bluuebunny/crossref_metadata_2025 huggingface dataset:
Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA
Authors: Gidoni D , Scheller A , Barnet B , Hantzopoulos P , Oren M , Prives C
Published: January 2020

In various permissive monkey cell lines infected with simian virus 40 there are two major forms of large T antigen which differ in their rate of sedimentation through sucrose gradients. The lighter (5 to 7S) form sedimented slightly more rapidly than the 4S tRNA marker, whereas the heavier (16S) form sedimented slightly more slowly than the 18S rRNA marker. The small t antigen did not form complexes which sedimented as rapidly as those formed by the large T antigen. The 16S T antigen form was converted to the slowly sedimenting 5 to 7S form in the presence of 1.0 M NaCl. The majority of large T antigen synthesized in cell-free protein-synthesizing systems primed by mRNA isolated from infected cells sedimented as the 5 to 7S form even when premixed with excess quantities of cellular T antigen. The formation of the 16S form in infected cells did not require ongoing viral or cellular DNA replication because considerable quantities of this T antigen class were produced in the presence of DNA synthesis inhibitors, such as cytosine arabinoside. Both 5 to 7S and 16S forms could be isolated separately and, therefore, each could be analyzed as to its individual properties. The 5 to 7S T antigen form bound more efficiently and tightly to DNA and had specific affinity for sequences at the viral origin of replication, whereas the 16S form bound less efficiently to DNA and exhibited very little specificity for origin-containing DNA sequences. It is therefore likely that the active DNA-binding species of T antigen isolated from infected cells is the 5 to 7S form.
Sequences of ribosome binding sites from the large size class of reovirus mRNA
Authors: Kozak M
Published: January 2020

Ribosome-protected fragments from two of the large-sized reovirus mRNAs were recovered from sparsomycin-blocked 80S initiation complexes. The sequence of each protected oligonucleotide was determined. The ribosome binding site of each message includes the m7G cap and a centrally positioned AUG codon. An adenine residue occurs three nucleotides upstream from the initiator codon, thus conforming to the pattern [AG]NNAUG observed for nearly all eucaryotic initiation sites.
Detection and characterization of agarose-binding, capsid-like particles produced during assembly of a bacteriophage T7 procapsid
Authors: Serwer P , Watson R H , Hayes S J
Published: January 2020

It has previously been shown that: (i) during infection of its host, the DNA bacteriophage T7 assembles a DNA-free procapsid (capsid I), a capsid with an envelope differing physically and chemically from the capsid of the mature bacteriophage, and (ii) capsid I converts to a capsid (capsid II) with a bacteriophage-like envelope as it packages DNA. Lysates of phage T7-infected Escherichia coli contained a particle (AG particle) which copurified with capsid II during buoyant density sedimentation, velocity sedimentation, and solid support-free electrophoresis, but was distinguished from capsid II by its apparent diversity during electrophoresis in agarose gels. Treatment of AG particles with trypsin converted most of them to particles that comigrated with trypsin-treated capsid II during electrophoresis in agarose gels. Irreversible binding of AG particles to agarose gels was shown to contribute to the apparent diversity of AG particles during agarose gel electrophoresis. The results of quantitation of AG particles and of capsid I and capsid II in lysates of a nonpermissive host infected with T7 amber mutants suggested that, in site of their capsid II-like properties, most AG particles were produced during assembly of capsid I and not during DNA packaging. The presence of AG particles in T7 lysates explains contradictions in previous data concerning the pathway of T7 assembly.
Tumorigenicity of partial transformation mutants of Rous sarcoma virus
Authors: Kahn P , Nakamura K , Shin S , Smith R E , Weber M J
Published: January 2020

