bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and morphological characterization of mycobacteriophage R1
Authors: Soloff B L , Rado T A , Henry B E , Bates J H
Published: January 2020

Large-scale propagation of mycobacteriophage R1 in broth culture has allowed the isolation of quantities of virus sufficient for characterization of its nucleic acid and lipid components as well as investigation of its ultrastructural attributes. Analysis of R1 DNA indicates that it is double stranded and possesses a molecular weight of 2.5 X 10(7) and a guanine-plus-cytosine content of 65.7 +/- 0.5%. The lipid fraction of R1 accounts for 14% of the total dry weight of the virus, 20% of which was identified as free or esterified sterols. A rapid loss of viral titer occurred after seconds of exposure to organic solvents. This result suggests that the lipid fractions of R1 is essential for its infectivity. Electron microscopic investigation of solvent-extracted R1 showed extensive deterioration of its normal morphology, including nucleocapsid disintegration and base plate separation. Routine phosphotungstate preparations demonstrated a particle with an oval head and a noncontractile tail. Altering the pH of the phosphotungstate negative stain from neutrality damage the viral particles. Uranyl formate-contrasted specimens displayed an elongated hexagonal nucleocapsid with a neck region; the cross-striated tail possessed a starlike base plate.
Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants
Authors: Yanagi K , Talavera A , Nishimoto T , Rush M G
Published: January 2020

Herpes simplex virus type 1 DNA synthesis and infections progeny production were studied in five different conditional hamster (BHK-21) cell cycle mutants. At the nonpermissive temperature (39.5 degrees C), both events were strongly inhibited in four of these cell lines. The degree of inhibition was a reproducible characteristic of each cell mutant and in two cases was dependent upon the multiplicity of infection. Experiments involving shifts to the nonpermissive temperature at least 3 h postinfection at 33.5 degrees C suggested that the defects in viral replication were not due to faulty adsorption, penetration, or uncoating, whereas experiments involving shifts of infected cells from the nonpermissive temperature to 33.5 degrees C revealed the reversible nature of the inhibition.
Effect of rhesus or vervet cytomegalovirus infection of DNA synthesis in untreated and 5-iodo-2'-deoxyuridine-arrested cells
Authors: Waner J L , Budnick J E , Albrecht T B
Published: January 2020

Infection of permissive cells with either rhesus or vervet cytomegalovirus resulted in suppression of cellular DNA synthesis, only viral DNA was resolved by isopycnic centrifugation after 24 h postinoculation. Infection of 5-iodo-2'-deoxyuridine (IUdR)-arrested cells with either of the simian cytomegaloviruses, however, induced synthesis of cellular DNA of normal density; synthesis of cellular DNA substituted with IUdR as evidenced by resolution of a heavy DNA peak after isopycnic centrifugation was not observed. Stimulation of DNA synthesis in IUdR-arrested cells was not observed with virus inactivated with UV light.
In vitro transcription of mouse mammary tumor virus RNA into DNA
Authors: Taylor J M , Illmensee R , Young H A
Published: January 2020

The in vitro transcription of the B-type mouse mammary tumor virus RNA into DNA is similar to that obtained with certain C-type viruses: synthesis can be initiated on an RNA primer molecule approximately the size of a tRNA, and a unique linkage (rA-dC) exists between this primer and the DNA product.
Spring viremia of carp virus RNA and virion-associated transcriptase activity
Authors: Roy P , Clewley J P
Published: January 2020

An RNA-dependent RNA polymerase activity has been demonstrated for spring viremia of carp virus (SVCV). The optimal temperature for in vitro synthesis of RNA was 20 to 25 degrees C. The SVCV enzyme activity was stimulated when the methyl donor S-adenosyl-L-methionine was included in the reaction mixture. S-adenosyl-L-methionine was not particularly effective in stimulating the virion RNA polymerase activity of vesicular stomatitis virus or pike fry rhabdovirus. The 5' nucleotide of the SVCV viral RNA is pppAp.
Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex
Authors: Chinnadurai G , Chinnadurai S , Green M
Published: January 2020

The infectivity of adenovirus type 2 DNA and a DNA-protein complex was studied in 293 cells, a human embryonic kidney cell line transformed by sheared adenovirus type 5 DNA, and in human KB cells. Adenovirus type 2 DNA was more infectious (up to about 40-fold) in 293 cells than in KB cells, whereas a DNA-protein complex (prepared by a rapid procedure) had about the same infectivity in both cell lines. These data may mean that a factor present in 293 cells (perhaps a viral-coded protein) enhances the infectivity of free viral DNA. The infectivity of DNA and the DNA-protein complex was increased up to fivefold by brief treatment of cell monolayers with 25% dimethyl sulfoxide after transfection. Under these conditions, (i) the infectivity of native adenovirus type 2 DNA ranged from 400 to 1,300 PFU/microgram of DNA in 293 cells and from about 9 to 14 PFU/microgram of DNA in KB cells, and (ii) the infectivity of the DNA-protein complex was 6 X 10(3)to 2 X 10(4) PFU/microgram in 293 cells and 1.4 X 10(4) to 1.6 X 10(4) PFU/microgram in KB cells.
Presence of 5'-terminal cap structures in virus-specific RNA from feline leukemia virus-infected cells
Authors: Thomason A R , Friderici K H , Velicer L F , Rottman F
Published: January 2020

