bluuebunny/crossref_metadata_2025 huggingface dataset:
Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrilich ascites tumor cells: further characteristics of the phenomenon
Authors: Sen G C , Shaila S , Lebleu B , Brown G E , Desrosiers R C , Lengyel
Published: January 2020

We reported earlier that the methylation of unmethylated reovirus mRNA (reo mRNAU) by the cellular methylating enzymes is impaired in extracts of uninfected, interferon-treated Ehrilich ascites tumor cells (S30INT). We find now that after the methylation of reo mRNAU has stopped in S30INT, the RNA can be reisolated and further methylated in an extract of control cells (S30C). Thus the impairment of methylation in S30INT cannot be due to cleavage or irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is due to the depletion of S-adenosylme thionine (the methyl donor), the accumulation of S-adenosylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is not due to the depletion of S-adenosylmethionine (the methyl donor), the accumulation of S-adenoxylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of the methylating enzymes. It may be due, however, to the unavailability of reo mRNAU for methylation. The extent of the impairment of reo mRNAU methylation in S30INT decreases with an increasing concentration of reo mRNAU but is not affected by added poly (U), ribosomal RNA, or encephalomyocarditis virus RNA (an mRNA that is probably not capped or methylated at its 5' end). The methylation of reo mRNAU is also impaired in an extract from cells that have not been treated with interferon but with the interferon inducer poly(I) - poly(C). The inhibitor is apparently a macromolecule that is inactivated during incubation. It decreases the methylation at the 7 position of the 5' terminal guanylate residue. In vitro, the rate of reo mRNA synthesis by reovirus cores in the presence of S30INT is the same as in the presence of S30C. However, the methylation of the de novo synthesized reo mRNA by the core-associated methylating enzyme(s) in vitro is inhibited by S30INT but not by S30C. The relevance of these phenomena to the inhibition of reovirus replication in interferon-treated cells remains to be established.
Evidence for a diffusible T4 bacteriophage protein governing the initiation of delayed early RNA synthesis
Authors: Linder C H , Sköld O
Published: January 2020

Two forms of prereplicative phage RNA can be discerned in Escherichia coli early after infection with bacteriophage T4, immediate early and delayed early RNA. The transition from immediate early to delayed early RNA synthesis is inhibited by chloramphenicol. The present work presents evidence for the existence of a phage-specific protein, which effects this transition. Delayed early RNA formation was measured by a cistron-specific enzyme-forming-capacity method, in which RNA synthesized in the presence of chloramphenicol was allowed to express itself into enzyme activity after (i) the addition of rifampin to inhibit further transcription and (ii) subsequent removal of chloramphenicol. As representatives of delayed early transcription, the two phage-specific enzymes dCTPase and deoxynucleotide kinase were chosen. Primary infection with a phage mutant defective in one of these two enzymes was found to induce a diffusible factor, which in the presence of chloramphenicol could effect the formation of delayed early RNA corresponding to the missing enzyme, upon superinfection with wild-type phage. The activity of this factor, acting in trans, was abolished by the amino acid analogue ethionine. Mutation in the suA gene of the host did not relieve phage of the apparent need for protein synthesis in the transition from immediate early to delayed early phage RNA synthesis.
In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification
Authors: White J L , Murakishi H H
Published: January 2020

A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form tobacco mosaic virus-infected tobacco callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. The viral RNA synthesis in vitro proceeded linearly for 30 min and required the four nucleotide triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digested by RNase in 0.1X SSC. Analysis of the product by polns) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized enzyme. The solubulized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.
Isolation of recombinants between T7 and T3 bacteriophages and their use in vitro transcriptional mapping
Authors: Beier H , Golomb M , Chamberlin M
Published: January 2020

A variety of T3 X T7 recombinants were isolated from crosses between T3 and T7 parental phages carrying amber markers in various genes (gene 1 to gene 19). The genetic constitution of these recombinants was determined by reference to the selected markers and also directly by analysis of the proteins translated from the T3 X T7 recombinants in vivo. Although T3 and TM phages are closely related, most T3 and T7 proteins differ slightly in size, and hence the genetic origin of a gene can be determined by protein analysis. The major transcripts read by T3 and T7 RNA polymerases from T3 X T7 recombinant phage DNAs vary, depending on which regions of the T3 or T7 chromosome are present. T7 RNA polymerase is unable to utilize major promoter sites employed by T3 polymerase at an appreciable rate, and the converse is also true. Hence the transcriptional pattern for a recombinant phage DNA obtained with the T3 or T7 polymerase allows a determination of the identity of the different promoter sites on the genome. The transcriptional analysis of T3 X T7 recombinant DNAs together with earlier observations has been used to map the promoter sites for five out of seven major T3 and T7 RNA species on the genetic maps of T3 and T7. The promoter sites for the T7 and T3 RNA species IIIa, IV, and V originate at the beginning of genes 7, 9, and 10, respectively; the promoter sites for the T7 and T3 RNA species I and II are located to the left of gene 11 and gene 13, respectively. No T3 X T7 recombinants were found for which the specificity of the phage RNA polymerase was not correlated with the corresponding promoter sites for species IIIa and I (the transcription of which covers 60% of the genome). That means that the RNA polymerase specified by the recombinant genome is able to read all of the information encoded in sequences read normally from major promoters by the enzyme on the wild-type phage genome. This suggests that the in vitro specificity for promoter site selection by the phage polymerases is also maintained in vivo.
Electron microscopic studies of circular DNA in mouse embryo fibroblasts infected by Rauscher leukemia virus
Authors: Kakefuda T , Lovinger G G , Gilden R V , Hatanaka M
Published: January 2020

Using electron microscopy, a closed circular form of DNA (4.3 mum in contour length) was detected in the nucleus of mouse embryo fibroblasts 2.5 h after infection by Rauscher murine leukemia virus. These circles were distinguishable from mitochondrial DNA by various criteria, including size, absence of secondary features, and resistance to EcoRI endonuclease.
Proteins of bovine leukemia virus. I. Characterization and reactions with natural antibodies
Authors: Deshayes L , Levy D , Parodi A L , Levy J P
Published: January 2020

The bovine leukemia virus (BLV) was purified from a chronically infected fetal lamb kidney cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this virus revealed the presence of eight distinguishable viral components with molecular weights ranging from 80,000 to 11,000. The major component is a non-glycosylated protein having a molecular weight of 24,000 (p24). At least three heavier polypeptides were found, one of them representing a glycoprotein (gp 60). In addition, four minor polypeptides with respective molecular weights of 19,000, 16,000, 13,000, and 11,000 were identified. In a complement fixation assay using naturally occurring antibodies of a leukemic cow, four polypeptides, which included gp 60, p35, p24, and p16, were found to be reactive.
UV-induced alleviation of K-specific restriction of bacteriophage lambda
Authors: Day R S
Published: January 2020

A partial release of K-specific restriction of phage lambda grown in Escherichia coli C was observed when E. coli K strains AB1157 (having wild-type repair of UV-produced DNA damage) and AB1886 (uvrA) were irradiated with UV light before infection. The effect occurred in AB1886 at lower UV fluences than it did in AB1157. Little or no release of restriction was observed when AB2463 (recA) or AB2494 (lex-1) was used. Such release of restriction appears to be another of the UV-induced phenomena associated with "SOS" repair.
Factors affecting composition and thermostability of mengovirus virions
Authors: Bond C W , Swim H E
Published: January 2020

The composition of mengovirus virions produced by infected cells varies with the incubation temperature. Virons produced at 37.0 or 39.5 degrees contain four major polypeptides (alpha, beta, gamma, and delta) and one minor polypeptide (beta'). Virons produced at 31.5 degrees C contain two additional polypeptides (D1 and E). The virions of two temperature-sensitive (ts) and thermolabile mutants of mengovirus (ts25 and ts88) contain an increased amount of polypeptide beta', with a corresponding decrease in polypeptide beta when compared with the wild-type mengovirus.
Regulation of Sendai virus transcription: evidence for a single promoter in vivo
Authors: Glazier K , Raghow R , Kingsbury D W
Published: January 2020

The synthesis of Sendai virus RNA species was examined after UV irradiation of cells late in infection. Compared with the inactivation of 50S genomic RNA synthesis, the synthesis of the group of mRNA species that sediments at 18S was inactivated at an average rate consistent with a process of sequential transcription from a single promoter. The rates of inactivation of the synthesis of individual mRNA's separated by polyacrylamide gel electrophoresis confirmed this and, with the aid of additional data, suggested that the order of genes in the Sendai virus genome is: 3'-NP-Fo-M-P-HM-L-5'.
Divergence of baboon endogenous type C virogenes in primates: genomic viral RNA in molecular hybridization experiments
Authors: Donehower L , Wong-Staal F , Gillespie D
Published: January 2020

RNA purified from two related RNA tumor viruses, one isolated from a baboon, Papio anubis, and the second from cultured blood leukocytes of a patient with acute myelogenous leukemia, was labeled with 125I and hybridized to DNA from different primates. RNA from both viruses showed maximum sequence homology with genes in baboons and little homology with genes of humans. The results confirm earlier suggestions that both viruses originated by transcription of baboon virogenes, and that one was transmitted to humans in nature. Hybridization of the viral RNA to cell DNA followed complicated kinetic patterns, indicating the presence of both repeated and infrequent virogene elements. This conclusion was verified in experiments using varied DNA:RNA ratios. It is proposed that virogenes, though composed of genes repeated 10 times or more, consist of some sequences more preferentially conserved than others. The non-uniformity of virogene sequence conservation limits the use of viral probes in studies concerning certain aspects of virogene evolution.
Homogeneity and complexity of avian oncornavirus proviral DNA determined by molecular hybridization
Authors: Evans R M , Shoyab M , Drohan W N , Baluda M A
Published: January 2020

The homogeneity of DNA complementary to the 35S RNA subunit of avian myeloblastosis virus (AMV) has been demonstrated by single or multistep hybridization. For multistep hybridizations, 35S AMV RNA was preselected for its ability to hybridize either to unfractionated leukemic DNA or to leukemic DNA enriched for unique or for reiterated sequences. These experiments indicate that the viral genome is complementary to DNA sequences with a low reiteration frequency. Competition experiments confirm the absence of fast-hybridizing sequences in viral DNA. Computer analyses of the data reveal that there are two to four copies of viral DNA in infected cells.
Isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the C3H and RIII mouse strains
Authors: Drohan W , Kettmann R , Colcher D , Schlom J
Published: January 2020

Radioactive 60-70S RNA from the mouse mammary tumor virus (MMTV) produced by the C3H mouse mammary tumor cell line (Mm5mt) hybridized to a greater extent, and at a lower Cot1/2 value, to the DNA of C3H mammary tumor cells than to the DNA of C3H liver cells. The 125I-labeled MMTV (C3H) 60-40S RNA was annealed to a vast excess of DNA from C3H livers, and single-stranded RNA was eluted from hydroxylapatite and recovered. This "recycled RNA" did not hybridize to the DNA of the apparently normal organs tested from normal or from mammary tumor-bearing C3H mice, but hybridized extensively to both the DNA from the C3H mammary tumor cell line and the DNA from spontaneous C3H mammary tumors. This hybridization could be competed out by the addition of unlabeled MMTV 60-70S RNA but was unaffected by the addition of unlabeled 60-70S RNA of C3H type C virus. Similar experiments were conducted with the RIII mouse strain. We therefore report on the isolation of the sequences of the RNA genomes of the MMTVs from C3H and RIII mice that are transmitted by some mechanism other than via the germ line. These studies further define the differences, via molecular hybridization, between the MMTV-S and the MMTV-L in both C3H and RIII mice.
Evidence that herpes simplex virus DNA is transcribed by cellular RNA polymerase B
Authors: Costanzo F , Campadelli-Fiume G , Foa-Tomasi L , Cassai E
Published: January 2020

In herpes simplex virus type 1 (HSV-1)-infected HEp-2 cells, amanitin added before or at various times after infection always reduced viral multiplication. Also, the three waves of transcription of HSV-1 DNA, which led to the synthesis of alpha, beta-, and gamma-polypeptides, were all sensitive to amanitin in HEp-2 cells, and the amanitin-sensitive RNA polymerase activities of isolated nuclei were equally sensitive to the inhibitor before and during the infection. On the contrary, HSV-1 DNA transcription was totally unaffected by amanitin in AR1/9-5B cells, a mutant subline of CHO cells that possesses an amanitin-resistant RNA polymerase B. Together, these results strongly suggest that HSV-1 DNA utilizes for its transcription a polymerase undistinguishable from host cell RNA polymerase B with respect to its sensitivity to amanitin.
Specific inhibition of DNA polymerase-associated RNase H by DNA
Authors: Modak M J , Marcus S L
Published: January 2020

The RNase H activity associated with several RNA-directed DNA polymerases is inhibited by the addition of DNA, in contrast to RNase H activity from enzymes devoid of polymerizing activity. Kinetic investigations of the inhibitory effect of DNA, using purified Rauscher leukemia virus DNA polymerase as a test enzyme, revealed that the addition of DNA to an ongoing RNase H reaction causes an immediate cessation of RNase H activity. Concomitant initiation of DNA synthesis by inhibitory DNA can occur, provided that appropriate substrate and primer is available. Thus, in addition to providing a simple test for the distinction between polymerase-associated and polymerase-independent RNase H activity, this study strongly supports the concepts that (i) RNase H activity expressed by several mammalian oncoviral reverse transcriptases is an integral part of that molecule, and (ii) that the catalytic site of RNase H activity is also involuved in template-primer binding.
Viral proteins expressed on the surface of murine leukemia cells
Authors: Ledbetter J , Nowinski R C , Emery S
Published: January 2020

Leukemic cells of AKR mice contain as constituents of their membranes the murine leukemia virus envelope protein gp70 and the precursor polyprotein of the viral internal (core) structural proteins. Both gp70 and the core polyprotein are represented on the cell surface as glycoproteins, as evidenced by incorporation of [3H]glucosamine into their structure and the binding of these proteins to lectins. The glycosylated core polyprotein exists in at least two serologically distinguishable forms: the 95,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30, p12, and p10, whereas the 85,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30 and p12, but not p10. Additional heterogeneity in these cell surface polyproteins has been observed wtih leukemias induced by exogenous leukemia viruses. Spontaneous leukemia cells of AKR mice invariably express gp70 and the core polyprotein on their cell surface; normal thymocytes of young AKR mice express gp70, but not the core polyprotein on their surface.
Properties of permissive monkey cells transformed by UV-irradiated simian virus 40
Authors: Gluzman Y , Davison J , Oren M , Winocour E
Published: January 2020

African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.
Friend strain of spleen focus-forming virus: a recombinant between mouse type C ecotropic viral sequences and sequences related to xenotropic virus
Authors: Troxler D H , Boyars J K , Parks W P , Scolnick E M
Published: January 2020

The genome of the Friend strain of the spleen focus-forming virus (SFFV) has been analyzed by molecular hybridization. SFFV is composed of genetic sequences homologous to Friend type C helper virus (F-MuLV) and SFFV-specific sequences not present in F-MuLV. These SFFV-specific sequences are present in both the Friend and Rauscher strains of murine erythroleukemia virus. The SFFV-specific sequences are partially homologous to three separate strains of mouse xenotropic virus but not to several cloned mouse ecotropic viruses. Thus, the Friend strain of SFFV appears to be a recombinant between a portion of the F-MuLV genome and RNA sequences that are highly related to murine xenotropic viruses. The implications of the acquisition of the xenotropic virus-related sequences are discussed in relation to the leukemogenicity of SFFV, and a model for the pathogenicity of other murine leukemia-inducing viruses is proposed.
Spatial relationships of the proteins of vesicular stomatitis virus: induction of reversible oligomers by cleavable protein cross-linkers and oxidation
Authors: Dubovi E J , Wagner R R
Published: January 2020

To delineate the proximity and spatial arrangement of the major structural proteins of intact vesicular stomatitis (VS) virions, protein complexes formed by oxidation or by bivalent cross-linkers were analyzed by two-dimensional electrophoresis on polyacrylamide slab gels. H2O2 oxidation of VS virions produced an N-polypeptide dimer (molecular weight, approximately equal to 110,000) on a first dimension gel that could be reduced to N monomers (molecular weight, approximately equal to 50,000). Proteins extracted from unreduced and unoxidized VS virions contained dimeric and trimeric forms of M-protein complexes as well as a heterodimer of M and N protein. Qualitatively similar VS viral protein complexes were generated by exposing VS virions to the reversible protein cross-linkers methyl-4-mercaptobutyrimidate (MMB), tartryl diazide (TDA), and dithiobis(succinimidyl proprionate) (DTBSP); cross-linked complexes on first-dimension gels were cleaved by reduction with 2-mercaptoethanol (MMB or DTBSP cross-linked) or by periodate oxidation (TDA cross-linked). In addition to covalently linked homodiamers of M and N proteins and a protein M-N heterodimer, the protein cross-linkers also generated homo-oligomers of G protein and a G-M heterodimer. These data suggest that the glycoprotein spike of VS virus is composed of more than one G protein. The existence of N-M and G-M heterodimers is consistent with the hypothesis that the matrix (M) protein may serve as a bridge between the G and N proteins in assembly of the VS virion.
Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes
Authors: Hiebsch R , Center M S
Published: January 2020

After infection of Escherichia coli with bacteriophage T7, the parenteral DNA forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Bruij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000 and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.
Alteration of the Intracellular Energetic and Ionic Conditions by Mengovirus Infection of Ehrlich Ascites Tumor Cells and Its Influence on Protein Synthesis in the Midphase of Infection
Authors: Egberts Egbert , Hackett Perry B. , Traub Peter
Published: January 2020

Mengovirus infection of Ehrlich ascites tumor cells caused a change of the intracellular ATP concentration. It increased by 35% within the first 3 h postinfection and then declined to zero within the next 5 h. The decrease in the ATP concentration was due, at least in part, to leakage of ATP into the medium, where it could be demonstrated by the luciferin-luciferase assay. Gross leakage of ATP was observed at 4.5 h postinfection, concomitant with the production of the first intracellular, infectious virus particles. A similar concentration decrease was detected for Mg
2+
, the polyamines, and K
+
, whereas an increase in the Na
+
concentration was observed. The intracellular Mg
2+
concentration varied synchronously with the ATP level, rising by 16% during the first 3 h postinfection and then progressively falling to lower values in the late period of the infectious cycle. After an initial slight enhancement, the putrescine, spermidine, and spermine concentrations declined at about 1.5 h postinfection. Wherease the intracellular K
+
concentration increased by 17% during the first hour postinfection, the Na
+
concentration diminished by the same value within the same time period, leaving the internal ionic strength unchanged early in infection. Three hours after the beginning of virus infection, there was a rapid decline of K
+
and enhancement of Na
+
within the cell. These alterations of the intracellular energetic and ionic conditions seem to be, at least in part, responsible for the cessation of virus-specific protein synthesis in mengovirus-infected Ehrlich ascites tumor cells commencing 3 to 3.5 h postinfection.
Isolation of specialized lambda transducing bacteriophages for flagellar genes (fla) of Escherichia coli K-12
Authors: Komeda Y , Shimada K , Iino T
Published: January 2020

Specialized transducing lambda phages carrying the region III flagellar genes (fla) of Escherichia coli K-12 were isolated by a new method. A strain carrying both a cryptic lambda prophage near the his genes and a deletion of the attlambda gene was used as a starting strain. The lysogen of lambdacI857pga18-bio69 was isolated in which the prophage was integrated within the lambda cryptic genes by means of recombination with the residual lambda DNA. The strains with deletions starting within the prophage and ending in these fla genes were selected from among the heat-resistant survivors of the lysogen. They were then infected with heat-inducible and lysis-defective lambda phages and, thus, specialized transducing phage lines for hag and fla were obtained. High-frequency transfer lines of rare phages carrying the fla genes were isolated by inducing a strain carrying a heat-inducible lambda prophage near the his genes and selecting by transduction of a fla deletion strain. Preliminary characterization of these transducing phages is also reported.
Episomal Viral DNA in a
Herpesvirus saimiri
-Transformed Lymphoid Cell Line
Authors: Werner Fred-Jochen , Bornkamm Georg W. , Fleckenstein Bernhard
Published: January 2020

The lymphoid cell line #1670 has been derived from the infiltrated spleen of a tumor-bearing marmoset monkey infected with
Herpesvirus saimiri.
The cells contain both types of
H. saimiri
DNA, unique light (L-) DNA (36% cytosine plus guanine) and repetitive heavy (H-) DNA (71% cytosine plus guanine), without producing infectious virus. Viral DNA was found to persist in these cells as nonintegrated circular DNA molecules. Closed circular superhelical viral DNA molecules were isolated by three subsequent centrifugation steps: (i) isopycnic centrifugation in CsCl, (ii) sedimentation through glycerol gradients, and (iii) equilibrium centrifugation in CsCl-ethidium bromide. The isolated circles had a molecular weight of 131.5 ± 3.6 × 10
6
. This is significantly higher than the molecular weight of linear DNA molecules isolated from purified
H. saimiri
virions (about 100 × 10
6
). Partial denaturation mapping of circular molecules from #1670 lymphoid cells showed uniform arrangement of H- and L-DNA sequences in all circles. All denatured molecules contained two L-DNA regions (molecular weights of 54.0 ± 1.8 × 10
6
and 31.5 ± 1.3 × 10
6
) and two H-DNA regions (molecular weight of 25.6 ± 1.9 × 10
6
and 20.0 ± 0.8 × 10
6
) of constant length. Maps of both L-regions suggested that the sequences of the shorter L-DNA region were a subset of those of the longer region. The sequences of both L-regions had the same orientation. Circular molecules from
H. saimiri
-transformed lymphoid cell line #1670 appeared to represent defective genomes, containing only 75% of the genetic information present in L-DNA of
H. saimiri
virions.
Electrophoretic analysis of the molecular weight of murine mammary tumor virus RNA
Authors: Dion A S , Heine U I , Pomenti A A , Korb J , Weber G H
Published: January 2020

Molecular weight determinations of native and subunit RNAs of murine mammary tumor virus (MuMTV), a type B oncornavirus, were performed by polyacrylamide gel electrophoresis and compared with molecular weights of well-characterized avian cellular RNAs and tobacco mosaic virus RNA. From extrapolations of semilog plots of the molecular weights of the standard RNAs versus relative electrophoretic mobilities and Ferguson plots, the subunit and native RNAs of MuMTV were found to possess molecular weights of 2.93 X 10(6) and 6.45 X 10(6), respectively. These data support the assumption that two subunit molecules comprise the native RNA of MuMTV.
Adenovirus DNA-binding protein in cells infected with wild-type 5 adenovirus and two DNA-minus, temperature-sensitive mutants, H5ts125 and H5ts149
Authors: Ginsberg H S , Lundholm U , Linné T
Published: January 2020

Studies have been done to characterize further H5ts125, an adenovirus type 5 conditionally lethal, temperature-sensitive (ts) mutant defective in initiation of DNA synthesis and to investigate whether the single-strand-specific DNA-binding (72,000 molecular weight) protein is coded by the mutated viral gene. When H5ts125-infected cells were labeled with [35S]methionine at 32 degrees C and then incubated without isotope at 39.5 degrees C, the mutant's nonpermissive temperature, the 72,000 molecular weight polypeptide was progressively degraded. Immunofluorescence examination of cells infected with wild-type virus, H5ts125, and H5ts149 (a second, unique DNA-minus mutant) showed that immunologically reactive DNA-binding protein was barely detectable in H5ts125-infected cells at 39.5 degrees C, whereas this protein was present in wild-type- and H5TS149-infected cells, that the protein made at 32 degrees C in H5ts125-infected cells lost its ability to bind specific DNA-binding protein antibody when the infected cells were shifted to 39.5 degrees C, and that if H5ts125-infected cells were shifted from the restrictive temperature to 32 degrees C, even in the presence of cycloheximide to stop protein synthesis, immunologically reactive DNA-binding protein reappeared.
Distribution of Mason-Pfizer Virus-Specific Sequences in the DNA of Primates
Authors: Drohan W. , Colcher D. , Schochetman G. , Schlom J.
Published: January 2020

Iodinated Mason-Pfizer virus (MPV) 60-70
S
RNA has been used in molecular hybridization experiments to determine the distribution of MPV-specific proviral sequences in the DNAs of primates. Approximately 20% of the MPV genome is present as endogenous provirus in rhesus monkeys. Competitive hybridization experiments showed no homology between MPV 60-70
S
RNA and the 60-70
S
RNAs of M7, RD-114, and the simian sarcoma virus. No MPV-specific proviral sequences were detected in the DNAs of apparently normal tissues of various species of New World monkeys, apes, and humans. The part of the MPV genome that is endogenous to rhesus is also endogenous to the other species of Old World monkeys examined: baboon, African green, and patas. This was determined as a result of the following observations: (i) C
0
t
1/2
values and final extent of hybridization were the same for all four species. (ii)
T

m

values of MPV 60-70
S
RNA and DNA of all four species were identical. (iii) The removal of MPV sequences endogenous to rhesus tissues by recycling against rhesus DNA resulted in the loss of any hybridizable MPV RNA to the DNAs of baboon, African green, and patas tissues. (iv) Mixing experiments of rhesus, African green, and baboon DNAs resulted in the same kinetics of hybridization as did rhesus DNA alone, when hybridized with MPV 60-70
S
RNA. These findings demonstrate that sequences that constitute an integral part of the MPV genome are conserved in the DNAs of several different species of Old World monkeys.
Glycosidase susceptibility: a probe for the distribution of glycoprotein oligosaccharides in Sindbis virus
Authors: McCarthy M , Harrison S C
Published: January 2020

Intact Sindbis virus and Triton-solubilized viral glycoprotein were treated with alpha-mannosidase and with a preparation of mixed glycosidases from Diplococcus pneumoniae to probe the accesibility of carbohydrate units on the viral surface. The products of glycosidase attack on Triton-solubilized virus showed that mose carbohydrate units of the glycoproteins are good substrates for these enzymes. The relative resistance of most of the viral oligosaccharides in intact virus particles showed that much of the carbohydrate is not accessible to glycosidases, probably because it is not exposed at the viral surface. The only completely accessible carbohydrate units on Sindbis glycoproteins were the type A oligosaccharides of E2. This differential accessibility of Sindbis oligosaccharides is discussed in relation to the organization of the viral surface.
E proteins of bacteriophage P22. I. Identification and ejection from wild-type and defective particles
Authors: Israel V
Published: January 2020

Of the nine proteins found in the virion of phage P22, four are ejected into the cell after adsorption. The four ejected proteins, termed E proteins, are gp16, gp20, gp26, and gp7. This was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled phage that had been adsorbed to cells and then eluted off the surface with distilled water. Phage particles that lack gp7 (7- particles) or gp20 (20- particles) successfully eject all their E proteins. The 16- particles do not eject gp7. Analysis of phage ghosts showed that they lack gp16, gp20, and gp7, but they have gp26 in close to normal quantities. Our results suggest roles for gp16 and gp26 in DNA and E protein ejection. All four E proteins are possible candidates for roles in helping the phage DNA cross the plasma membrane.
Characterization of Molecular Species Carrying Gross Cell Surface Antigen
Authors: Snyder Harry W. , Stockert Elisabeth , Fleissner Erwin
Published: January 2020

The Gross cell surface antigen (GCSA), associated with expression of endogenous Gross-type murine leukemia virus (G-MuLV) in tissues of mice, is defined by the cytotoxic reaction of a C57BL/6 antiserum, anti-AKR spontaneous leukemia K36, with cells of the Gross virus-induced C57BL/6 leukemia, E♂G2. Sequential lactoperoxidase-catalyzed radioiodination of E♂G2 cells, Nonidet P-40 lysis, precipitation with anti-K36 serum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified molecules with properties of polyproteins encoded by the
gag
region of the viral genome. These cell surface species could also be labeled by in vitro culturing of E♂G2 with radioactive glucosamine. The viral specificity of these molecules and their participation in the GCSA typing system were established as follows. (i) Absorption of anti-K36 serum with GCSA
+
, but not GCSA
−
, leukemias led to a marked decrease in precipitation of these proteins. (ii) The same E♂G2 cell surface proteins were also precipitated by antisera against the MuLV virion proteins p30 and p15. (iii) Anti-K36 was shown to possess antibodies against Gross virus p30 and p15. (iv) “Clearing” the E♂G2 lysate of molecules reactive with anti-p30 or anti-p15 sera removed molecules reactive with anti-K36 serum. (v) Absorption of anti-K36 serum with disrupted G-MuLV virions or with Gross p30 or p15 removed GCSA cytotoxic antibodies; partial absorption was achieved with disrupted Rauscher-MuLV (R-MuLV) or with R-MuLV p30, and no absorption was found with R-MuLV p15. These data show that E♂G2 cells express, on their surfaces, MuLV core polyproteins that apparently can be glycosylated and on which the determinants of GCSA are located.
Identification of the Gross cell surface antigen associated with murine leukemia virus-infected cells
Authors: Ledbetter J , Nowinski R C
Published: January 2020

The Gross cell surface antigen (GCSA) is produced by cells that are either exogenously infected with murine leukemia virus (MuLV) or are expressing endogenous MuLV genomes. In immune precipitation assays, GCSA was resolved into two serologically distinct 85,000- and 95,000-dalton viral proteins. These antigenic components are glycosylated forms of the polyprotein precursors of the MuLV internal structural proteins.
Cell-free synthesis of herpes simplex virus-coded pyrimidine deoxyribonucleoside kinase enzyme
Authors: Preston C M
Published: January 2020

The incubation of a cell-free protein-synthesizing system prepared from rabbit reticulocytes with cytoplasmic RNA from herpes simplex virus (HSV)-infected cells resulted in increased thymidine kinase activity. This enzyme activity was specifically inhibited by anti-HSV antiserum and was relatively unaffected by TTP, an inhibitor of cellular thymidine kinases. Induction of the new activity was prevented by addition of inhibitors of eucaryotic protein synthesis, and no new activity was detected when RNA from cells infected with pyrimidine deoxyribonucleoside kinase-deficient mutants, instead of wild-type HSV, was added. An increased deoxycytidine kinase activity with similar properties to the HSV-specified enzyme activity was also present in cell-free systems incubated with RNA from HSV-infected cells. Phosphorylation of thymidine and deoxycytidine at 30 degrees C continued for longer than 11 h. The findings are consistent with the accurate synthesis in vitro of enzymically active HSV-specified pyrimidine deoxyribonucleoside kinase.
Bacteriophage production by doubly lysogenic Corynebacterium diphtheriae
Authors: Groman N , Laird W
Published: January 2020

Parental and recombinant phage production by tandem, double lysogens of Corynebacterium diphtheriae was studied in strains in which the coupling of prophage markers and the order of prophage was established. The results from studies of mass lysates and single bursts showed that the recombinant class of phage, designated R1, was predominant in UV-induced lysates followed by the parental, P1 class and to a lesser extent the P2 and R2 classes. Single bursts of UV-treated cells contained phage from one to all four of the phage classes, and this appeared to reflect the action of two excision processes. The data indicate that recombinant phages R1 and R2 are formed by a process of general recombinational excision and that this is the primary event leading to phage production in both UV-irradiated and spontaneously induced double lysogens. This process, which depends on exchange between homologous genes and is reciprocal, accounts for the excision of R1 phage from the host chromosome. A second excision process, probably site-specific excision, also occurs in many of the same cells and accounts for the excision of P1, P2, and R2 phages. The significance of these results for the spread of toxinogenicity in strains of C. diphtheriae is discussed.
Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase
Authors: Kozloff L. M. , Lute M. , Crosby L. K.
Published: January 2020

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp
td
) and the T4D-induced dihydrofolate reductase (gp
frd
) are baseplate structural components. With regard to phage
td
it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with
td
+
, whereas particles produced after infection with
td
−
had no measurable enzymatic activity; (ii) a mutation of the T4D
td
gene from
td
ts
to
td
+
simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both
td
ts
and
frd
ts
genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage
frd
it has been found that two spontaneous revertants each of two different T4D
frd
ts
mutants to
frd
+
not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the
td
gene and/or the
frd
gene indicate that these particles still retain some characteristics associated with the presence of both the
td
and the
frd
molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.
Differential effect of arabinofuranosylthymine of the replication of human herpesviruses
Authors: Miller R L , Iltis J P , Rapp F
Published: January 2020

The thymidine analog 1-beta-arabinofuranosylthymine (ara-T) has previously been found to selectively inhibit herpes simplex virus replication. At a relatively nontoxic conentration (50 microgram/ml), ara-T reduced herpes simplex virus yields by 4 to 5 log10. Ara-T was also effective in inhibiting the replication of varicellazoster virus (VZV) in vitro in human embryo fibroblasts, completely preventing VZV-specific cytopathic effects. The inhibition of VZV was reversible upon drug removal at 48 h after addition but was not reversible after 5 days of treatment. ara-T also reduced cell-free virus infectivity and the plaque-forming cell yield of VZV. Compared with the untreated controls, which demonstrated a 1-log10 increase over input plaque-forming cells at 24 h after infection, 50 microgram of ara-T per ml resulted in a 1-log10 decrease. In contrast to herpes simplex virus and VZV, cytomegalovirus replication was relatively resistant to ara-T. Neither cytopathic effects nor the incorporation of [3H]thymidine into acid-insoluble material in cytomegalovirus-infected cells was markedly affected. Analysis of the newly synthesized labeled DNA by CsCl buoyant density determinations indicated that the same relative proportions of cell and virus DNA were synthesized with or without added drug. Interpretation of these results with regard to virus-induced deoxypyrimidine kinase is discussed.
Simian virus 40-specific ribosome-binding proteins induced by a nondefective adenovirus 2-simian virus 40 hybrid
Authors: Jay G , Jay F T , Friedman R M , Levine A S
Published: January 2020

We have studied the intracellular distribution of the two simian virus 40-specific proteins, with apparent molecular weights of 56,000 and 42,000, detectable in human KB cells infected by a nondefective adenovirus 2-simian virus 40 hybrid, Ad2+ND2. After a 20-min pulse of [35S]methionine, about two-thirds of the newly synthesized 56K protein and one-third of the 42K protein were found localized on the plasma membrane. The remainder of each protein was found in the cytoplasm, whereas the nuclear fraction was virtually free of either component. A significant portion of both proteins present in the cytoplasmic fraction was complexed to the 40S ribosomal subunits and was not removed by treatment with 0.5 M KCl. Moreover, the portion that was found free in the cytoplasm could bind preferentially and quantitatively to purified 40S ribosomes in vitro, leading us to propose that these simian virus 40 proteins may act as translational control elements in cells.
Effect of interferon on dimethyl sulfoxide-stimulated Friend erythroleukemic cells: ultrastructural and biochemical study
Authors: Luftig R B , Conscience J F , Skoultchi A , McMillan P , Revel M , Ruddle F H
Published: January 2020

Treatment of dimethyl sulfoxide-stimulated Friend erythroleukemic cells (clone 745) with mouse interferon (50 U/ml) led to the following changes: (i) a net decrease (40 to 60%) in both the total number of apparently newly synthesized virion particles per cell section and in the average number of cell sections containing one or more virion particles; (ii) a large decrease (80 to 90%) in the number of particles released into the supernatant fluid, as assayed by reverse transcriptase activity; (iii) an initial increase in the number of "immature" or "enveloped A-type" virions followed by an increase in the accumulation of empty, core shell-like particles; and (iv) a decrease in the number of cytoplasmic vacuolar structures, which have been implicated as a major site of virus production and which we show here by serial sectioning to be, in several instances, invaginations of the plasma membrane. The effects on virus production were noticeable 2 h after interferon addition and reached their full extent by 13 h. We conclude from these observations that interferon acts upon the late stage(s) of virion maturation, leading both to a decrease in virion production as well as to the formation of defective particles. In contrast, a small but significant increase in the rate at which globin mRNA and hemoglobin accumulate is observed after interferon treatment.
Evidence for Simian Virus 40 (SV40) Coding of SV40 T-Antigen and the SV40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus Type 2-SV40 Hybrid Viruses
Authors: Mann Kristine , Hunter Tony , Walter Gernot , Linke Hawley
Published: January 2020

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2
+
ND1, Ad2
+
ND2, Ad2
+
ND4, and Ad2
+
ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2
+
ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2
+
ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.