Chicken embryo cells infected with partial transformation mutants of Rous sarcoma virus were tested for tumor-forming ability in chickens and in nude mice. Cells transformed by each of these partial transformation mutants display different combinations of transformation parameters. They therefore present a potentially favorable system for analyzing which properties of transformed cells are necessary for tumor formation. We found that the relative tumorigenicity of the virus mutants was generally similar in chickens and in nude mice, except that certain temperature-conditional mutants appeared to be sensitive to the differences in body temperature of the two experimental animals. (The body temperature of nude mice is 4 to 5 degrees C lower than that of chickens). Thus, the nude mouse appears to be a suitable system for testing the tumorigenicity of transformed chicken cells. Because mice are nonpermissive for Rous sarcoma virus infection and replication, it was possible to recover the transformed chicken cells from the tumors in this host and to determine what phenotypic changes they had undergone during tumor development. We also examined the relationship between various cellular properties of the virus-infected chicken cells in vitro and their tumorigenicity in nude mice. The combined results of these two studies indicated that anchorage independence and plasminogen activator production were highly correlated with the tumor-forming ability of these cells, whereas loss of fibronectin did not correlate with tumorigenicity. Furthermore, the inability of the least tumorigenic virus mutant to stimulate the phosphorylation of a 36,000-Mr target of pp60src raises the possibility that the 36,000-Mr protein plays a role in tumor formation.
Bovine coronavirus structural proteins
Authors: King B , Brian D A
Published: January 2020

The tissue culture-adapted strain (Mebus) of bovine coronavirus was grown in the presence of isotopically labeled amino acids, glucosamine, or orthophosphate for the purpose of analyzing the virion structural proteins. Five species of polypeptides were identified when purified virions were solubilized in urea and sodium dodecyl sulfate and resolved by polyacrylamide gel electrophoresis. Four species were glycosylated and had apparent molecular weights of 140,000, 120,000, 100,000, and 26,000. The glycoproteins were susceptible to proteolytic cleavage and enzymatic iodination when intact virions were studied and are thus at least partially external to the virion envelope. The 140,000-molecular-weight glycoprotein is apparently a dimer of 65,000-molecular-weight glycopolypeptides held together by disulfide linkages. Species 5 was phosphorylated and had an apparent molecular weight of 52,000. In the intact virion, it was unaffected by protease and was not enzymatically iodinated. It is therefore apparently an internal protein.
Identification of the initiation site of poliovirus polyprotein synthesis
Authors: Dorner A J , Dorner L F , Larsen G R , Wimmer E , Anderson C W
Published: January 2020

The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVP00. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VP0, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of the tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of the poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.
Effects of monensin on morphogenesis and infectivity of Friend murine leukemia virus
Authors: Srinivas R V , Melsen L R , Compans R W
Published: January 2020

The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious virus was inhibited by treatment of Friend murine leukemia virus-infected Eveline cells with the sodium ionophore monensin. Virus yields were reduced more than 50-fold by 10(-5) M monensin, whereas particle production was reduced by 50% in monensin-treated cells. The resulting particles failed to incorporate newly synthesized gp70 and p15(E), whereas the other structural proteins, p30, p15, p12, and p10, were incorporated into virions. However, monensin did not inhibit the incorporation into virions of preformed gp70. A reduction in the efficiency of cleavage of the PrENV glycoprotein precursor and a defect in the processing of simple endo-H-sensitive to complex endo-H-resistant oligosaccharides suggest that intracellular transport of gp70 may be blocked before its entry into the Golgi apparatus. Fewer particles were found to bud from the cell surface, but intracellular vacuoles with budding virions were detected. Ferritin labeling and pulse-chase studies suggested a cell surface origin for these vacuoles. These experiments indicate that monensin inhibits the transport of Friend murine leukemia virus glycoproteins at an early stage, with a resultant block in the assembly and release of infectious virus.
Cloning of Two Genetically Transmitted Moloney Leukemia Proviral Genomes: Correlation Between Biological Activity of the Cloned DNA and Viral Genome Activation in the Animal
Authors: Chumakov Ilya , Stuhlmann Heidi , Harbers Klaus , Jaenisch Rudolf
Published: January 2020