The F-422 line of feline thymus tumor cells, chronically infected with the Rickard strain of feline leukemia virus (R-FeLV), was labeled with 32P, and the total cytoplasmic RNA was isolated. The RNA was centrifuged through sucrose gradients, and R-FeLV virus-specific RNA (vRNA) was located by hybridization of portions of the gradient fractions to R-FeLV complementary DNA. vRNA classes with average sedimentation coefficients of approximately 36S, 28S, 23S, and 15S were identified. Each class of RNA was recovered by hybridized with mercurated R-FeLV complementary DNA, and the hybrids were chromatographed on columns of sulfhydryl-Sepharose to separate them from unhybridized cellular RNA. Although insufficient amount of 36S and 28S vRNA were obtained for further analysis, the 23S and 15S VRNA classes were analyzed to determine the nature of their 5' termini. Each of these vRNA classes was found to contain stoichiometric amounts of cap structures per unit length of RNA, consistent with the presence of one cap per molecule. The structure of the 23S vRNA cap was found to be m7G5'ppp5'GmpAp, whereas that of the 15S vRNA cap was m7G5'ppp5'GmpGp.
Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei
Authors: Kedinger C , Brison O , Perrin F , Wilhelm J
Published: January 2020

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.
Role of the simian virus 40 gene A product in regulation of DNA synthesis in transformed cells
Authors: Butel J S , Soule H R
Published: January 2020

Cells transformed by tsA mutants of simian virus 40 (SV40) are temperature sensitive for the maintenance of the transformed phenotype. The kinetics of induction of DNA synthesis were determined for hamster cell transformants shifted to the permissive temperature after a 48-h serum arrest at the nonpermissive temperature. DNAsynthesis was initiated in the tsA transformants by 8 h after shiftdown was maximal by 12 h. The presence or absence of fetal bovine serum at the time of temperature shift had no effect on the kinetics of initiation of DNA synthesis. Analysis of TTP in tsA transformants revealed similar levels of incorporation of [3H]thymidine into TTP at both permissive and nonpermissive temperatures. Autoradiography revealed that by 12 h after a shift to the permissive temperature, approximately 50% of the cells exhibited labeled nuclei after a 60-min pulse with [3H]thymidine, indicating that a majority of the cells were actively synthesizing DNA. By 8 to 12 h after a shiftup of confluent tsA transformants to the nonpermissive temperature, the number of labeled nuclei was reduced to approximately 16%, regardless of serum concentration. These data indicate that the SV40 gene A product, either directly or indirectly, regulates cellular DNA synthesis in transformed cells.
Isolation of the envelope of vesicular stomatitis virus
Authors: Taube S E , Rothfield L I
Published: January 2020

Vesicular stomatitis virus was disrupted by a combination of freezing and thawing, osmotic shock, and sonic treatment. Subviral components were separated by isopycnic centrifugation. The low-density, lipid-rich fractions were pooled and shown to contain primarily viral glycoprotein. Further purification of this material resulted in the isolation of a preparation of vesicles which contained only the G protein and the same phospholipids as in the intact virions and exhibited spikelike structures similar to those on intact vesicular stomatitis virions. We conclude that we have isolated fragments of native vesicular stomatitis virus envelopes.
Evidence for a herpes simplex virus-specific factor controlling the transcription of deoxypyrimidine kinase
Authors: Leung W C
Published: January 2020

A cistron-specific, enzyme-forming-capacity method was used to study the control of herpes simplex virus (HSV)-specific deoxypyrimidine kinase (dPyK) mRNA synthesis. A virus-specific factor was formed by a primary infecting virus, and this factor effected the transcription of dPyK mRNA of a superinfecting virus in the presence of cycloheximide, suggesting that the factor acted in "trans" and was a diffusible one. After the addition of actinomycin D to prevent further transcription and upon removal of cycloheximide, the dPyK mRNA was allowed to express into dPyK activity. A factor from HSV-1 could effect the transcription of dPyK mRNA of both HSV-1 and HSV-2. Amino acid analogs, canavanine or ethionine, inhibited the action of this factor, suggesting that a protein was involved. This protein factor was shown to belong to the alpha (or immediate-early) group of HSV-Specific polypeptides in preductively infected cells.
Synthesis of a mammalian parvovirus in Brij-58-lysed cells
Authors: Gautschi M , Reinhard P
Published: January 2020

An in vitro system was developed, prepared by lysis of parvovirus LuIII-infected cells with Brij-58. In this subcellular system, DNA of viral genome size and synthesized and subsequently packaged into particles with physicochemical properties of mature LuIII virions.
Murine leukemia virus (T-8)-transformed cells: identification of a precursor polyprotein containing gag gene-coded proteins (p15 and p12) and a nonstructural component
Authors: Sacks T L , Reynolds F H , Deobagkar D N , Stephenson J R
Published: January 2020

Mink cells nonproductively transformed by the T-8 strain of mink cell focus-inducing virus express two type C viral amino terminal gag gene-coded structural proteins, p15 and p12, in the form of a 90,000 to 110,000 molecular weight polyprotein that lacks detectable immunological reactivity with other known type C virus-coded translational products. The observation concurs with the previous demonstration of similar high-molecular-weight precursor polyproteins in cell lines nonproductively transformed by either of two other mammalian sarcoma viruses also limited in virus-coded structural protein expression to p15 and p12.
Purification and characterization of an early protein (E14K) from adenovirus type 2-infected cells
Authors: Persson H , Oberg B , Philipson L
Published: January 2020

One adenovirus type 2 (Ad2) early protein, with an apparent molecular weight of 14,000 in sodium dodecyl sulfate-polyacrylamide gels (E14K), was purified to homogeneity. Purification involved fractionation of cytoplasmic extracts, precipitation at low pH, and DEAE-cellulose, phosphocellulose, and hydroxylapatite chromatography. The yield was around 12 microgram of purified protein per 10(9) HeLa cells. The two Ad2 DNA binding proteins with molecular weights of 75,000 and 45,000 (E75K and E45K) were purified by the same procedure. Tryptic peptide analyses indicated that the E14K protein is unrelated to the DNA binding proteins. The purified E14K protein has a high content of basic amino acids and a sedimentation coefficient of 5.5S in the native state, corresponding to a molecular weight of around 95,000. Pulse-chase experiments suggest that the E14K polypeptide is a primary translation product. Immunoprecipitation with a monospecific antiserum against the E14K protein revealed that it is exclusively localized in the cytoplasm of infected cells. E14K started to be synthesized at 2 hpostinfection, with a maximal rate of synthesis at 4 to 6 h postinfection. Immunoprecipitation of cell extracts from four different Ad2-transformed hamster embryo cell lines revealed that only one (Ad2HE4) of them expresses this protein. The adenovirus-simian virus 40 hybrid virus (Ad2ND1) does not express this protein, suggesting that the gene for the E14K protein is located in the part of the Ad2 genome which is deleted in this hybrid virus.
Characterization of the mRNA's for the polyoma virus capsid proteins VP1, VP2, and VP3
Authors: Hunter T , Gibson W
Published: January 2020

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.
Identification of Sendai virus mRNA species
Authors: Jones K , Pridgen C , Kingsbury D W
Published: January 2020

Cell-free translation of separated Sendai virus mRNA species identified the message for polypeptide M and suggested the identity of the message for polypeptide NP.
Peptide-chain initiation with Lsc poliovirus is intrinsically more resistant to hypertonic environment than is peptide-chain initiation with Mahoney virus and deletion mutants of Mahoney virus
Authors: Tershak D R
Published: January 2020

Peptide-chain initiation with LSc poliovirus was more resistant to hypertonic medium than peptide-chain initiation with Mahoney poliovirus. Protein synthesis of LSc virus retained its relative resistance to high osmolarity created by the addition of excess NaCl to the medium in cells coinfected with Mahoney virus. The data indicate that peptide-chain initiation with LSc virus is intrinsically more resistant to high osmolarity than that of Mahoney virus rather than reflecting different permeability changes in cells after infection. Two Mahoney virus mutants harboring deletions at the 5' end of the viral chromosome exhibited the same sensitivity to excess NaCl as parent virus, suggesting that the original chromosomal region for peptide-chain initiation has not been severely altered by the deletions.
Susceptibility of influenza A viruses to amantadine is influenced by the gene coding for M protein
Authors: Lubeck M D , Schulman J L , Palese P
Published: January 2020

Influenza A virus recombinants derived from "resistant" and "sensitive" parental viruses were examined for susceptibility to inhibition by amantadine. Correlation of gene constellation and amantadine susceptibility revealed that the gene coding for M protein influences sensitivity or resistance to amantadine. All recombinants which derived an M protein from an amantadine-resistant parent were found to be resistant to amantadine. All amantadine-sensitive recombinants derived an M gene from the amantadine-sensitive parent. However, a few amantadine-resistant recombinants which derived an M gene from the sensitive parent were also isolated, suggesting that the expression of amantadine sensitivity in these recombinants may be influenced by other genes.
Anatomy of Herpes Simplex Virus DNA XII. Accumulation of Head-to-Tail Concatemers in Nuclei of Infected Cells and Their Role in the Generation of the Four Isomeric Arrangements of Viral DNA
Authors: Jacob Robert J. , Morse Lawrence S. , Roizman Bernard
Published: January 2020

Previous reports (H. Delius and J. B. Clements, J. Gen. Virol.
33
:125-134, 1976; G. S. Hayward, R. J. Jacob, S. C. Wadsworth, and B. Roizman, Proc. Natl. Acad. Sci. U.S.A.
72
:4243-4247, 1975; B. Roizman, G. S. Hayward, R. Jacob, S. W. Wadsworth, and R. W. Honess, Excerpta Med. Int. Congr. Ser.
2
:188-198, 1974) have shown that herpes simplex virus DNA extracted from virions accumulating in the cytoplasm of infected cells consists of four populations of linear molecules differing in the orientation of the covalently linked large (L) and small (S) components relative to each other. Together, these four isomeric arrangements of viral DNA display four different termini and four different L-S component junctions. In the studies reported in this paper, we analyzed with restriction endonucleases the newly replicated viral DNA shortly after the onset of viral DNA synthesis, the progeny DNA accumulating in the nuclei late in infection, and rapidly sedimenting DNA present in nuclei of infected cells at 8 h after infection. In each instance the nuclear viral DNA contained a decreased concentration of all four terminal fragments and an increase in the concentration of fragments spanning the junction of L and S components relative to the concentration of other DNA fragments. The results are consistent with the hypothesis that the viral DNA accumulating in the nuclei consists of head-to-tail concatemers arising from the replication of DNA by a rolling-circle mechanism. A model is presented for generation of all four isomeric arrangements of herpes simplex virus DNA from one arrangement based on excision and repair of unit length DNA from head-to-tail concatemers and known features of the sequence arrangement of viral DNA.
Reverse transcriptase from avian myeloblastosis virus
Authors: Houts G E , Miyagi M , Ellis C , Beard D , Beard J W
Published: January 2020

From lots of 20 to 30 g of avian myeloblastosis virus RNA-dependent DNA polymerase was obtained in preparations of purity greater than 95% by using a two-step column chromatographic procedure employing DEAE (DE 52) and carboxymethylcellulose (CM 52.). Yields of RNA-dependent DNA polymerase varied from approximately 20,000 to 35,000 U/g of virus. Specific activity of the enzyme was about 35,000 to 60,000 U/mg of protein. Free of detectable RNase activity, the product exhibited a molecular weight of about 160,000, an isoelectric point of 6.5, and approximately 2 mol of fatty acid per mol of enzyme.
Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein
Authors: Khan A S , Stephenson J R
Published: January 2020

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.
Some Physiochemical Properties of Yaba Poxvirus Deoxyribonucleic Acid
Authors: Yohn David S. , Gallagher John F.
Published: January 2020

Yaba tumor poxvirus deoxyribonucleic acid (DNA) has a density of 1.6905 in CsCl and its T
m
value in 0.015
m
citrate in saline is 82.3. The guanine plus cytosine content estimated from these properties was taken to be 32.5 ± 0.5%, a value 2 to 3% less than for DNA from other poxviruses reported to date.
Infection by Bacteriophage P1 and Development of Host-controlled Restriction and Modification and of Lysogenic Immunity
Authors: Werner Elizabeth R. , Christensen J. R.
Published: January 2020

Shigella dysenteriae
cells were infected with phage P1 or P1cl. The outcome of superinfection of these cells with phage T1.Sh or T1.Sh(P1) or P1cl was studied as a function of time after the initial infection. Cells undergoing either a lytic response or a lysogenic response to the primary infection develop the ability to specifically restrict T1.Sh between 30 and 45 min. Between 15 and 30 min, the cells seem to develop the ability to produce T1.Sh(P1) after infection by T1.Sh. However, reasons are given for believing that this apparent time difference is consistent with a simultaneous development of the two capacities (restriction and modification) within the cell. This development occurs between 30 and 45 min. Cells infected with P1cl and superinfected 45 or more min later with T1.Sh(P1) can yield both P1cl and T1. Cells infected with P1 become resistant to infection by P1cl within 5 to 10 min. It is argued that this early immunity is not necessarily different in mechanism from true lysogenic immunity.
Site- and Gene-specific Limited Heterocatalytic Expression in Bacteriophage T4-infected
Escherichia coli
Authors: Snustad D. Peter
Published: January 2020

Genetic evidence for site- and gene-specific variation in limited heterocatalytic expression in phage T4-infected
Escherichia coli
is reported, and the implications of such variation are discussed.
Nature of
Escherichia coli
B(P1) Yielder Cells at the Time of Infection with Restricted T1
Authors: Winstead Laura , Drexler Henry
Published: January 2020

In the infection of
Escherichia coli
B(P1) with restricted T1, it was shown that yielder cells consist of both special and nonspecial cells. Special or predetermined yielders occurred only among the earliest yielders. In most instances, yielder-cell formation was most easily explained by assuming that the first step was a chance escape of the restricted phage DNA from the degrading enzyme of the restricting cell.
Biochemical characterization of the type C retrovirus associated with lymphoproliferative disease of turkeys
Authors: Yaniv A , Gazit A , Ianconescu M , Perk K , Aizenberg B , Zimber A
Published: January 2020

Turkeys inoculated with spleen extracts from lymphoproliferative disease (LPD)-affected birds developed viremia, followed by typical LPD lesions. Electron microscopy and biochemical characterization established that the virus present in the blood of infected turkeys is a type C retrovirus. The viral particles possess a buoyant density of 1.17 g/ml in sucrose gradients; they contain high-molecular-weight RNA and an RNA-instructed DNA polymerase with efficient exogenous and endogenous activity. The LPD virus polymerase is preferentially activated by magnesium ions. Cross nucleic acid hybridization assays revealed no sequence homology between the viral genome of LPD and avian myeloblastosis virus or reticuloendotheliosis virus, thus indicating that the LPD virus belongs to a distinct group unrelated to the avian leukosis-sarcoma virus complex or to the reticuloendotheliosis virus group.
Simian Virus 40-Host Cell Interaction During Lytic Infection
Authors: Gershey Edward L.
Published: January 2020

Both exponentially growing and serum-arrested subcloned CV-1 cell cultures were infected with simian virus 40 (SV40). By 24 h after infection 96% of the nuclei of these permissive cells contained SV40 T-antigen. Analysis of the average DNA content per cell at various times after infection indicated that by 24 h most of the cells contained amounts of DNA similar to those normally found in G
2
cells. Analysis of cell cycle distributions indicated that a G
2
DNA complement was maintained by over 90% of the cells in the infected populations 24 to 48 h postinfection. Cells continued to synthesize SV40 DNA during the first 50 h after infection, and cytopathic effect was first observed 60 h after inoculation. After infection the number of mitotic cells that could be recovered by selective detachment decreased precipitously and was drastically reduced by 24 h. A study of the kinetics of decline in the number of mitotic cells suggests that this decline is related to an event during the cell cycle at or near the G
1
-S-phase border upon which commencement of SV40 DNA replication apparently depends. It was concluded that after SV40 infection, stationary cells are induced to cycle, and cycling cells complete one round of cellular DNA synthesis but do not divide. Although the infected cells continue to synthesize viral DNA, they do not appear able to reinitiate cellular DNA replication units. These results imply that the abundance of T-antigen (produced independently of cell cycle phase) in the presence of the enzymes required for continued DNA synthesis is not sufficient for reinitiation of cellular DNA synthesis.
gag-Related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms
Authors: Edwards S A , Fan H
Published: January 2020

Immunoprecipitation of labeled extracts from murine leukemia virus-infected cells with antisera specific for internal structural (gag) proteins yields three major gag-related polyproteins with molecular weights of 180,000 (Pr180gag-pol), 80,000, and 65,000 (Pr65gag). It has been shown by others that Pr65gag is the immediate precursor of the internal structural (gag) protein, and that Pr180gag-pol is the precursor to reverse transcriptase. In studies reported here, the 80,000-dalton gag-related polyprotein from Moloney strain murine leukemia virus (M-MuLV)-infected cells was found to be glycosylated by the following criteria: (i) incorporation of [3H]mannose, (ii) a change in electrophoretic mobility upon digestion with endoglycosidase H, and (iii) a change in electrophoretic mobility when glycosylation was inhibited by treatment of the cells with tunicamycin during labeling. The 80,000-dalton gag polyprotein has therefore been designated GpP80gag. The unglycosylated form of GpP80gag was a polypeptide of 75,000 daltons. A comparison of [3H]mannose and [3H]galactose labeling experiments suggested that GpP80gag is further glycosylated to yield a glycopolypeptide of 95,000 daltons. This 95,000-dalton polypeptide is relatively rapidly cleaved to yield two glycopeptides of 55,000 and 40,000 daltons which are released into the cell culture fluid, as soluble proteins. Cell-free translation of M-MuLV genomic RNA resulted in two major gag-related products of 75,000 and 65,000 daltons. The 65,000-dalton gag-related cell-free translation product comigrated with Pr65gag, and the 75,000-dalton cell-free product comigrated with the unglycosylated form of GpP80gag. Both of the gag-related cell-free translation products could be labeled with [35S]formyl methionine, which is incorporated only as the N-terminal amino acid during translation. Other investigators have shown that GpP80gag and Pr65gag differ at their N-termini, and these results combined with those reported here suggest that GpP80gag and Pr65gag are translated from two separate initiation sites in M-MuLV RNA.
Induction of mouse mammary tumor virus RNA in mammary tumors of BALB/c mice treated with urethane, X-irradiation, and hormones
Authors: Michalides R , van Deemter L , Nusse R , Hageman P
Published: January 2020

The involvement of mouse mammary tumor virus (MTV) in the development of mammary tumors of nonviral etiology in BALB/c mice was studied by measuring the levels of MTV RNA, MTV DNA, and MTV proteins in spontaneously arising and hormonally, chemically, and/or physically induced mammary tumors of BALB/c females. The following results were obtained. (i) Spontaneous mammary tumors contained very low levels of MTV RNA; 4 X 10(-6)% of the the cytoplasmic RNA was MTV RNA. No MTV proteins could be demonstrated by using sensitive radioimmunoassays for MTV proteins p27 and gp52. (ii) Mammary tumors induced by treatments with urethane or X-irradiation alone contained higher levels of MTV RNA; these tumors contained 3- and 19-fold more MTV RNA, respectively, compared with spontaneous mammary tumors. (iii) Mammary tumors induced by combined treatment with urethane and X-irradiation expressed high levels of MTV RNA in the mammary tumors; a 1,724-fold increase in MTV RNA content compared with spontaneous mammary tumors was observed. However, very low levels of MTV proteins gp52 and p27 were detected, suggesting some kind of impairment at the translation of the MTV RNA. MTV RNA was also induced by this treatment in mammary glands and spleens, but not in the livers of tumor-bearing animals. (iv) Balb/c females continuously exposed to prolactin contained high levels of MTV RNA and MTV proteins in stimulated mammary glands and in the hormonally induced mammary tumors. These findings suggest that MTV is not responsible for the maintenance and probably also not for the development of all murine mammary cancers.
Host cell DNA chain initiation protein requirements for replication of bacteriophage G4 replicative-form DNA
Authors: Dumas L B , Goltz S , Benesh D T
Published: January 2020

Bacteriophages G4ev1 and G4bs1 are simple temperature-resistant derivatives of wild-type G4 as demonstrated by restriction endonuclease analyses. The rate of replication of the duplex replicative-form DNA of these phages was normal in dnaB and dnaC mutants of the host, whereas the rate was markedly reduced in a dnaG host mutant at the restrictive temperature. We conclude that G4 duplex DNA replication requires the host cell dnaG protein, but not the dnaB and dnaC proteins. The reasons for the differences between our conclusions and those based on previously published data are documented and discussed.
Gs, an allele of chickens for endogenous avian leukosis viral antigens, segregates with ev 3, a genetic locus that contains structural genes for virus
Authors: Astrin S M , Robinson H L
Published: January 2020

Gs is an allele of chickens for the expression of endogenous avian leukosis virus-related core (gs) and envelope (chf) antigens. Progeny of a genetic cross in which Gs was segregating were analyzed for endogenous viral DNA as well as for the expression of endogenous viral antigens. Viral genetic information was identified by cleavage of embryo DNA with restriction endonucleases, electrophoretic separation of the resulting fragments, and identification of bands containing viral sequences by hybridization of the DNA to 32P-labeled viral RNA. Four different chromosomal sites of residence of endogenous viral sequences were identified by this method. These sites were the same as those previously assigned to the endogenous viral loci ev 1, ev 3, ev 4, and ev 5. ev 1 was present in all of the progeny of the cross. ev 3, ev 4, and ev5 were present in various combinations with ev 1. ev 3 cosegregated with the gs+chf+ phenotpye. Cells which did not contain ev 3 but contained ev 1, ev 4, and/or ev 5, did not express detectable levels of viral antigens. We suggest that Gs contains the structural genes for endogenous virus which reside at ev 3 and that these structural genes code for gs and chf in gs+chf+ cells.
Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA's of B77 Avian Sarcoma Virus
Authors: Stoltzfus C. Martin , Kuhnert Lee K.
Published: January 2020

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5′-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5′ sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5′ terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3′ terminal half of the 38S RNA. The entire 101-base 5′ terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5′-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5′ sequences was similar to the fraction of RNA containing sequences identical to the extreme 3′ terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5′-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5′-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5′-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5′-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5′ leader sequence or additional splicing in the terminal 101 bases.
Analysis of the DNAs from seven varicella-zoster virus isolates
Authors: Richards J C , Hyman R W , Rapp F
Published: January 2020

The 32P-labeled DNAs from seven different clinical isolates of human varicella-zoster virus (VZV) were independently digested with five site-specific restriction endonucleases, EcoRI, HindIII, SmaI, BamHI, and AvaI. The digestion products were analyzed by electrophoresis on 0.5% agarose gels followed by autoradiography of the dried gels. Evaluation of the restriction enzyme cleavage patterns revealed small variations among the VZV DNAs. The VZV DNAs were also compared based on their buoyant densities in CsCl. No significant buoyant density differences were detected among the VZV DNAs.
In vitro association of empty adenovirus capsids with double-stranded DNA
Authors: Tibbetts C , Giam C Z
Published: January 2020

Several lines of evidence suggest that empty adenovirus capsids are preassembled intermediates in the pathway of virion assembly. We have observed that purified empty capsids of subgroup B adenoviruses have a remarkable affinity for DNA in vitro. The products of capsid-DNA association are sufficiently stable, once formed in low-salt solution, to permit purification and characterization in CsCl density gradients. Neither virions nor the DNA-containing incomplete particles of subgroup B adenoviruses can give rise to such in vitro reaction products. The average molecular weight of the empty adenovirus capsids is about 123 X 10(6), consistent with the absence of viral core peptides and a small deficiency of exterior shell polypeptides. Electron microscopy of negatively stained capsids and the capsids bound to DNA reveals a typical adenovirus size and architecture. The particles appear with a surface discontinuity that is presumed to expose the DNA binding site(s). The DNA molecules associated with the empty capsids are susceptible to the actions of DNase and restriction endonucleases. The dependence of rate of capsid-DNA association on DNA length suggests randomly distributed binding sites on the DNA molecules. Although the DNA molecules can successively acquire additional empty capsids, the empty particles themselves are restricted to interactionwith only one DNA molecule. Electron microscopy of the capsid-DNA complexes spread in cytochrome c films shows that the particles are bo-nd along the contour of extended duplex DNA. The amount of DNA within each bound particle appears to be less than 300 base pairs, as estimated by the length of the DNA molecules visible outside of the bound particle. The empty capsid-DNA association product described in this report provides an interesting substrate for further investigation of the DNA packaging process in a defined in vitro system, with extracts or purified components from infected cells.
Effect of Canavanine on Murine Retrovirus Polypeptide Formation
Authors: Murphy Edwin C. , Arlinghaus Ralph B.
Published: January 2020

Canavanine is an arginine analog which is widely used to inhibit proteolytic processing of viral polyproteins. Certain results obtained with canavanine have suggested that it may have other effects. Therefore, we examined the effects of canavanine on the cell-free synthesis of murine retrovirus proteins. It was found that the electrophoretic mobility of the major
gag
-related cell-free product of both Rauscher murine leukemia virus (R-MuLV) and Moloney murine sarcoma virus 124 (Mo-MuSV-124) RNA was dependent on the concentration of canavanine used during translation. As the canavanine concentration was increased up to 4 mM, the apparent size of the major
gag
-related polypeptide also increased from 65,000 (R-MuLV RNA) or 63,000 (Mo-MuSV-124 RNA) to approximately 80,000 daltons. Additional increases in the canavanine concentration up to 12 mM did not increase the size of the
gag
gene product beyond 80,000 daltons. This change in electrophoretic mobility appeared to be due to a substitution of canavanine for arginine residues in the polypeptides, not to a change in their actual size. If amber suppressor tRNA and canavanine were used together during translation of Mo-MuSV-124 RNA and Mo-MuLV RNA, the results were also in agreement with this proposal. Translation experiments done with ovalbumin mRNA and mengovirus 35S RNA indicated that canavanine incorporation caused a shift in the electrophoretic mobility of ovalbumin from 43,000 to 45,000 daltons and caused the appearance of two slightly larger polypeptides in the 155,000- and 115,000- dalton regions of the mengovirus RNA cell-free product.
Synthesis of thymine and alpha-putrescinylthymine in bacteriophage phi W-14-infected Pseudomonas acidovorans
Authors: Maltman K L , Neuhard J , Lewis H A , Warren R A
Published: January 2020

Host DNA synthesis stopped about 10 min after the infection of Pseudomonas acidovorans with bacteriophage phi W-14, but host DNA was not degraded to acid-soluble fragments. The synthesis of host but not of phage DNA was inhibited by 5-fluorodeoxyuridine. The nucleotide pools of infected cells did not contain dTTP, and infection resulted in the appearance of dTTPase activity. Although ornithine labeled the alpha-putrescinylthymine residues of phi W-14 DNA, ornithine-labeled nucleotides were not detected in infected cells. A new deoxynucleoside triphosphate did appear in infected cells, but it was not labeled by ornithine. It is concluded that the thymine and alpha-putrescinylthymine in phi W-14 DNA are synthesized at the polynucleotide level.
RNA-dependent DNA polymerase of an endogenous type C virus of mice: purification and partial characterization
Authors: Hizi A , Yaniv A
Published: January 2020

An RNA-dependent DNA polymerase was isolated from purified virions of endogenous oncornaviruses released by the MOPC-315 murine myeloma cell line. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme was found to consist of two major polypeptides with molecular weights of about 28,000 and 26,500. The active enzyme had a molecular weight of approximately 56,000, as calculated from its sedimentation on glycerol density gradients, indicating that it is probably a dimer of the two subunit polypeptides. The isolated MOPC-315 virus polymerase exhibited all three activities known to be found in the DNA polymerase from oncornaviruses, namely, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and an RNase H. The RNA-dependent polymerase activity showed a prounced preference for Mn2+ over Mg2+, whereas the DNA-dependent and RNase H reactions were catalyzed by these two cations to an almost equal extent. The purified polymerase was found to be immunologically related to the polymerase of Rauscher murine leukemia virus.
Characterization of virus produced by a lymphoma induced by inoculation of AKR MCF-247 virus
Authors: Pedersen F S , Buchhagen D L , Chen C Y , Hays E F , Haseltine W A
Published: January 2020

We report the characterization of the virus produced by a lymphoid cell line derived from a lymphoma of an AKR mouse after injection of the polytropic AKR virus MCF-247. The virus displays polytropic host range properties and is indistinguishable from MCF-247 as judged by analysis of the large RNase T1-resistant oligonucleotides of the RNA genome. Restriction enzyme analysis of cellular DNA revealed the presence of sequences homologous to MCF-247 genomic RNA. The EcoRI cleavage fragments were characteristic of MCF-247 DNA provirus cleavage products.
Cleavage of phi X174 single-stranded DNA by gene A protein and formation of a tight protein-DNA complex
Authors: Eisenberg S
Published: January 2020

The gene A protein cleaves phi X174 single-stranded DNA (ssDNA). The cleavage appears to be stoichiometric, whereby a gene A protein molecule breaks a phosphodiester bond and binds to the 5' end. The enzyme introduces mostly a single break in a circular ssDNA molecule. However, at high enzyme-to-DNA ratios, more than one break in the DNA could be observed. The cleavage of the ssDNA by gene A protein renders the DNA sensitive to the action of terminal transferase to incorporate [alpha -32P]ATP. Thus, the 3'OH end is free. All attempts to label the 5' end by T4-induced polynucleotide kinase and [gamma-32P]ATP failed. The formation of a gene A-ssDNA complex was demonstrated directly by using 3H-labeled gene A protein and 32P-labeled ssDNA in the reaction. Such a complex is resistant to treatments with 0.2 M NaOH, banding in CsCl, and boiling in 2.5% sodium dodecyl sulfate. Only treatment with a nuclease released the bound protein. Also, after cleaving [32P]ssDNA by gene A protein, followed by either DNase I or micrococcal nuclease digestion, a fraction of the 32P label remained resistant to nuclease treatment and comigrated with gene A protein on polyacrylamide gels.
Characterization of intracellular viral RNA in interferon-treated cells chronically infected with murine leukemia virus
Authors: Salzberg S , Bakhanashvili M , Bari S , Berman I , Aboud M
Published: January 2020

We have recently found that Moloney murine leukemia virus assembles within cytoplasmic vacuoles of chronically infected NIH/3T3 cells rather than at their surface (submitted for publication). In the present study we found that if these cells were treated with interferon (IF) for 24 to 48 h the intracellular virus particles accumulated at a two- to threefold-higher level than that observed in untreated cells. Nevertheless, despite this accumulation, no difference between IF-treated and untreated cells was observed in the amount of the total cytoplasmic viral RNA or in its 35S or 21S species. When cellular virions were sedimented from the cytoplasmic fraction, a markedly higher amount of viral RNA was detected in the viral pellet of IF-treated cells than was detected in untreated cells, whereas the amount of viral RNA left in the virus-free cytoplasm of IF-treated cells was much lower than that in the untreated cells. Furthermore, the amount of the cytoplasmic polyriboadenylic acid-containing viral RNA was also remarkably higher in the IF-treated cells. Viral polyribosomes appeared to be fully functional in IF-treated cells, since no effect of IF on viral protein synthesis could be detected. Analysis of the nuclear viral RNA showed no difference between IF-treated and untreated cells after 24 h of IF treatment. Both contained a comparable amount of 35S viral RNA. However, at 48 h a significant accumulation of viral RNA was observed in the nucleus of the IF-treated cells as compared with the untreated cells, although in both cases only 35S species were evident. This accumulation appeared to activate a degradation process which destroyed nuclear viral RNA, since a dramatic shift toward smaller-sized molecules of viral RNA and a remarkable reduction in its amount were observed after 72 h of IF treatment.