bluuebunny/crossref_metadata_2025 huggingface dataset:
Biological properties of "partial" transformation mutants of Rous sarcoma virus and characterization of their pp60src kinase
Authors: Anderson D D , Beckmann R P , Harms E H , Nakamura K , Weber M J
Published: January 2020

We have isolated mutants of Rous sarcoma virus from an unmutagenized stock of the Schmidt-Ruppin strain of Rous sarcoma virus. These mutants induce only a "partial" transformation, and the transformation properties induced show unusual properties or combinations. Cells infected with mutant CU2 have a unique "blebby" morphology, have lost surface fibronectin, form very small colonies in soft agar, and are nearly normal with respect to adhesiveness and hexose transport. Cells infected with mutant tsCU11 have a nearly normal morphology, but grow well in soft agar. Cells infected with mutant CU12 have a fusiform morphology, intermediate levels of hexose transport and fibronectin, and form very large colonies in soft agar. Because the appearance of the different parameters of transformation is dissociated in these mutant-infected cells, these data are interpreted as supporting a model in which the transforming protein pp60src interacts with more than one primary target in generating the transformed phenotype. All of the mutants display levels of pp60src kinase activity less than that of the wild type. In the case of mutant CU12, the lower kinase activity is in part a consequence of a lower steady-state amount of pp60src inside the cell.
Cellular transformation by subgenomic feline sarcoma virus DNA
Authors: Barbacid M
Published: January 2020

The genome of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) is a 4.3-kilobase-pair (kbp) RNA molecule that contains a 1.5-kbp cellular insertion (fes gene) flanked by feline leukemia virus sequences at its 5' end (1.6 kbp) and 3' end (1.2 kbp) (Sherr et al., J. Virol. 34:200-212, 1980). DNA transfection techniques have been utilized to determine the regions of the ST-FeSV genome involved in malignant transformation. I have found that the 3.7-kbp 5'-end fragment of the ST-FeSV provirus (which corresponds to the 3.4-kbp 5'-end fragment of the viral genome) is sufficient to transform NIH/3T3 fibroblasts. Enzymes that cleave the ST-FeSV provirus DNA within the feline leukemia virus gag gene sequences or within the fes gene abolished the transforming activity. Preservation of the proviral large terminal repeats was also required for transformation. Transformed NIH/3T3 cells obtained by transfection of total or subgenomic ST-FeSV DNA expressed normal levels of the ST-FeSV gene product ST P85 and of its associated protein kinase activity. Furthermore, these cells contained high levels of phosphotyrosine residues, a biochemical marker associated with cellular transformation induced by certain retroviruses including ST-FeSV. These results, taken together, strongly support the concept that only those ST-FeSV proviral sequences necessary for ST P85 expression are involved in malignant transformation.
Structure of Bacillus subtilis bacteriophage SPP1 DNA in relation to its transfection activity
Authors: Humphreys G O , Trautner T A
Published: January 2020

The availability of a detailed restriction map of SPP1 DNA allowed defined manipulations of such molecules. These were performed to investigate structural requirements for SPP1 transfection. (i) The transfection activity of SPP1 DNA was destroyed by degradation with restriction enzymes. Biological activity could be regenerated when transfection was performed with a combination of two different restriction endonuclease digests, provided that such digests generated widely overlapping DNA fragments. (ii) Unique DNA molecules were constructed from the natural population of circularly permuted SPP1 DNA molecules by using genetic engineering techniques. Such molecules had the same specific transfection activity as did the circularly permuted SPP1 DNA. These results are discussed in the context of current models of DNA processing in transfection.
Linkage map of the fragments of herpesvirus papio DNA
Authors: Lee Y S , Tanaka A , Lau R Y , Nonoyama M , Rabin H
Published: January 2020

Herpesvirus papio (HVP), an Epstein-Barr-like virus, causes lymphoblastoid disease in baboons. The physical map of HVP DNA was constructed for the fragments produced by cleavage of HVP DNA with restriction endonucleases EcoRI, HindIII, SalI, and PvuI, which produced 12, 12, 10, and 4 fragments, respectively. The total molecular size of HVP DNA was calculated as close to 110 megadaltons. The following methods were used for construction of the map; (i) fragments near the ends of HVP DNA were identified by treating viral DNA with lambda exonuclease before restriction enzyme digestion; (ii) fragments containing nucleotide sequences in common with fragments from the second enzyme digest of HVP DNA were examined by Southern blot hybridization; and (iii) the location of some fragments was determined by isolating individual fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. Terminal heterogeneity and internal repeats were found to be unique features of HVP DNA molecule. One to five repeats of 0.8 megadaltons were found at both terminal ends. Although the repeats of both ends shared a certain degree of homology, it was not determined whether they were identical repeats. The internal repeat sequence of HVP DNA was found in the EcoRI-C region, which extended from 8.4 to 23 megadaltons from the left end of the molecule. The average number of the repeats was calculated to be seven, and the molecular size was determined to be 1.8 megadaltons. Similar unique features have been reported in EBV DNA (D. Given and E. Kieff, J. Virol. 28:524-542, 1978).
Cytoplasmic viral DNA synthesis in exogenous infection of murine leukemia virus: effect of interferon and cycloheximide
Authors: Aboud M , Moldovan-Levin D , Salzberg S
Published: January 2020