The
Mov-7
and
Mov-9
substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at the
Mov-7
locus and
Mov-9
locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying the
Mov-9
locus but is not activated in animals carrying the
Mov-7
locus. Consequently, only
Mov-9
mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to the
Mov-7
and
Mov-9
loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that the
Mov-7
and
Mov-9
loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10
−5
XC plaques per genome equivalent were induced by the
Mov-9
clone, whereas only 10
−9
XC plaques per genome equivalent were induced by the
Mov-7
clone. Moreover, NIH 3T3 cells transfected with the
Mov-9
clone produced NB-tropic M-MuLV, whereas cells transfected with the
Mov-7
clone did not produce infectious virus. The results suggest that M-MuLV integrated at the
Mov-7
locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression in
Mov-7
and
Mov-9
mice correlates with the biological properties of the respective clones. Genomic DNA from
Mov-9
mice was not infectious in the transfection assay (specific infectivity < 10
−7
PFU per genome equivalent). As the only difference between the genomic and the cloned
Mov-9
DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.
Nucleotide sequence analysis of the long terminal repeat of integrated simian sarcoma virus: evolutionary relationship with other mammalian retroviral long terminal repeats
Authors: Devare S G , Reddy E P , Law J D , Aaronson S A
Published: January 2020

Nucleotide sequence analysis of the long terminal repeat (LTR) of the integrated simian sarcoma virus showed that the simian sarcoma virus LTR comprised 504 nucleotides with an inverted repeat of seven bases at its 5' and 3' termini. At the site of simian sarcoma virus integration, cellular flanking sequences adjacent to the proviral LTR contained a direct repeat of four bases. A 13-base sequence after the 5' LTR was found to be complementary to prolyl tRNA, suggesting that tRNAPro may serve as the primer for reverse transcription of simian sarcoma virus RNA. The U5 and R regions, derived respectively from the 5' end and terminally redundant sequences of the viral RNA, were found to have similar organization and sequence homology close to that of Moloney murine sarcoma virus or Moloney murine leukemia virus. These results indicate that regions within LTRs with known functionally important sequences have been most well conserved during retrovirus evolution.
Rapid method for the preparation of encephalomyocarditis virus protease from rabbit reticulocyte lysates
Authors: Shih C Y , Naseer N , Shih D S
Published: January 2020

Fractionation of the reticulocyte lysate translation products of encephalomyocarditis virus RNA by ultracentrifugation showed that the viral proteins were distributed differentially in the supernatant and the ribosomal pellet fractions. The viral noncapsid proteins C and D, which contain the viral protease sequence, sedimented preferentially with the pellet fraction. Incubation of the resuspended pellet and subsequent centrifugation of the suspension resulted in cleavage of the protease from proteins C and D and separation of the enzyme from reticulocyte particulate proteins. Preparations thus obtained contained only three encephalomyocarditis virus proteins and were almost devoid of reticulocyte proteins.
Cloning and characterization of DNA complementary to the measles virus mRNA encoding hemagglutinin and matrix protein
Authors: Rozenblatt S , Gesang C , Lavie V , Neumann F S
Published: January 2020

Since cloning and characterization of DNA complementary to measles virus mRNA encoding for the nucleocapsid protein (M. Gorecki and S. Rozenblatt, Proc, Natl. Acad, Sci, U.S.A. 77:3686--3690, 1980), two additional measles-specific clones containing different classes of sequences have been characterized. The cloned plasmids contain inserts of 480 and 530 base pairs as shown by agarose gel electrophoresis and electron microscopy. The sizes of the mRNA species complementary to these inserts are 1,700 and 1,550 nucleotides, respectively as determined by the Northern technique. The cloned DNA fragments were further identified as reverse transcripts of the mRNA coding for the glycoprotein and matrix protein of measles virus. The major cell-free translation products of mRNA selected by hybridization to the individual cloned DNAs comigrated with the 70K in vitro products and matrix proteins. One of the cell-free translation products (70K) was also immunoprecipitated specifically with monoclonal antibodies against measles virus glycoprotein.
Mapping of an adenovirus function involved in the inhibition of DNA degradation
Authors: Lai Fatt R B , Mak S
Published: January 2020

A function involved in the inhibition of DNA degradation has been assigned through complementation tests to a product of region E1b of the adenovirus genome (between 4.5 and 10.5 map units). DNA degradation induced by the adenovirus type 12 (Ad12) cyt mutant H12cyt70 and the Ad5 early deletion mutant dl313 (with the deletion between 3.5 and 10.7 map units) was inhibited by coinfection with Ad5 region E1a (between 0 and 4.5 map units) mutants dl312 and hr1 and region E1b mutant hr6. The defect of inhibition of DNA degradation in Ad5 dl313 was also complemented in 293 cells. This DNase-inhibitory function does not appear to involve polypeptide IX or the 58,000-dalton polypeptide. Wild-type Ad12 induced DNA degradation in hamster embryo cells, suggesting that the DNase-inhibitory function is not expressed in these nonpermissive cells. Additional evidence suggests the involvement of a second viral product which positively influences the DNase activity and which appears to be an early function.
Molecular cloning and characterization of murine leukemia virus-related DNA sequences from C3H/HeN mouse DNA
Authors: Roblin R , Young J M , Mural R J , Bell T E , Ihle J N
Published: January 2020

Ten murine leukemia virus (MuLV)-related DNA sequences were isolated from C3H/HeN mouse genomic DNA by cloning of EcoRI fragments in a Charon 4A vector. Detailed restriction endonuclease maps of four of the clones were developed by using AKR MuLV [32P]cDNA as a probe. C3H clone 14-9 contains approximately 7 kilobase pairs of MuLV-related DNA, one copy of an MuLV long terminal repeat-like sequence, and a region of flanking mouse DNA. C3H clones 34.2 and 36.1 contain approximately 2 kilobase pairs of MuLV-related DNA, one copy of a MuLV LTR-like sequence, and differing lengths of flanking mouse DNA sequences. C3H clone 8.13 was found to contain an insert of 5.7 kilobase pairs of MuLV-related DNA with two long terminal repeat-like regions and sequences which are partially homologous to AKv-1. Comparison fo the restriction endonuclease cleavage maps of these C3H clones with maps recently developed for ecotropic and xenotropic MuLV DNAs indicates that C3H clone 14-9 corresponds to the 5'-terminal portion of a genomic DNA sequence related to xenotropic MuLVs, whereas C3H clones 34.2 and 36.1 correspond to the 3' terminal portions of genomic DNA sequences related to xenotropic MuLVs. Clone 8.13 represents a deleted, xenotropic MuLV-related provirus. C3H clones 14-9, 34.2, 36.1, and 8.13 provide defined DNA sequence probes with which to characterize the organization and expression of endogenous MuLV-related DNA sequences in the mouse genome.
Identification of a unique mouse mammary tumor virus in the BALB/cNIV mouse strain
Authors: Puma J P , Fanning T G , Young L J , Cardiff R D
Published: January 2020

We examined the genetic structure, in terms of restriction endonuclease recognition sites, of the milk-transmitted, low-oncogenic mouse mammary tumor virus (MuMTV) of the BALB/cNIV mouse strain. An analysis with EcoRI documented the presence of acquired cNIV proviruses in the mammary tumor DNAs of BALB/cNIV animals. A comparison of tumor DNAs digested with PstI showed that both the cNIV MuMTV and C3Hf MuMTV proviruses lacked the 4.3- and 1.1-kilobase pair fragments characteristic of C3H MuMTV patterns. An examination of mammary tumor and normal, nonmammary tissue DNAs with BamHI supported the idea that the cNIV MuMTV is identical to the C3Hf MuMTV and demonstrated that these two low-oncogenic proviruses are identical to the high-oncogenic C3H MuMTV provirus with respect to a pair of BamHI sites which define a 1.3-kilobase pair fragment. For each of the three MuMTV strains, we also mapped DNAs generated in isolated virions by reverse transcription of their genomic RNAs. Our results showed that cNIV and C3Hf MuMTV are distinct entities by virtue of an additional PstI site within the cNIV long terminal repeat sequence. Another unique feature of cNIV MuMTV revealed by the analysis of virion-generated DNAs was the existence of a family of genomes within the cNIV population. We concluded that cNIV is distinct from its presumptive C3Hf MuMTV predecessor.
Characterization of the DNA of a Nonoccluded Baculovirus, Hz-1V
Authors: Huang Yuan-Shen , Hedberg M. , Kawanishi C. Y.
Published: January 2020

The DNA of the nonoccluded baculovirus (Hz-1V) obtained from the IMC-Hz-1 cell line was characterized by physicochemical and restriction endonuclease techniques. Hz-1V DNA isolated from purified virus had buoyant densities of 1.58 and 1.54 g/ml in CsCl-ethidium bromide density gradients, which corresponded to supercoiled and to relaxed circular and linear DNA, respectively. Neutral CsCl equilibrium centrifugation indicated that the Hz-1V DNA had a buoyant density of 1.7024 g/ml, which corresponded to a guanine-plus-cytosine (G+C) content of 43%. Thermal denaturation indicated a high G+C domain(s) in the Hz-1V genomic DNA. The domain(s), which included about 11% of the total genomic DNA, exhibited a

T
m

of 97°C. The remaining portion (89%) of the DNA had a

T
m

of 86.5°C. The

T
m

s corresponded to G+C contents of 42 and 67%, respectively. The mean genetic complexity of Hz-1V DNA determined by DNA reassociation kinetic analysis was found to be 152 × 10
6
. A possible rapidly reassociating component comprising approximately 13% of the genome was observed. The mean molecular weights from restriction endonuclease digests were 159 × 10
6
for both
Hind
III and
Eco
RI. Genomic heterogeneity was found in both the wild-type Hz-1V stock and in two plaque isolates. Of 12 single-plaque isolates, 3 basic restriction endonuclease DNA fragment patterns were observed. The molecular size estimates from electron microscopic contour lengths of uncloned viral DNA ranged from 70 to 158 megadaltons, and the mode was the 130- to 140-megadalton class.
Filamentous structures associated with Epstein-Barr virus-infected cells
Authors: Moore C L , Griffith J D , Shaw J E
Published: January 2020

After the onset of Epstein-Barr virus DNA and protein synthesis 10 h after superinfection of Raji cells (a cell line containing Epstein-Barr virus DNA but not producing virus), filamentous structures 25 nm in diameter and 0.2 to 1.4 micrometers in length could be detected in the cell cytoplasm by electron microscopy. These structures banded in metrizamide gradients with viral DNA and proteins, but at a density different from that of virions or nucleocapsids. These filaments, enriched in a 155,000-dalton protein similar in size to a major nucleocapsid protein of Epstein-Barr virus, may represent intermediates in viral nucleocapsid assembly.
Inhibitory effect of E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on herpes simplex virus replication and DNA synthesis
Authors: Descamps J , Sehgal R K , De Clercq E , Allaudeen H S
Published: January 2020

The effect of E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on herpes simplex virus (HSV) replication was examined and compared with that of E-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd). The 50% inhibitory dose against HSV type 1 (HSV-1) was 0.1 microgram/ml compared with 0.008 microgram/ml for BVdUrd; the antimetabolic 50% inhibitory dose of BVaraU ranged from 20 to 95 micrograms/ml. The addition of 50 micrograms of BVaraU per ml to HSV-1-infected Vero cells decreased the synthesis of viral and cellular DNA by 37 and 28%, respectively. The 5'-triphosphate (BVaraUTP) competed with dTTP in DNA synthesis by the herpes-viral and cellular DNA polymerases; the apparent Ki values of HSV-1 DNA polymerase, DNA polymerase alpha, and DNA polymerase beta were 0.14, 0.32, and 5 microM, respectively. Thus, BVaraU was a less effective antiherpesvirus agent than BVdUrd; unlike BVdUrd, it did not appear to be internally incorporated into replicating DNA in virus-infected cells.
Bovine Papilloma Virus Transcription: Polyadenylated RNA Species and Assessment of the Direction of Transcription
Authors: Amtmann Eberhard , Sauer Gerhard
Published: January 2020

The bovine papilloma virus type 1 (BPV-1)-specific RNA species were identified in virus-induced bovine warts, hamster tumors, and transformed hamster and mouse cells. In each case two major species were present (1.1 and 1.3 kilobases [kb]). Also two species of 1.6 and 1.8 kb appearing in variable amounts were found. Only in the keratinized periphery of the warts, where virus replication takes place, was it possible to reveal an additional 2-kb RNA species. In this tissue, however, the 1.6-kb species was not detected. The basal part of a bovine wart contained an additional minor, 2.9-kb, BPV-1-specific RNA sequence. By hybridization with purified defined BPV-1 DNA fragments it was shown that most of the coding sequences of the 2-kb species were transcribed from a region between 0.02 and 0.19 map units. The majority of the coding sequences of the smaller species in transformed cells were located in the region between 0.31 and 0.61 map units. The putative 5′ ends mapped between 0.72 and 0.96 map units. Oligodeoxythymidylic acid-primed [
32
P]cDNA was synthesized from various RNA preparations to generate probes for the detection of 3′ termini of the polyadenylated BPV-1 RNAs. By hybridization across the BPV-1 genome only one signal between the map positions 0.30 and 0.40 was obtained when RNA from transformed cells and from a tumor was used as a template. In contrast, RNA from the periphery of a wart led to the detection of an additional signal which was confined to the region between 0.96 and 1.00 map units. From the arrangement of both the 3′ termini and the coding areas along the viral genome it appears that several RNA species are transcribed from one DNA strand.
New physical map of bacteriophage T5 DNA
Authors: Rhoades M
Published: January 2020

The locations of 103 cleavage sites, produced by 13 restriction endonucleases, were mapped on the DNA of bacteriophage T5. Single- and double-digest fragment sizes were determined by agarose gel electrophoresis, using restriction fragments of phi X174 DNA and lambda DNA as molecular weight standards. Map coordinates were determined by a computer-based least-squares procedures (J. Schroeder and F. Blattner, Gene [Amst] 4:167-174, 1978). The fragment sizes predicted by the final map are all within 2% of the measured values. Based on this analysis, T5st(+) DNA contains 121,300 base pairs (Mr, 80.3 X 10(6) and has a terminal repetition of 10,160 base pairs (Mr, 6.7 X 10(6)). Restriction endonuclease analysis after treatment with exonuclease III and a single-strand-specific endonuclease allowed precise localization of five of the natural single-chain interruptions in T5 DNA. Revised locations for several T5 deletions were also determined.
Adenovirus DNA is associated with the nuclear matrix of infected cells
Authors: Younghusband H B , Maundrell K
Published: January 2020

Viral DNA was found to be tightly associated with the nuclear matrix from HeLa cells lytically infected with human adenovirus type 5. The bound viral DNA, like cell DNA, was resistant to nonionic detergent and to extraction with high-salt (2 M NaCl) solution. However, whereas over 95% of the cell DNA was recovered in the matrix fraction, the amount of associated viral DNA varied during infection. Throughout the lytic cycle, the amount of matrix-associated adenovirus type 5 DNA increased until it reached a plateau level at 20 to 24 h after infection. At this stage, the matrix-bound DNA represented 87% of the total viral DNA; after this stage, additional newly synthesized viral DNA accumulated as non-matrix-associated DNA. DNase digestion studies revealed that all viral DNA sequences were equally represented in the matrix-bound DNA both early and late in infection; thus, unlike cell DNA, there seem to be no preferred attachment sites on the viral genome. An enrichment of viral DNA relative to cell DNA was found in the matrix-associated DNA after extensive DNase I digestion. This finding, together with an in situ hybridization study, suggests that the viral DNA is more intimately associated with the nuclear matrix than is cell DNA and probably does not exist in extended loops.
Characterization of a cell culture persistently infected with the DA strain of Theiler's murine encephalomyelitis virus
Authors: Roos R P , Richards O C , Green J , Ehrenfeld E
Published: January 2020

We established a persistent infection in L 929 cells with the DA strain of Theiler's murine encephalomyelitis virus. Our studies showed that only a small number of cells in the cultures contained infectious virus or viral antigen. A role for interferon in the maintenance of persistence was suggested. Viral isolates from the cultures were not temperature sensitive, nor did they contain viral capsid polypeptide mutations or defective interfering particles. T1 oligonucleotide maps showed evidence of mutation in two of three isolates.
Detailed characterization of an apparently unspliced beta herpes simplex virus type 1 gene mapping in the interior of another
Authors: Draper K G , Frink R J , Wagner E K
Published: January 2020

We precisely localized the coding region and determined the nucleotide sequence of a 1.2-kilobase beta herpes simplex virus type 1 mRNA which underlies the 3' region of the 5.2-kilobase beta mRNA mapping in HindIII fragment K. This mRNA, which lacks readily detectable splices, has its own promoter by the criteria of identification of putative herpes simplex virus type 1 control sequences and in vitro transcription by a Manley polymerase system.
Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR
Authors: Ensinger M J
Published: January 2020

One hundred temperature-sensitive mutants of vaccinia virus WR were isolated from virus that had been mutagenized with 5-bromodeoxyuridine or N-methyl-N'-nitro-N-nitrosoguanidine. A rapid screening procedure based on the ability of vaccinia virus to form plaques under liquid overlay medium was used to identify potential mutants among randomly picked plaque isolates or plaques preselected for their small size after temperature shift-up. The preselection technique resulted in a sixfold increase in the number of successful mutant isolations relative to the number of plaques picked. All of the mutants had efficiencies of plating at 39.5 degrees C relative to that at 33 degrees C of 10(-4) or less, and 33 of 40 produced 10% or less of the amount of virus at the nonpermissive temperature (39.5 degrees C) relative to that at the permissive temperature (33 degrees C). Experiments with the fluorescent DNA binding dye Hoechst 33258 demonstrated that 6 of the 100 mutants failed to form characteristic cytoplasmic DNA factories at 39.5 degrees C. To facilitate the functional grouping of such a large number of mutants, a rapid infectious center assay was developed. Thirty of the mutants were assigned to 16 or 17 complementation-recombination groups by using this assay. Recombination experiments have allowed the construction of a genetic map representing 22 mutants in 12 of these groups.
Physical and biological features of polyoma virus mutants able to infect embryonal carcinoma cell lines
Authors: Vasseur M , Katinka M , Herbomel P , Yaniv M , Blangy D
Published: January 2020

Three new polyoma mutants were selected for their ability to grow on the embryonal carcinoma cell line F9. These mutants share in common an insertion of two nucleotides, a thymine and an adenine, in the noncoding region located on the late side of the origin of replication. We have found that these insertions exist in all of the other polyoma virus mutants able to grow on F9 cells (Fujimura et al., Cell 23:809-814, 1981; Katinka et al., Nature (London) 290:720-722, 1981; K. Sekikawa and A. J. Levine, Proc. Natl. Acad. Sci. U.S.A. 78:1100-1104, 1981). The region containing these insertions could be folded into a stable secondary structure which included a guanine plus cytosine (G + C)-rich stem. The adenine and thymine were inserted in such a way that they maintained the palindrome in the G + C-rich stem and were complementary in the putative secondary structure that we present here. Another class of polyoma virus mutants selected on a multipotential carcinoma cell line (PCC4-Aza) were characterized by a more complex rearrangement (deletion and duplication) which occurred in the same region. This arrangement preserved the G + C-rich palindrome and also yielded a sequence which still allowed the folding of another type of stable secondary structure. The significance of these findings is discussed.
Asynchronous bidirectional replication of polyoma virus DNA
Authors: Buckler-White A J , Krauss M R , Pigiet V , Benbow R M
Published: January 2020

The structure of polyoma virus replicative intermediates isolated from infected 3T6 cells was analyzed by two-dimensional agarose gel electrophoresis (Sundin and Varshavsky, Cell 21:103-114, 1980) and quantitative electron microscopy (Krauss and Benbow, J. Virol. 38:815-825, 1981). DNA replication was initiated at a single site (ori) in essentially all of the replicative intermediates. Most of the early replicative intermediates were formed by unidirectional synthesis in the direction of early transcription. Most mid- and late replicative intermediates contained two replication forks which had traveled unequal distances from the origin. Asynchronous initiation of the two growing forks was postulated to account for these observations.
Molecular cloning of a highly leukemogenic, ecotropic retrovirus from an AKR mouse
Authors: Lenz J , Crowther R , Klimenko S , Haseltine W
Published: January 2020

SL3-3 is a leukemogenic, ecotropic retrovirus produced by a T-cell line derived from a spontaneous lymphoma of an AKR mouse. We have isolated a molecular clone of its DNA provirus from infected NIH 3T3 fibroblasts. Cloned proviral DNA produced infectious virus upon transfection onto NIH 3T3 cells. Virus derived by transfection induced lymphomas at high frequency in AKR/J, C3H(f)/Bi, CBA/J, and NFS/N mice. Heteroduplex and RNase T1 fingerprinting analyses showed that the genomes of SL3-3 and the non-leukemogenic virus, Akv, contain no major substitutions relative to one another and differ by only a few base changes. These results unambiguously show that SL3-3 is a highly leukemogenic virus and that major rearrangements of the genome relative to Akv are not required for virulence.
Hybrid Selection of Small RNAs by Using Simian Virus 40 DNA: Evidence that the Simian Virus 40-Associated Small RNA Is Synthesized by Specific Cleavage from Large Viral Transcripts
Authors: Alwine James C.
Published: January 2020

The simian virus 40 (SV40)-associated small RNA (SAS-RNA), approximately 64 nucleotides, is virally encoded within a region of the viral late (+) DNA strand which encodes no known protein. The SAS-RNA arises in abundance late in SV40 lytic infection. Previous data indicate that the synthesis of the SAS-RNA may be under the control of the normal late viral promoter; i.e., inhibition of transcription from the late promoter results in cessation of SAS-RNA synthesis. The synthesis of SAS-RNA was examined to determine whether the SAS-RNA is the product of cleavage from noncoding regions of nuclear late transcripts or an independent transcription product like 5S RNA, or the adenovirus VA-RNAs. The data described below suggest that SAS-RNA is cleaved from large late transcripts. In vitro transcription of DNA fragments containing the SAS-RNA coding region yielded no SAS-RNA synthesis; this result was supported by DNA sequence analysis, which indicated no promoter-like regions either within or flanking the SAS-RNA coding region. In support of a cleavage mechanism, the SAS-RNA has a 3′-phosphate end, an occurrence which is indicative of nuclease cleavage. In addition, 5′-end labeling of the SAS-RNA was possible only after calf alkaline phosphatase treatment; this indicates that the SAS-RNA is not capped. Hybrid selection analysis was used to demonstrate that separation of the SAS-RNA coding region from the normal late promoter resulted in elimination of SAS-RNA synthesis. This was demonstrated in SV40-transformed cells in which integration of a single copy of SV40 breaks the continuity of the late coding region, so that the SAS-RNA coding region is physically separated from the normal late promoter. The lack of SAS-RNA synthesis indicates that the SAS-RNA coding region cannot function as a primary transcription unit. The same result and conclusion were obtained by using a permissive cell line transformed by SV40 (COS-1 cells); here it was found that the integrated SAS-RNA coding region was not expressed even during a viable lytic infection in which the SAS-RNA could be expressed from the infecting viral genomes. The simplest conclusion drawn from the data is that the SAS-RNA is cleaved from larger late transcripts which initiate at the normal late promoter. This conclusion suggests that many of the small RNAs found in normal eucaryotic cells may be synthesized by specific cleavage rather than by primary transcription. In the course of these studies several small cellular RNAs were detected, due to their specific hybrid selection, by using SV40 DNA. Primary mapping and characterization data of these RNAs are also presented.
Molecular analysis of the c-myc locus in normal tissue and in avian leukosis virus-induced lymphomas
Authors: Neel B G , Gasic G P , Rogler C E , Skalka A M , Ju G , Hishinuma F , Papas T , Astrin S M , Hayward W S
Published: January 2020

We isolated molecular clones of the provirus-host cell junctions (tumor junction fragments) from two avian leukosis virus-induced lymphomas and compared the structures of these clones with a clone of the normal c-myc gene. