection and at nearly the same concentrations. Thus, variations in leukemogenicity can be determined by a genetic property of an ecotropic leukemia virus, and this property is expressed in some manner more subtle than simple control of replication.
Adenovirus type 2 early polypeptides immunoprecipitated by antisera to five lines of adenovirus-transformed rat cells
Authors: Wold W S , Green M
Published: January 2020

We have identified adenovirus type 2 (Ad2)-induced early polypeptides (EPs) and have attempted to determine which EPs are coded by each of the four early gene blocks. [35S]methionine-labeled EPs were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cycloheximide pretreatment followed by labeling in hypertonic medium (210 to 250 mM NaCl) facilitated the detection of EPs. Seven major (reproducible bands in autoradiograms) EPs were detected with molecular weights of 74,000 (74K), 21K, 19K, 15K, 13.5K, 11.5K, and 11K. Minor (weaker bands) EPs of 55K, 52K, 42K, 18K, 12K, 8.8K, and 8.3K were also often seen. To identify and map the genes for virus-coded EPs, we prepared antisera against five lines of adenovirus-transformed cells that retain different fractions of the viral genome. The lines were F17, 8617, F4, and T2C4 transformed by Ad2 virions and 5RK (clone I) transformed by transfection with the Ad5 HsuI-G fragment (map position 0 to 8). The early gene blocks retained and expressed (in part) as RNA in these cells were as follows: 5RK(I), block 1 (70% of left 8% of genome); F17, block 1; 8617, blocks 1 and 4; F4 blocks 1, 2, and 4; T2C4, blocks 1, 2, 3, and 4. The following major EPs were immunoprecipitated: 15K by all antisera; 53K and 14.5K by F17, T2C4, 8617, and F4 antisera; 11.5K by T2C4, 8617, and F4 antisera; 44K, 42K, 19K, and 13.5K by T2C4 antisera; 11K by 8617 antisera. Minor EPs of 28K, 18K, and 12K were precipitated by all antisera except 5RK(I). The 53K and 15K EPs were precipitated also from Ad2 early infected monkey cells by the F17 antiserum and by sera from hamsters bearing tumors induced by Ad1-simian virus 40. The relationships between some of the immunoprecipitated EPs were investigated by the partial proteolysis procedure. All 53K EPs are the "same" (i.e., highly related), all 15K EPs are the "same," and all 11.5K EPs are the "same." The 15K EP is highly related to the 14.5 K EP. Although less certain, all 28K EPs appeared related, as did all 18K EPs. The T2C4-specific 44K EP is probably a dimer of the 21K glycopolypeptide. The T2C4-specific 13.5K EP and the 8617-specific 11K EP appear unrelated to any other polypeptides. These immunoprecipitation data provide evidence that early gene block I (map position 1 to 11) may encode major 53K, 15K, and 14.5K polypeptides, and minor 28K, 18K, and 12K polypeptides, and that all or some of the gene for 15K and 14.5K lies within map position 1 to 8. The surprisingly complex pattern of polypeptides coded by early gene block I raises the possibility that some polypeptides may be coded by overlapping "spliced" mRNA's. The possible block locations of the genes for the 21K, 13.5K, and 11.5K polypeptides are discussed.
Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2
Authors: Jariwalla R J , Aurelian L , Ts'o P O
Published: January 2020

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).
Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus
Authors: Polos P G , Gallaher W R
Published: January 2020

The role of membrane components in the interaction of cells with Newcastle disease virus (NDV) was studied using a ricin-resistant mutant of Chinese hamster ovary cells (CHO-15B), in which there is a deficiency in distal saccharides at the plasma membrane. Compared to the parental wild type, the mutant was shown to be 4- to 10-fold less sensitive to either fusion from without or fusion from within induced by NDV. The mutant and wild type were nearly indistinguishable with respect to other interactions with NDV. Viral attachment was investigated with 125I-labeled NDV and found to be comparable in both lines. Functionally equivalent amounts of hemagglutinin were produced, as measured by the fraction of cells positive for hemadsorption, or by the number of erythrocytes adsorbed per cell. No significant differences in the morphogenesis or yield of progeny virus were seen. The ability of the mutant to produce a fusion factor was measured by transfer of infected cells to uninfected monolayers. Infected CHO-15B cells were capable of inducing fusion normally in the indicator wild-type monolayers, but were incapable of inducing fusion in mutant monolayers. These results suggest that the insensitivity of the CHO-15B mutants to fusion may be due to inhibition of an early virus-cell interaction subsequent to viral attachment, whereas other events in infection appear to be unaffected by the cell surface mutation.
Are class I (pre-early) proteins of bacteriophage T5 sufficient to induce abortive infection of ColIb+ Escherichia coli?
Authors: Glenn J , Cheung A K , Duckworth D H
Published: January 2020

When T5 bacteriophage infect a colicin Ib-containing host, a variety of membrane changes and inhibition of macromolecular synthesis occur. This work shows that all these changes also occur when a mutant of T5 that can only inject 8% of its DNA is used. This indicates that all the information necessary for the abortive infection is present on this 8% (first-step-transfer) DNA.
Uncoating of adenovirus type 2
Authors: Mirza M A , Weber J
Published: January 2020

The uncoating of adenovirus type 2 and a temperature-sensitive mutant, tsl, was studied. HEp-2 cells were infected with 32P- OR 125I-labeled purified virions for various lengths of time, and the nuclear and cytoplasmic fractions were analyzed by sucrose gradient velocity sedimentation and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Within 1 h of infection, virions were converted into three subviral structures: (1) subviral structures in the cytoplasm with a density greater than virions but which qualitatively still contained all virus polypeptides; (ii) corelike structures associated with both the nuclear and cytoplasmic fractions and composed of viral DNA and polypeptides VIa2, V and PVII; and (iii) putative DNA-terminal protein complexes in the nuclei. The kinetic and compartmentalization studies suggested that the DNA-terminal protein complex is the end product of uncoating. The virions which were synthesized by tsl at the nonpermissive temperature and contained the precursor polypeptides PVI and PVII were found to be blocked in uncoating at the corelike stage. This block in uncoating provides the explanation for the lack of infectivity of these virions. A model for the uncoating of adenovirus is proposed.
Capsid protein precursor is one of two initiated products of translation of poliovirus RNA in vitro
Authors: Humphries S , Knauert F , Ehrenfeld E
Published: January 2020

Previous studies in our laboratory have demonstrated that cell-free systems translating the Mahoney strain of poliovirus type I RNA utilize two unique initiation sites. In this study, defective-interfering particles of poliovirus, which contain deletions in the region encoding the capsid proteins, are shown to initiate translation of proteins in vitro at these same two sites. Both the standard virus and the defective-interfering virus RNA direct the synthesis of two polypeptides labeled with n-formyl-methionine (fmet) at their amino termini. The size of the smaller fmet polypeptide synthesized in vitro by the defective virus appears identical in size to that of the standard virus. However, the larger-molecular-weight fmet polypeptide is reduced in size from 115,000 to 69,000 daltons. This correlates exactly with the reduced size of the precursor to the capsid proteins synthesized by the defective virus in vivo and with the size of the deletion in the defective virus RNA (1,200 bases). This provides genetic evidence that the 115,000-dalton fmet polypeptide synthesized into vitro by the standard virus is NCVP1a, the precursor to the coat proteins. Although the identity of the small (5,000 to 10,000 daltons) fmet polypeptide is not clear, several lines of evidence enable us to exclude the possibility that it is VP4, the smallest viral capsid protein.
Multiple integration sites for Moloney murine leukemia virus in productively infected mouse fibroblasts
Authors: Bacheler L T , Fan H
Published: January 2020

The integration sites for viral DNA in cells infected with Moloney murine leukemia virus (M-MuLV) were studied by restriction endonuclease cleavage of cellular DNA followed by electrophoresis in agarose gels, blot transfer to nitrocellulose, and detection by M-MuLV-related sequences by hybridization with high-specific-activity 32P-labeled M-MuLV complementary DNA. When EcoRI was used to cleave cellular DNA, numerous DNA fragments with sequence homology to M-MuLV were detected in uninfected mouse cell DNA. These endogenous sequences are mouse specific since they are not detectable in rat cell DNA, and are related to the 38S genomic RNA of M-MuLV. Infected cells contain additional M-MuLV-specific DNA fragments which are not detected in uninfected cells. Different patterns of M-MuLV-specific DNA fragments were detected in each cloned infected line examined. These data suggest the existence of multiple sites for integration of M-MuLV DNA in infected mouse fibroblasts. Cleavage of infected cell DNA with BamHI, which cleaves M-MuLV viral DNA at least twice, released the internal BamHI B fragment from each infected line, confirming the presence of integrated M-MuLV DNA sequences in each infected cell line which retain some features of the sequence organization of unintegrated M-MuLV DNA.
Effect of Temperature-Sensitive Mutation on Activity of the RNA Transcriptase of Vesicular Stomatitis Virus New Jersey
Authors: Szilágyi J. F. , Pringle C. R.
Published: January 2020

The virion-associated RNA transcriptase activity of vesicular stomatitis virus New Jersey temperature-sensitive (
ts
) mutants was assayed in vitro at the permissive (31°C) and restrictive (39°C) temperatures. RNA synthesis at 39°C by the RNA-negative
ts
A1 and the RNA-positive
ts
C1 and
ts
D1 mutants was similar to that of wild-type virus. The RNA-negative
ts
B1 synthesized only small amounts of RNA in vitro at 39°C. The three mutants of complementation group E were dissimilar in the amounts of RNA they synthesized at 39°C:
ts
E1 synthesized very little RNA,
ts
E2 synthesized moderate amounts, and RNA synthesis by
ts
E3 was not inhibited. The two mutants of group F were also dissimilar, since
ts
F1 synthesized very little RNA at 39°C, whereas
ts
F2 synthesized as much RNA as wild-type virus. The revertant clones
ts
B1/R1,
ts
E1/R1, and
ts
F1/R1 synthesized RNA at 39°C in amounts comparable to wild-type virus, indicating that the heat sensitivity of the transcriptase activity of the mutants
ts
B1,
ts
E1, and
ts
F1 was associated with temperature sensitivity. Similar heat sensitivities were observed when transcribing nucleoprotein complexes were used in the assays, showing that the mutated polypeptides were part of the viral core. The heat stability of the mutant
ts
B1 was similar to that of wild-type virus, and in vitro RNA synthesis was fully restored when the temperature was lowered to 31°C after 30 min of preincubation at 39°C, showing that the inhibition was due to reversible configurational change of the mutated polypeptide. When virions of the mutant
ts
E1 were heated for 5 h at 39°C, their infectivity and transcriptase activity were as stable as those of the wild-type virus, whereas transcriptase activity became very heat labile after disruption of the viral coat with a neutral detergent. This suggests an interaction between the mutated polypeptide and a coat polypeptide which stabilizes the activity of the transcriptase. The RNA transcriptase activity of the mutant
ts
F1 was also heat labile, although to a lesser extent than that of
ts
E1. Thus, the defects in transcriptase activity of groups B, E, and F suggest that all three polypeptides of the virus core, polypeptides L, N, and NS, are involved in the transcription. In addition, we postulate that the mutated gene products of groups E and F are multifunctional, being required both in transcription and replication, and that the gene product of group E may also be involved in some late stage of virus development.
M viral RNA segment of bunyaviruses codes for two glycoproteins, G1 and G2
Authors: Gentsch J R , Bishop D L
Published: January 2020

Tryptic peptide digests of the two viral glycoproteins (G1 and G2) of snowshow hare (SSH) virus, La Crosse, La Crosse (LAC) virus, and an SSH/LAC recombinant virus which has a large (L)/medium (M)/small (S) RNA segment genome composition of SSH/LAC/SSH were analyzed by ion-exchange column chromatography. The analyses prove that the M RNA species of bunyaviruses codes for the two viral glycoproteins.
Program of bacteriophage gh-1 DNA transcription in infected Pseudomonas putida
Authors: Jolly J F
Published: January 2020

The program of transcription in phage gh-1-infected Pseudomonas putida was examined. It was found that the host P. putida RNA polymerase transcribes early RNA from the L strand of gh-1 DNA during the initial stages of infection. The host RNA polymerase is also undoubtedly responsible for transcription of complementary RNA late in the infectious cycle because complementary RNA was not transcribed when rifampin was added to the infected cell culture. The gh-1-induced RNA polymerase transcribes late RNA from the L strand of gh-1 DNA late in the infectious cycle. The P. putida RNA polymerase transcribed only early RNA from primarily the L strand of gh-1 DNA in vitro when the molar ratio of enzyme to gh-1 DNA was 0.5. When the molar ratio was 50 the P. putida RNA polymerase transcribed RNA from the H strand of gh-1 DNA as well as complementary RNA. Thgh-1 RNA polymerase transcribed only the L strand of gh-1 DNA in vitro but transcribed both early and late RNA.
Neuroblastoma Cell Fusion by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus
Authors: Hughes Joseph V. , Dille Bruce J. , Thimmig Roberta L. , Johnson Terry C. , Rabinowitz Stanley G. , Dal Canto Mauro C.
Published: January 2020

A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 39°C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39°C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion.
Polyoma DNA synthesis in isolated nuclei: evidence for defective replication forks
Authors: Bjursell G , Munck V , Therkelsen A J
Published: January 2020

Nuclei, isolated from polyoma virus-infected mouse 3T6 cells, were incubated under condtions suitable for polyoma DNA synthesis. By using electrom microscopy and standard regression statistics, it was shown that replication is mainly unidirectional in a large number of molecules, indicating the presence of inactive replication forks. The replication forks were inactivated randomly, and the defect seemed to be present from the beginning of the in vitro incubation.
Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus
Authors: Payne L G
Published: January 2020

HeLa, SIRC, and RK-13 cells were compared as to their production of intracellular naked vaccinia virus (INV) and extracellular enveloped vaccinia virus (EEV) after infection with vaccinia strains WR and IHD-J. IHD-J produced more EEV from all three cell lines than did WR, although both strains produced approximately the same quantity of INV. The most efficient EEV release was from RK-13 cells infected with IHD-J, which was 200 times more than from WR-infected SIRC cells. This permitted for the first time the purification of milligram quantities of EEV that contained much fewer cell protein contaminants than could be obtained from HeLa or SIRC cells. The INV surface proteins 200K, 95K, 65K, and 13K were present in both HeLa and RK-13 cell-derived INV but were absent in SIRC cell INV. These proteins were absent in EEV from all three cell lines. Four glycoproteins of molecular weights 210 x 10(3) (210K), 110K, 89K, and 42K and five glycoproteins in the 23K to 20K range plus a nonglycosylated protein of 37K were detected in EEV from the hemagglutinin-positive IHD-J vaccinia strain. The 89K glycoprotein was not present in EEV or membranes from cells infected with the hemagglutinin-negative vaccinia strain IHD-W. Antisera to IHD-W lacking hemagglutinin-inhibiting antibodies did not precipitate the 89K glycoprotein of IHD-J. The only glycoprotein that specifically attached to rooster erythrocytes was the 89K glycoprotein. This evidence indicates that the 89K glycoprotein is the vaccinia hemagglutinin.
Recombination in bacteriophage T1 in the presence of host restriction
Authors: Kotval J S , Potts T V , Christensen J R
Published: January 2020

When unmodified phage T1 infects restricting host cells at high multiplicities of infection, there is an increase in recombination frequency in all regions of the T1 map compared to the level of recombination in standard crosses when short distances are examined. The enhancement of recombination frequency is not uniform for all regions but is greatest for markers near the center of the map and not so great for markers near the ends. Crosses between markers at the extremities of the map show that there is no increase in recombination frequency under restriction conditions. An examination of phage T1 heterozygotes suggests that an increase of ends created by the process of P1 restriction increases recombination. When T1 crosses are done in the absence of host restriction, recombination defects in the host have no effect on phage recombination and we conclude that phage T1 codes for its own recombination genes. Host recombination functions are also dispensable for the recombination occurring during infection of restricting host cells by unmodified phage at high multiplicities of infection.
Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant
Authors: Yamada Y , Silnutzer J , Nakada D
Published: January 2020

Upon infection with bacteriophage T7, a newly isolated mutant strain of Escherichia coli, Y49, produces T7-specific macromolecules including DNA almost normally. However, concatemeric T7 progeny DNA molecules, synthesized in Y49 cells, are later cleaved abnormally, resulting in an accumulation of DNA molecules shorter in size than the T7 genome and a poor production of progeny phage (Y. Yamada, J. Silnutzer, and D. Nakada, J. Mol. Biol. 121:95-111, 1978). The abnormal cutting of concatemeric T7 DNA in Y49 cells is accompanied by a simultaneous accumulation of large amounts of two types of phage head-related particles, proheads and newly found "X particles." Lysates from normal T7 infection of parental cells also contain X particles, although to a lesser amount. Electron microscopic examination of phage head-related particles (i.e., proheads, X particles, and empty heads), gel electrophoretic analysis of proteins in these particles, and kinetic studies on the appearance and fate of these particles suggest that X particles are likely to be intermediary structures between proheads and phage heads probably derived from proheads during the process of T7 DNA packaging. Our data also suggest that empty heads are not precursors to phage heads but are derived from proheads as by-products probably due to an abortive attempt to package T7 DNA. The host mutation in Y49 strain appears to block a step of T7 DNA processing and packaging pathway after generation of X particles from proheads.
Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein
Authors: Mosher R A , Mathews C K
Published: January 2020

Dihydrofolate reductase plays a dual role in bacteriophage T4, first, as an enzyme of thymidylate metabolism, and second, as a protein component of the tail baseplate. Antibody to the purified enzyme has been used to study its synthesis and intracellular turnover. The antibody specifically precipitates one protein from T4D-infected cell extracts. This has been identified as dihydrofolate reductase, although the polypeptide molecular weight (22,000) is lower than that earlier determined for this enzyme. The protein comigrates on gels with pY, a genetically undefined protein component of the baseplate. However, it is not pY, for pY is synthesized late in infection, whereas virtually no dihydrofolate reductase synthesis occurs later than 10 min after infection at 37 degrees C. Dihydrofolate reductase, once formed, is neither degraded nor converted to proteins of higher or lower molecular weight. Thus, it is probably incorporated into virions at the same molecular weight as that of the soluble enzyme. 125I-radiolabeled antibody binds to the wedge substructure of the baseplate, and this binding is blocked by preincubation with purified T4 dihydrofolate reductase. Thus, the enzyme protein seems to be a component of the wedge.
Growth state of the cell early after infection with simian virus 40 determines whether the maintenance of transformation will be A-gene dependent or independent
Authors: Seif R , Martin R G
Published: January 2020

The existence of both temperature-sensitive (N) and temperature-insensitive (A) rat transformants, isolated after infection with simian virus 40 tsA mutant, is reported. Both types can be isolated as dense foci. Foci appearing after infection of rapidly growing cells were temperature sensitive. Infection of cells arrested at confluence gave rise to foci that were temperature insensitive. Transformants isolated by the agar assay (conditions under which normal cells are unable to grow) were also temperature-insensitive. N-transformants remained temperature sensitive upon entering the resting state at the restrictive temperature and upon re-entering the growth cycle at the permissive temperature. They also remained temperature sensitive under a variety of conditions restrictive for nontransformed cells. Thus, the state of the cell in the first few days after infection fixes the cells. Thus, the state of the cell in the first few days after infection fixes the cell as an N- or A-transformant. Various models for transformation are discussed, including one proposing that the virus interacts in two ways with a central cell mechanism controlling growth. The maintenance of the transformed phenotype would be dependent on T-antigen in N-transformants but independent of T-antigen in A-transformants.
Isolation and localization of herpes simplex virus type 1 mRNA abundant before viral DNA synthesis
Authors: Holland L E , Anderson K P , Stringer J R , Wagner E K
Published: January 2020

Herpes simplex virus type 1 (HSV-1) DNA covalently bound to cellulose was used as a reagent to isolate viral RNA transcripts for size analysis on denaturing agarose gels. Nuclear and polyribosomal RNA isolated at 2 h postinfection (p.i.) migrated with sizes between 1,500 and 5,500 nucleotides. At 6 h p.i. (when viral DNA synthesis is underway), viral polyribosome-associated polyadenylated RNA showed different discrete sizes of species predominating, with RNA larger than 5,500 nucleotides clearly present. Nearly 50% of the newly made viral RNA found in the nucleus at 6 h p.i. was from 5,000 to 10,000 nucleotides in length. A high-resolution transcription map of the viral mRNA abundant at 2 h p.i. was compiled from the hybridization of Southern blots of HSV-1 DNA restriction fragments to both sizes of fractionated polyribosomal polyadenylated RNA and 3' complementary DNA probe made to this size of fractionated RNA. We have identified and mapped 16 mRNA species abundant at 2 h p.i. These RNAs range in size from 1,500 to 5,300 nucleotides and map throughout the HSV-1 genome. In some instances, a direction of transcription can be suggested. Further, about one-third of this number of mRNA's has been found in cells infected with a DNA-negative temperature-sensitive mutant (tsB2) and grown at the nonpermissive temperature (39 degrees C).
Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells
Authors: Chang C , Simmons D T , Martin M A , Mora P T
Published: January 2020

Two new species of antigens were detected in simian virus 40-transformed mouse cells, in addition to the large (94,000 daltons) and small (20,000 daltons) tumor antigens. These antigens were immunoprecipitated from cell extracts by using anti-T serum and not normal, nonimmune serum. One of these was a protein with a molecular weight of approximately 130,000 and was present in some but not all SV40-transformed mouse cells. The other, which we have named Tau antigen, has a molecular weight of 56,000 as estimated by electrophoresis through acrylamide gels and was found in all virus-transformed cells examined. The 13,000-daltons antigen contained about 15 methionine-tryptic peptides which were also present in the large SV40 tumor antigen as determined by ion-exchange chromatography. This strongly suggested that the protein was virus coded. The 56,000-dalton Tau antigen appeared to share only two methionine-tryptic peptides with the large species of SV40 tumor antigen, as determined by ion-exchange and paper chromatographies. Our results are compatible with a cellular origin for Tau antigen. However, our data do not exclude the possibility that this protein contains sequences specified by the virus DNA.
Method for determining the extent and copy number of overlapping and nonoverlapping segments of integrated viral genomes
Authors: Patch C T , Hauser J , Lewis A M , Levine A S
Published: January 2020

We analyzed the method of exhaustive hybridization of single-stranded DNA and derived a general relationship between the fraction of the probe DNA hybridized and the sizes and copy numbers of the segments of the viral genome integrated in cellular DNA. The equations employed can be used to analyze integrated DNA comprised of overlapping and nonoverlapping segments of the viral genome. Using these equations, we analyzd the adenovirus type 2 DNA content of a series of hamster cell lines transformed by adenovirus type 2 and several adenovirus type 2-simian virus 40 hybrid viruses. We found no eividence that the integrated viral DNA is comprised of overlapsping segments. However, the number of copies of the integrated segments varies between lines cloned from the same transformed isolate, and copy numbers change during in vivo passage of transformed cells.
New classes of viable deletion mutants in the early region of polyoma virus
Authors: Griffin B E , Maddock C
Published: January 2020

Viable mutants of polyoma virus have been isolated which have deletions in defined parts of the early region of the genome. One class of mutants has deletions (less than 1% of viral genome length) located between 71.5 and 73.5 on the physical map of polyoma virus DNA, near the origin of replication. These mutants appear to grow and to transform cells in a manner indistinguishable from wild-type virus. A second type of mutant with deletions (about 2% of viral genome length) located between about 88 and 94.5 units on the physical map of polyoma virus DNA have altered transformation properties. One of the latter (which maps between 88 and 91.5 units) also has altered growth characteristics, whereas another (which maps between 91.5 and 94.5 units) resembles wild-type virus in its growth properties. The regions with deleted sequences have been defined by cleaving mutant DNAs with restriction endonucleases and analyzing pyrimidine tracts.
Adenovirus type 2 terminal protein: purification and comparison of tryptic peptides with known adenovirus-coded proteins
Authors: Harter M L , Lewis J B , Anderson C W
Published: January 2020

The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with [35S]methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to [35S]methionine during the "intermediate" period of 13 to 21 h postinfection, suggesting that the protein is synthesized during this interval. The tryptic peptides of the terminal protein were compared with those of several known adenovirus-coded proteins and found to be unrelated. In particular, the terminal protein is not related to the 38-50K early proteins encoded by the leftmost 4.4% of the adenovirus genome, one region essential for the transforming activity of the virus. Neither is it related to the 72K single-strand-specific DNA binding protein, the minor virion component IVa2, or the major capsid component hexon.
Regulation of persistent infection with herpes simplex virus in vitro by hydrocortisone
Authors: Nishiyama Y , Rapp F
Published: January 2020

About 1% of Raji cells showed sensitivity to herpes simplex virus type 2 (HSV-2) infection when tested by infectious center assays or immunofluorescence tests, and the percentage did not change during cell passage. The addition of hydrocortisone to Raji cells persistently infected with HSV-2 caused a marked increase in virus production and in the number of HSV-producing cells. In the case of HSV-1, it was shown that the addition of hydrocortisone was required to maintain persistent infection. These observations suggest that control of replication of HSV-1 and HSV-2 in these cells is regulated by different mechanisms.
Characterization of the proteins of intracisternal type A and extracellular oncornavirus-like particles produced by MOPC-460 myeloma cells
Authors: Robertson D L , Jhabvala P S , Godefroy-Colburn T , Thach R E
Published: January 2020

The mouse plasmacytoma cell line, MOPC-460, produces both intracisternal and intracytoplasmic A-type particles when grown as a solid tumor. When these cells are grown either as an ascites tumor or in tissue culture, a third type of particle is produced extracellularly. This particle, the "myeloma-associated virus," is closely related to, and probably an alternate form of, the intracisternal A-type particle. The proteins present in these two types of particles were compared by tryptic peptide mapping. Both types of particles were found to contain essentially the same major proteins of 76,000 (p76), 68,000 to 70,000 (p68-70), and 45,000 (p45) daltons, in addition to varying amounts of smaller proteins. The relative proportions of all these proteins varied from preparation to preparation in an unpredictable way. The p45, p68, and p70 proteins all contained sequences found in p76, suggesting precursor-product relationships of p76 leads to p70 leads to p45 for solid tumor A-type particles and p76 leads to p68 leads to p45 for extracellular myeloma-associated virus. In addition, immune precipitation experiments have established that p76 contains at least some of the antigenic determinants characteristic of murine leukemia virus p30. This confirms earlier nucleic acid hybridization studies which indicated a moderate degree of relatedness between MOPC-460 A-type particles and several standard murine leukemia and sarcoma viruses. Taken together, our results provide evidence supporting the concept that MOPC-460 A-type particles may represent aberrant forms of C-type murine viruses.
Electron microscopic mapping of RNA transcribed from the late region of polyoma virus DNA
Authors: Manor H , Wu M , Baran N , Davidson N
Published: January 2020

The polyoma virus (Py) RNA species transcribed from the L DNA strand of the "late" region of the Py genome in Py-infected mouse cells have been mapped by hybridization with specific fragments of Py DNA followed by electron microscopic visualization of the hybrids. Total cellular polyadenylated Py-specific RNA molecules having an S value in the range of 16S to 20S were purified by oligodeoxythymidylic acidcellulose column chromatography, preparative hybridization with Py DNA, and sucrose gradient centrifugation. Cytoplasmic Py-specific RNA was similarily purified, except that it was not fractionated by sucrose gradient centrifugation. Hybrids of these RNA molecules and Py DNA fragments were spread for electron microscopy by either the cytochrome c technique or the bacteriophage T4 gene 32 protein method. The polyadenylic acid at the 3'-end of the RNA in the hybrids was identified by labeling with simian virus 40 DNA circles to which polybromodeoxyuridylic acid tails had been covalently attached. These experiments revealed the presence of three L DNA strand transcripts in both RNA preparations. Two of these RNA molecules were found to be spliced from chains transcribed from two noncontiguous parts of the late region. The third molecule either is a continuous transcript of the entire late region or contains a splicing feature which is too small to be reliably observed by the electron microscope methods used. The 5'-ends of the three RNA species map within a region extending from 68 to 70 map units on the Py restriction endonuclease map. Each of the two spliced molecules contains a 5'-terminal leader sequence transcribed from a DNA segment with an estimated length of 60 to 110 nuvleotides. The 3'-ends of the leaders map at 66.7 +/- 1.0 and 66.4 +/- 0.50 map units. In these molecules the 5'-ends of the other part (the main body) map at 59.4 +/- 0.90 and 49.4 +/- 2.0 map units, respectively. The 3'-termini of all three RNA species map at 24 to 25 map units.
Physical map of biologically active Harvey sarcoma virus unintegrated linear DNA
Authors: Goldfarb M P , Weinberg R A
Published: January 2020

BALB/c JLS V9 cells recently infected with Harvey sarcoma virus-murine leukemia virus (HSV-MuLV) complex contained unintegrated HSV linear DNA of 6.0-kilobase pair mass. The cells also contained two HSV closed circular DNA species along with MuLV-encoded linear and closed circular DNA species. HSV 6.0-kilobase pair linear DNA induced focal transformation upon transfection of NIH 3T3 mouse fibroblasts, and the biological activity of HSV DNA did not require helper MuLV functions. A physical map of restriction endonuclease cleavage sites along HSV 6.0-kilobase pair linear DNA was derived. Comparison of this map with one for Moloney MuLV DNA showed that the HSV and Moloney MuLV genomes are identical near their viral RNA 3' ends.
Assembly of vesicular stomatitis virus nucleocapsids in vivo: a kinetic analysis
Authors: Hsu C H , Kingsbury D W , Murti K G
Published: January 2020

Pulse-chase labeling and cell fractionation were used to examine the pathways taken by the three nucleocapsid polypeptide species of vesicular stomatitis virus into nucleocapsids and then into virions. An improved method of polyacrylamide gel electrophoresis resolved nucleocapsid polypeptides N and NS from cellular actin, facilitating accurate quantitation of the viral polypeptides. Contrary to previous belief, the rate of NS synthesis was found to be a constant fraction of total virus protein synthesis throughout infection, indicating a consistent mechanism of virus protein synthesis regulation. In the kinetic studies, each polypeptide species displayed the following characteristic behavior. (i) Structural polypeptide N was the only species that entered a metabolically active soluble pool before assembly into nucleocapsids. The size of this pool increased with time after infection, causing an increasing delay in the appearance of pulse-labeled N molecules in nucleocapsids. (ii) Throughout infection, the entire complement of L molecules entered nucleocapsids immediately after their synthesis, without diversion through a soluble pool. (iii) Although 75% of newly synthesized molecules of the transcriptase-associated protein NS entered a soluble pool, they never emerged from the compartment. At all times after infection, about 25% of the NS molecules bypassed the soluble pool and entered nucleocapsids directly after their synthesis, as if in concert with L. These results indicate that VSV nucleocapsid assembly in vivo is a stepwise process, comprising an initial condensation of N with the viral RNA, followed by attachment of L and NS, analogous to the stepwise assembly of Sendai virus nucleocapsids. (D. W. Kingsbury, C.-H. Hsu, and K. G. Murti. Virology 91:86-94, 1978). About half of the intracellular nucleocapsids were recovered in a form that sedimented at anomalously low centrifugal forces, reflecting an association with large cellular organelles. This attachment was mediated mainly by electrostatic forces, since these "bound" nucleocapsids were released by elevated salt concentrations. The kinetic behavior of nucleocapsid polypeptides was the same in both fractions, providing no evidence for a division of nucleocapsid functions between cellular compartments.
Detection of polymorphism in BALB/c leukemia viruses with mouse antisera
Authors: Brown J P , Boiocchi M , Nowinski R C
Published: January 2020

Antisera produced in mice recognize primarily type-specific antigenic determinants on both the major core protein, p30, and the major envelope proteins, gp70 and p15(E), of the endogenous leukemia viruses (MuLV) of BALB/c mice. Three different mouse sera were investigated in detail. (i) Antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted with the gp70, p15(E), and p30 proteins of MuLV. Certain pools of the C57BL/6 anti-AKR K36 serum contained antibodies which serologically distinguished the p30 proteins of N-ecotropic, B-ecotropic, and xenotropic BALB/c MuLV. (ii) Antisera prepared in BALB/c mice against the BALB/c sarcoma 1315 contained antibodies that reacted with a type-specific antigen of the 1315 MuLV gp70 that is not found on other BALB/c MuLV. (iii) The normal sera of multiparous BALB/c mice contained antibodies that reacted with gp70 and p15(E) proteins of ecotropic MuLV. Sera from some of these mice contained antibodies that serologically distinguished the gp70 of N-ecotropic and B-ecotropic BALB/c viruses. These results emphasize the utility of mouse antisera in the serological typing of MuLV. Furthermore, the antigenic differences observed in the p30 and gp70 proteins should be of particular use in the future analysis of recombinant BALB/c MuLV.
Methylation of nuclear simian virus 40 RNAs
Authors: Aloni Y , Dhar R , Khoury G
Published: January 2020

Nuclear RNAs, pulse-labeled with [methyl-3H]methionine and [3H]uridine under optimal conditions, were prepared from cells infected with simian virus 40 at a late time after infection. The labeled RNAs were hybridized with restriction fragments of simian virus 40 DNA. Levels of methyl-3H-labeled RNA annealing with the early (E) or late (L) strands of a particular restriction fragment were compared with [3H]uridine-labeled RNA annealing with the same fragment. The methyl-3H-labeled RNA hybridizing to the various restriction fragments was eluted and analyzed for caps and internal 6-methyladenosine residues. Caps of transcripts of both the L and E strands were primarily located in a fragment containing the origin for viral DNA replication. The highest level of the internal 6-methyladenosine residues in simian virus 40 nuclear RNA was located on the L strand within a fragment from 83 to 0.0 map units and in RNA transcripts from the E strand within a fragment from 37 to 67 map units. Portions of both these regions represent intervening sequences not represented in cyotplasmic mRNA's. Lower levels of internal methylation were found in other locations as well. The potential role of internal 6-methyladenosine residues in RNA processing and transport from the nucleus to the cytoplasm is discussed.