Cytoplasmic viral DNA synthesis can be followed efficiently by [3H]thymidine labeling of cells exogenously infected with Moloney murine leukemia virus. Both the negative and the positive strands of viral DNA reached their maximal level in the cytoplasm at 3.5 h postinfection. Interferon treatment before infection markedly reduced the amount of viral DNA formed during the first 3.5 h, but led to a second major wave of viral DNA synthesis, peaking at 7.5 h postinfection. No such late cytoplasmic DNA synthesis occurred in the untreated control. Inhibition of protein synthesis by cycloheximide, on the other hand, stimulated cytoplasmic viral DNA synthesis during the first 3.5 h.
Defective interfering particles of Sindbis virus do not interfere with the homologous virus obtained from persistently infected BHK cells but do interfere with Semliki Forest virus
Authors: Weiss B , Schlesinger S
Published: January 2020

Defective interfering particles derived from wild-type Sindbis virus no longer interfere with the infectious virus cloned from BHK cells persistently infected with Sindbis virus for 16 months. These particles do interfere with the replication of Semliki Forest virus.
Two major DNA variants present in serially propagated stocks of the WR strain of vaccinia virus
Authors: Panicali D , Davis S W , Mercer S R , Paoletti E
Published: January 2020

Two major DNA variants have been isolated from serially propagated stocks of the WR strain of vaccinia virus. Restriction enzyme mapping of the two variants with HindIII, AvaI, XhoI, SstII, and SmaI revealed a 6.3-megadalton deletion in the smaller DNA variant. The deletion was mapped at ca. 6.8 megadaltons in from the left terminus, just beyond the inverted terminal repeat. The additional DNA present in the larger variant was found to represent unique viral sequences that were transcribed both in vitro and in vivo. One-step growth curves in HeLa cells revealed no difference in rat of replication or burst size when progeny was scored on CV-1 monolayers.
Structure of Sendai viral proteins in plasma membranes of virus-infected cells
Authors: Bowen H A , Lyles D S
Published: January 2020

Purified plasma membranes attached to polycationic polyacrylamide beads by their external surface were isolated from BHK cells infected with Sendai virus. Each of the viral proteins could be identified in the membranes of infected cells. Proteolysis with trypsin, which digests only the cytoplasmic surface of these membranes (because the external surface is protected by its attachment to beads), revealed that the internal proteins, L, P, NP, and M, were present on the cytoplasmic surface of the membrane and that small segments of the viral envelope glycoproteins, HN and F0, were partially exposed on the cytoplasmic surface. Since the major portions of HN and F0 are known to be present on the external membrane surface, these glycoproteins are transmembrane proteins before Sendai virus budding in infected cells.
Synthesis of Black Beetle Virus Proteins in Cultured
Drosophila
Cells: Differential Expression of RNAs 1 and 2
Authors: Friesen Paul D. , Rueckert Roland R.
Published: January 2020

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 × 10
6
(RNA 1) and 0.5 × 10
6
(RNA 2), respectively. Virions contained two proteins, β with a molecular weight of 43,000 (43K) and γ (5K), and traces of a third protein, α (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein α. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured
Drosophila
cells induced the synthesis of five new proteins:
A
, α, β, γ, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [
35
S]methionine-labeled cultures. All but protein γ could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein α and the coordinate appearance of proteins β and γ, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor α. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and α, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor α peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.
Analysis of the virogenes related to the rhesus monkey endogenous type C retrovirus in monkeys and apes
Authors: Tainsky M A
Published: January 2020

Molecular hybridization studies were carried out by using a [3H]complementary DNA (cDNA) probe to compare the endogenous type C retrovirus of rhesus monkeys (MMC-1) with other known retroviruses and related sequences in various primate DNAs. The genomic RNA of the endogenous type C retrovirus of stumptail monkeys (MAC-1) was found to be highly related to the MMC-1 cDNA probe, whereas the other retroviral RNAs tested showed no homology. Related sequences were found in Old World monkey DNAs and to a lesser extent in gorilla dn chimpanzee DNAs. No homology was detected between MMC-1 cDNA and DNA of gibbon, orangutan, or human origin. Restriction endonuclease analysis of genomic DNA indicated that many of the several hundred sequences related to MMC-1 in rhesus monkey DNA differed from that integrated into DNA of infected canine cells. Gorilla and chimpanzee DNAs contained a specific restriction endonuclease fragment of the MMC-1 genome.
Variation in nucleotide sequences coding for the N-terminal regions of the matrix and nonstructural proteins of influenza A viruses
Authors: Hall R M , Air G M
Published: January 2020

Nucleotide sequences have been determined for complementary DNA transcribed from the 3' ends of RNA segments 7 (matrix gene) and 8 (nonstructural gene) from a number of human influenza A viruses isolated over a period of 43 years and representing H0N1, H1N1, H2N2, and H3N2 subtypes. The pattern of nucleotide variation in both genes suggests that RNA segments 7 and 8 were conserved during the reassortment events which were responsible for the antigenic shifts H1N1 leads to H2N2 and H2N2 leads to H3N2. During the 23-year period between the isolation of A/PR/8/34(H0N1) and A/RI/5-/57(H2N2), substitutions have occurred at 7 of 230 nucleotides in RNA segment 7 and 13 of 220 nucleotides in RNA segment 8, and in 20 years A/RI/5-/57(H2N2) to A/Canberra Grammar/77(H3N2) substitutions have occurred at 5 of 230 nucleotides in RNA segment 7 and 12 of 220 nucleotides in RNA segment 8. These give rise to 2 of 67, 5 of 64, 1 of 67, and 5 of 64 amino acid changes, respectively. The number of nucleotide and amino acid changes observed is of the same order of magnitude as that which occurs over a comparable period of drift in RNA segments 4 and 6, which code for the variable antigenic determinants hemagglutinin and neuraminidase.
Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to murine coronaviruses A59 and JHM and human coronavirus OC43, but not to human coronavirus 229E
Authors: Gerdes J C , Klein I , DeVald B L , Burks J S
Published: January 2020

Two coronaviruses (SK and SD), isolated from fresh autopsy brain tissue from two multiple sclerosis patients, were compared with known human and murine coronaviruses. In plaque neutralization assays, antisera prepared against multiple sclerosis isolates SK and SD demonstrated significant cross-reactivity to each other and to murine coronavirus A59, weak cross-reactivity to murine coronavirus JHM, but no cross-reactivity to the human coronavirus 229E. Antiserum to SK or SD failed to inhibit hemagglutination of chicken erythrocytes by the human coronavirus OC43. However, OC43 antiserum neutralized both SD and SK. Specific coronavirus polypeptides were identified and compared by immunoprecipitation and polyacrylamide gel electrophoresis. Infected and mock-infected 17Cl-1 cells were pretreated with actinomycin D and labeled with [35S]methionine. Polypeptides in Nonidet P-40 cytoplasmic extracts were immunoprecipitated with homologous and heterologous antisera. Identical polypeptides were precipitated from A59-, SD-, or SK-infected cell extracts by SD, SK, OC43, or A59 antisera. The polypeptides of human virus 229E were antigenically distinct, with the exception of weak recognition of a polypeptide of 50,000 molecular weight. We conclude that the two multiple sclerosis virus isolates SK and SD are closely related serologically to the murine coronavirus A59 and the human coronavirus OC43.
Mutants of Fujinami sarcoma virus which are temperature sensitive for cellular transformation and protein kinase activity
Authors: Hanafusa T , Mathey-Prevot B , Feldman R A , Hanafusa H
Published: January 2020

Two temperature-sensitive mutants of Fujinami sarcoma virus were isolated and characterized. Cells infected with the mutants were temperature sensitive in focus formation, colony formation, increased sugar uptake, and synthesis of plasminogen activator. The changes between transformed and nontransformed states of cultures were completely reversible by shifting the temperature. A Fujinami sarcoma virus-specific protein of 130,000 daltons, p130, was synthesized in mutant-infected cells regardless of the temperature, but the immunoprecipitates of p130 from extracts of infected cells were active in protein kinase only when cells had been incubated at the permissive temperature. These results appear to indicate that p130 is the transforming protein of Fujinami sarcoma virus, and that its protein kinase activity plays a crucial role in cell transformation by this virus.
Inhibition of cellular DNA synthesis by vesicular stomatitis virus
Authors: McGowan J J , Wagner R R
Published: January 2020

DNA synthesis in mouse myeloma (MPC-11) cells and L cells was rapidly and progressively inhibited by infection with vesicular stomatitis virus (VSV). No significant difference in cellular DNA synthesis inhibition was noted between synchronized and unsynchronized cells, nor did synchronized cells vary in their susceptibility to VSV infection after release from successive thymidine and hydroxyurea blocks. Cellular RNA synthesis was inhibited to about the same extent as DNA synthesis, but cellular protein synthesis was less affected by VSV at the same multiplicity of infection. The effect of VSV on cellular DNA synthesis could not be attributed to degradation of existing DNA or to decreased uptake of deoxynucleoside triphosphates, nor were DNA polymerase and thymidine kinase activities significantly different in VSV-infected and uninfected cell extracts. Analysis by alkaline sucrose gradients of DNA in pulse-labeled uninfected and VSV-infected cells indicated that VSV infection did not appear to influence DNA chain elongation. Cellular DNA synthesis was not significantly inhibited by infection with the VSV polymerase mutant tsG114(I) at the restrictive temperature or by infection with defective-interfering VSV DI-011 (5' end of the genome), but DI-HR-LT (3' end of genome) exhibited initially rapid but not prolonged inhibition of MPC-11 cell DNA synthesis. DNA synthesis inhibitory activity of wild-type VSV was only slowly and partially inactivated by very large doses of UV irradiation. These data suggest that, as in the effect of VSV on cellular RNA synthesis (Weck et al., J. Virol. 30:746-753, 1979), inhibition of cellular DNA synthesis by VSV requires transcription of a small segment of the viral genome.
Coliphage which requires either the LamB protein or the OmpC protein for adsorption to Escherichia coli K-12
Authors: Beher M G , Pugsley A P
Published: January 2020

Either of two different proteins in the outer membrane of Escherichia coli K-12 (LamB and OmpC) can function in the constitution of receptor activity for a newly isolated T-even bacteriophage. This bacteriophage (SSI) differs from other T-even phages which use the OmpC protein as their receptors. The simple procedure used to isolate phage SSI may be suitable for the detection of bacteriophages with novel outer membrane receptor requirements.
Isolation of a protein kinase induced by herpes simplex virus type 1
Authors: Blue W T , Stobbs D G
Published: January 2020

We have isolated a new cyclic AMP-independent protein kinase activity induced in HeLa cells by infection with herpes simplex virus type 1. Induction of the enzyme does not occur in cells treated with cycloheximide at the time of infection, or in cells infected with UV-inactivated herpes simplex virus type 1. The amount of enzyme induced in infected cells is dependent upon the multiplicity of infection. An enzyme with identical properties to the appearing in infected HeLa cells is also induced by herpes simplex virus type 1 in BHK cells.
Reovirus inhibition of cellular DNA synthesis: role of the S1 gene
Authors: Sharpe A H , Fields B N
Published: January 2020

Type 3 reovirus inhibits L cell DNA synthesis, whereas type 1 reovirus exerts little or no effect on L cell DNA synthesis. By using recombinant viruses containing both type 1 and type 3 double-standard RNA segments, we determined that one double-stranded RNA segment, the reovirus type 3 S1 double-stranded RNA segment which encodes the viral hemagglutinin, segregates with and is responsible for the capacity of reovirus type 3 to inhibit L cell DNA synthesis.
One extra oligosaccharide chain of the high-mannose class in the E2 protein of a Sindbis virus isolate
Authors: Cancedda R , Bonatti S , Leone A
Published: January 2020

The number and nature of oligosaccharide chains in the E2 envelope protein (and its precursor PE2) from two strains of Sindbis virus, originally derived from the same HR strain, were investigated. The E2 glycoproteins (and the PE2 proteins) of the two strains had different electrophoretic mobilities on a sodium dodecyl sulfate-polyacrylamide gel. We observed that the glycopeptides from the E2 protein of one of the two strains included an extra oligosaccharide chain of the mannose-rich type. The altered pattern of glycosylation does not interfere with the intracellular migration of the protein and the envelopment and budding of the virus.
Genome structure of avian sarcoma virus Y73 and unique sequence coding for polyprotein p90
Authors: Yoshida M , Kawai S , Toyoshima K
Published: January 2020

The genome structure of a newly isolated sarcoma virus, Y73, was studied. Y73 is a defective, potent sarcomagenic virus and contains 4.8-kilobase (kb) RNA as its genome; in contrast, helper virus associated with Y73 had 8.5-kb RNA, similar to other avian leukemia viruses. Fingerprinting analysis these RNAs demonstrated that the 4.8-kb RNA contains a specific RNA sequence of 2.5 kb, which represents the transforming gene (yas) of Y73. This specific sequence was mapped in the middle of the genome and had at both ends 1- to 1.5-kb sequences in common with Y73-associated virus RNA. This structure is very similar to those of avian and mammalian leukemia viruses. In vitro translation of the 4.8-kb RNA and the immunospecificity of the products directly demonstrated that polyprotein p90, containing p19, is a product translated from capped 4.8-kb RNA and that the specific peptide portion is coded by the yas sequence. Protein 90, which was also found in cells transformed with Y73, was suggested to be a transforming protein.
Restriction alleviation by bacteriophages lambda and lambda reverse
Authors: Toothman P
Published: January 2020

Deletion analysis indicated that the phage lambda restriction alleviation gene(s) ral resides between the cIII and N genes. The Ral+ phenotype was expressed only when lambda ral+ carried a modification such that it was resistant to restriction by the host specificity system. Under these conditions, Ral function protected superinfecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoP1. Ral-protected phage DNA was not concomitantly K and B modified, but rather received only the modification specified by the system of the restricting host. Possible mechanisms for Ral action are discussed. Of the other lambdoid phages tested, the hybrid phage lambda rev had Ral activity, whereas phi 80vir and one lambda-P22 hybrid did not. The restriction alleviation activity of lambda rev called Lar, may be the same as the activity expressed in sbcA- strains of Escherichia coli, but it was functionally separable from exonuclease VIII activity (the product of the recE gene), which is also expressed in sbcA- strains.
Gene expression of herpes simplex virus. III. Effect of arabinosyladenine on viral polypeptide synthesis
Authors: Pedersen M , Talley-Brown S , Millette R L
Published: January 2020

Arabinosyladenine, an established antiherpetic drug, was used to block herpes simplex virus type 1 DNA synthesis quantitatively in infected xeroderma pigmentosum cells. Kinetic analyses of viral polypeptides synthesized in the presence and absence of this drug revealed that there were at least six distinct kinetic classes of polypeptides. These differed in time of appearance after infection, time of maximum rate of synthesis, kinetics of turnoff, and sensitivity to arabinosyladenine. This study showed that arabinosyladenine had the following three main effects on herpes simplex virus type 1 gene expression. (i) The turnon of immediate early and delayed early polypeptides (kinetic classes 1 and 2) was retarded. (ii) The turnoff of early (immediate early and delayed early) polypeptides (classes 1 through 3) was delayed. (iii) The synthesis of late polypeptides (class 4 through 6) was inhibited by arabinosyladenine, with class 6 severely (80 to 90%) inhibited. The kinetic data presented here, along with the findings of other workers on the effects of inhibition of viral DNA synthesis, suggest that viral DNA replication is required for optimum synthesis of late viral polypeptides.
Vesicular stomatitis virus RNA polymerase can read through the boundary between the leader and N genes in vitro
Authors: Herman R C , Lazzarini R A
Published: January 2020

Triphosphorylated in vitro transcripts of vesicular stomatitis virus were selected by mercury-Sepharose chromatography using adenosine-5'-O-(3-thiotriphosphate) as an affinity probe. Numerous RNAs ranging from less than 47 up to several hundred nucleotides in length were detected. Some of these contain the leader RNA covalently linked to transcripts of the N gene. A comparison of the published genomic sequences at the estimated termination sites of several of these RNAs reveals some homology with the sequence present at both the end of the leader and polymerase genes.
Identification of unintegrated forms of Kirsten murine sarcoma viral DNA and restriction endonuclease cleavage map of linear DNA
Authors: Tsuchida N , Kominami R , Hatanaka M , Uesugi S
Published: January 2020

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.
Restriction and Modification in
Bacillus subtilis
: Gene Coding for a
Bsu
R-Specific Modification Methyltransferase in the Temperate Bacteriophage Φ3T
Authors: Noyer-Weidner M. , Pawlek B. , Jentsch S. , Günthert U. , Trautner T. A.
Published: January 2020

The resistance of Φ3T DNA to degradation by the restriction enzyme
Bsu
R or its isoschizomer
Hae
III is due to obligatory modification of such DNA. Biochemical and genetical experiments indicate that Φ3T codes for a methyltransferase, which methylates Φ3T DNA itself or heterologous DNA at target sites 5′-GG
*
CC.
Gene-Coding Assignments of Rotavirus Double-Stranded RNA Segments 10 and 11
Authors: Dyall-Smith Michael L. , Holmes Ian H.
Published: January 2020

The gene-coding assignments for genome segments 10 and 11 of a simian virus and two human rotaviruses were determined. For those viruses having a “long” RNA gel pattern (electropherotype), segments 10 and 11 encoded proteins NS
3
and O
4
, respectively. The human virus with a “short” electropherotype had the opposite assignments and also differed in (enzyme-linked immunosorbent assay) serotype from the human virus with a long electropherotype.
Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40
Authors: Bender M A , Brockman W W
Published: January 2020

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.
α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected
Heliothis zea
Larvae and
Spodoptera frugiperda
Cells
Authors: Grula Marjori A. , Buller Patricia L. , Weaver Robert F.
Published: January 2020

[
3
H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of
Heliothis zea
larvae infected with
H. zea
nuclear polyhedrosis virus and from cultured
Spodoptera frugiperda
cells infected with
Autographa californica
nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [
3
H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from
H. zea
larval fat bodies and
S. frugiperda
cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in
S. frugiperda
cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II.
Coordinate regulation of two cytoplasmic RNA species transcribed from early region 2 of the adenovirus 2 genome
Authors: Goldenberg C J , Rosenthal R , Bhaduri S , Raskas H
Published: January 2020

Early region 2 (E2) of the adenovirus 2 genome specifies a 72,000-dalton DNA-binding protein that is required for viral DNA replication. Electron microscopy studies have detected two major forms of 20S E2 mRNA, one species with a 5' leader from map position 75 and a second form having a leader from position 72 (Chow et al., J. Mol. Biol. 134:265-303, 1979). Only the species with a leader from position 75 was detected at early times; however, both forms were found at late times. We have analyzed the temporal regulation of E2 expression by documenting mRNA accumulation in the cytoplasm. Kinetic studies of pulse-labeled RNAs demonstrated a peak of E2 cytoplasmic RNa synthesis at 10 to 12 h, coinciding with the time of maximal synthesis of the 72,000-dalton DNA binding protein and viral DNA. To estimate the relative abundances of the two major E2 RNA species at various times during infection, total E2 cytoplasmic and polysomal 20S RNAs were isolated by hybridization-selection with specific DNA probes. The leader sequences in the selected RNAs were then quantitated by further RNA-DNA hybridization. We found that the elevated accumulation rate for E2 cytoplasmic RNA at late times reflected an increase in formation of both major species. Moreover, for all time points examined 66% of the mRNA species had a 5' end from map position 75, and 33% had a 5' terminus from position 72. Continuous labeling experiments provided evidence that both RNA forms have comparable half-lives. The results suggest that the two major species encoded by E2 are regulated in a coordinate fashion late in infection.
Amino-terminal sequence analysis of alphavirus polypeptides
Authors: Welch W J , Sefton B M , Esch F S
Published: January 2020

The single late 26S mRNA of Semliki Forest virus (SFV) directs the synthesis of the four viral structural proteins, C, E3, E2, and E1, and the recently described nonstructural protein, 6K. We report here partial NH2-terminal amino acid sequences of the SFV polypeptides E3 and 6K and of p62, the precursor to E3 and E2. In addition, were have determined a partial NH2-terminal sequence of the Sindbis virus homolog of 6K, the 4.2K protein. p62 and E3 of SFV have identical NH2-terminal amino acid sequences. Comparison of the partial NH2-terminal sequences of 6K of SFV and 4.2K of Sindbis virus with the deduced amino acid sequence encoded by the 26S mRNA of each virus reveals that the genes for these peptides are located in each case between those for E2 and E1. The order of the genes on the 26S mRNA of the alphaviruses is therefore 5'-C-E3-E2-6K-E1-3'. We discuss two mechanisms by which the nascent viral glycoproteins may be inserted into the membrane of the endoplasmic reticulum.
Evidence that there exist four classes of RNA tumor viruses which encode proteins with associated tyrosine protein kinase activities
Authors: Patschinsky T , Sefton B M
Published: January 2020

The transforming protein of Rous sarcoma virus, p60src, the Abelson virus protein, p120, and the Y73 virus protein, p90, all have associated tyrosine protein kinase activities in vitro. Possible structural homology between these functionally related proteins was investigated by two-dimensional analysis of both methionine-containing and phosphate-containing tryptic peptides derived from biosynthetically labeled proteins. Marked differences were found between the maps of both [35S]methionine-labeled and 32P-labeled tryptic peptides. This suggests that the transforming gene of Rous sarcoma virus and the putative transforming genes of Abelson virus and Y73 virus are different. In addition, each of these genes has been shown previously to be unrelated to the putative transforming gene of Fujinami sarcoma virus, another virus which encodes a protein with associated tyrosine protein kinase activity. Therefore, it appears that there exist at least four distinct classes of functionally related RNA tumor viruses. Analysis of phosphorylated tryptic peptides did, however, reveal homology between one of the two phosphotyrosine-containing tryptic peptides of p90 of Y73 virus and the single phosphotyrosine-containing tryptic peptide of p60src of Rous sarcoma virus. Comigration of these two peptides in several different buffers and the identical mobility of their phosphorylated cleavage products after secondary digestion with protease V8 of Staphylococcus aureus indicated that p60src and p90 contain an identical site of tyrosine phosphorylation in vivo. The results are discussed with respect to the evolution of RNA tumor viruses which encode proteins with associated tyrosine protein kinase activities and the limitations of analysis of detecting homology between genes by both molecular hybridization and peptide mapping.
Purification and translation of murine mammary tumor virus mRNA's
Authors: Dudley J P , Varmus H E
Published: January 2020

We have studied the functions of the intracellular RNAs of mouse mammary tumor virus (MMTV) by purification and translation in vitro. Two major size classes of MMTV RNA, 35S and 24S RNA, were isolated from MMTV-infected rat (XC) cells and cultured mammary tumor cells by preparative hybridization of whole cell or polyadenylated RNA to cloned MMTV DNA covalently bound to chemically activated paper disks (diazobenzyloxymethyl paper). Genomic-length (35S) RNA was prepared free of 24S RNA by rate zonal sedimentation in sucrose gradients. Experiments using [3H]uridine-labeled cellular RNA indicated that the preparative annealing method was highly specific and capable of effecting a 300-fold enrichment for viral RNA; the recovered RNA appeared to be intact under denaturing conditions and directed synthesis of full-length gag and env polypeptides in vitro. The products of in vitro translation were identified by gel mobility, immunoprecipitation tests with antisera against gag and env products, and partial digestion with Staphylococcus V8 protease. The 35S RNA species directed synthesis of several gag-related polypeptides, including three previously reported in extracts of infected cells; 24S RNA directed synthesis of two polypeptides closely related to env proteins from infected cells. Therefore, 35S RNA includes mRNA's for gag and gag-pol, whereas 24S RNA is the mRNA for env. These results help establish the position of env on the physical map of the MMTV genome and bear upon the coding potential of the genome.
Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells
Authors: Lyles D S , McConnell K A
Published: January 2020

A scheme was developed for the subcellular fractionation of murine erythroleukemia cells transformed by Friend leukemia virus. The subcellular localization of the env-related glycoproteins was determined by immune precipitation with antiserum against gp70, the envelope glycoprotein of the helper virus, followed by gel electrophoresis. In cells labeled for 2 h with [35S]methionine, the glycoprotein encoded by the defective spleen focus-forming virus, gp55SFFV, was found primarily in the nuclear fraction and in fractions containing dense cytoplasmic membranes such as endoplasmic reticulum. A similar distribution was noted for gp85env, the precursor to gp70. The concentration of viral glycoproteins in the nuclear fraction could not be accounted for by contamination with endoplasmic reticulum. In pulse-chase experiments, neither glycoprotein underwent major redistribution. However, labeled gp85env disappeared from intracellular membranes with a half-time of 30 min to 1 h, whereas labeled gp55SFFV was stable during a 2-h chase. In plasma membrane preparations with very low levels of contamination with endoplasmic reticulum, gp70 was the major viral env-related glycoprotein detected; a minor amount of gp55SFFV and no gp85env could be detected. The unexpected result of these experiments is the amount of viral glycoproteins found in the nuclear fraction. Presence of viral proteins in the nucleus could be relevant to the mechanism of viral leukemogenesis.
Selective disappearance of two secreted host proteins in the course of Semliki Forest virus infection of Aedes albopictus cells
Authors: Reigel F , Koblet H
Published: January 2020

One secreted host protein of molecular weight 54,000 (SP 54) disappeared (from 24 to 48 h after infection) in Semliki Forest virus-infected Aedes albopictus cell clone C6/36 grown in both Mitsuhashi-Maramorosch basal medium and tissue culture medium 199 and reappeared when cells went into the permanently infected state. C6/36 is a high virus producer showing a cytopathic effect. A second secreted host protein of molecular weight 62,000 (SP 62) was prominent if cell clone C6/36 was grown in tissue culture medium 199. After infection in this medium, the protein showed a behavior similar to that described for SP 54. These secreted proteins were not affected in two original Aedes albopictus cell lines. SP 54 and SP 62 are monomeric proteins and structurally not related.
Isolation of monoclonal antibodies against avian oncornaviral protein p19
Authors: Greiser-Wilke I , Owada K M , Moelling K
Published: January 2020

For the production of monoclonal antibodies against pp60src and the gag precursor protein Pr76gag, the spleens of mice bearing tumors that had been induced by avian sarcoma virus Schmidt-Ruppin D-transformed cells were used. One hybridoma culture produced antibodies that were directed against the p19 portion of the gag precursor. However, no antibodies directed against pp60src could be detected in any of the hybridoma supernatants. The anti-p19-producing hybridoma culture was cloned twice in soft agar, and a stable clone was used for the production of high-titer ascites fluid in mice. The monoclonal antibodies belonged to the immunoglobulin G subclass 2b. The antibodies precipitated Pr76gag and the processed virion-associated p19, as well as the 75,000-molecular-weight gag fusion protein from avian erythroblastosis virus-transformed bone marrow cells. Also, viral ribonucleoprotein complexes were specifically precipitable, indicating that they contain p19 molecules.
Leakage induced in Escherichia coli cells by A protein-RNA complexes from bacteriophage f2
Authors: Cody J D , Conway T W
Published: January 2020

Complexes of f2 phage RNA and its A protein, or maturation protein, transfect Escherichia coli cells much better than does protein-free RNA. We used these complexes to introduce the bacteriophage f2 lysis gene into cells. The A protein-RNA complex was found to kill cells, probably by causing them to leak large macromolecules. Previously induced beta-galactosidase leaked from cells treated either with the A protein-RNA complex or with lethal but noninfectious complexes that had been treated with formaldehyde. This observation was consistent with an earlier finding that formaldehyde-treated f2 RNA stimulates the in vitro synthesis of a lysis protein. The complexes did not stimulate the rate of leakage of beta-galactosidase from a streptomycin-resistant mutant known to be lysis defective. On the other hand, the rate of leakage was increased in a double mutant resistant to both streptomycin and rifampin and which is lysed normally by f2 bacteriophage.
Cloning and expression of bacteriophage SP02 DNAZ polymerase gene L in Bacillus subtilis, using the Staphylococcus aureus plasmid pC194
Authors: Rutberg L , Rådén B , Flock J I
Published: January 2020

HindIII restriction endonuclease fragments of DNA from temperate Bacillus subtilis bacteriophage SP02 were cloned in B. subtilis by using the plasmid pC194. Three hybrid plasmids which permit growth of the mutant SP02 susL244 in suppressor-negative bacteria were isolated. SP02 gene L is thought to code for a DNA polymerase essential for autonomous replication of SP02 DNA. Extracts of bacteria carrying one of these hybrid plasmids, pC194-96, had 10- to 30-fold increased DNA polymerase activity. The plasmid-induced DNA polymerase activity differed from that of the known B. subtilis DNA polymerases in several respects. The results of the experiments support the idea that phage SP02 codes for a new DNA polymerase.
Loss of functional large T-antigen and free viral genomes from cells transformed in vitro by polyoma virus after passage in vivo as tumor cells
Authors: Lania L , Hayday A , Fried M
Published: January 2020

We have analyzed the state, arrangement, and expression of polyoma viral DNA sequences in a number of in vitro-transformed Fischer rat cells before and after growth in vivo as tumour cells. When the in vitro lines used to induce the tumors contained only a single insert of viral sequences and did not produce either a full-size 100,000-dalton (100K) large T-antigen or free viral genomes, no differences in the above-mentioned properties were observed. By contrast, in vitro cell lines containing multiple inserts of viral sequences, a functional 100K large T-antigen, and free viral genome induced tumor cells which displayed a reduced number of inserts of viral sequences and which did not produce either a functional 100K large T-antigen or free viral genomes. All of the in vitro lines and their tumor cell derivatives expressed the polyoma virus 55K middle and 22K small T-antigen species. Possible mechanisms for the selection in vivo against cells containing a functional 100K large T-antigen and consequently free viral genomes are discussed.
Identification of a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus
Authors: Adkins Becky , Hunter Tony
Published: January 2020

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60

src

-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.
Molecular biology of rotaviruses. I. Characterization of basic growth parameters and pattern of macromolecular synthesis
Authors: McCrae M A , Faulkner-Valle G P
Published: January 2020

The United Kingdom tissue-adapted bovine rotavirus growing in African green monkey kidney (BSC-1) cells was selected as a model system with which to study the detailed molecular virology of rotavirus replication. Study of the kinetics of infectious virus production revealed a fairly rapid replication cycle, with maximum yield of virus after 10 to 12 h at 37 degrees C. Progeny genome synthesis was first detected during the virus latent period at 2 to 3 h postinfection. Study of the kinetics of viral polypeptide synthesis showed that virus rapidly inhibited cellular polypeptide synthesis such that by 4 h postinfection, only virus-induced polypeptides, 15 of which were detected, were being synthesized. No qualitative changes in the pattern of viral polypeptide synthesis were observed during infection, although, based on kinetic synthesis, three quantitative classes of polypeptides were defined. Pulse-chase analysis revealed three post-translational changes in viral proteins, two of which were shown to be due to glycosylation. Tunicamycin inhibition studies were used to identify the putative non-glycosylated precursors of the two glycoproteins. Comparison of the infected-cell polypeptides with those present in purified virions revealed that mot of the virus-induced proteins were incorporated into virions, with only VP9 being a truly nonstructural protein. Some localization of the various polypeptides within the purified virion was achieved by producing viral cores.
Herpesvirus saimiri DNA in tumor cells--deleted sequences and sequence rearrangements
Authors: Desrosiers R C
Published: January 2020

Herpesvirus saimiri DNA in continuous lymphoblastoid cell lines obtained from viral induced tumors in marmosets has been analyzed by gel electrophoresis of restricted DNA. Southern transfer to nitrocellulose filters, and hybridization to 32P-labeled viral DNA or DNA fragments. The viral DNA fragments EcoRI-G, -H, -D, and -I, KpnI-A, and BamHI-D and -E were not detected in Southern transfers of DNA from the nonproducing 1670 cell line. For each restriction endonuclease, a new fragment appeared, consistent with a 13.0-megadalton deletion of viral DNA sequences. This deletion encompassed 35 to 48 +/- 0.6 megadaltons from the left end of the unique DNA region. A sequence arrangement map is presented for the major population of H. saimiri DNA sequences in the 1670 cell line. Although H. saimiri DNA in the nonproducing 70N2 cell line can be distinguished from viral DNA in the 1670 cell line by several criteria, the same sequences were found to be deleted in the major population of viral DNA molecules. Unlike 1670 and 70N2 cells, restricted DNA from the virus-producing cell lines 77/5 and 1926 contained all of the DNA fragments present in the parental virion DNA. DNA from 1670, 70N2, and 77/5 cells contained additional viral DNA fragments that did not comigrate with any virion DNA fragments. Most of these unexplained fragments were confined to or highly enriched in partially purified circular or linear DNA fractions. DNA from tumor cells taken directly from a tumor-bearing animal contained viral DNA indistinguishable from the parental virion DNA by the assay conditions used. These results indicate that viral DNA sequence rearrangements can occur upon cultivation of tumor cells in vitro and that excision of DNA sequences from the viral genome may play a role in establishing the nonproducing state of some tumor cell lines.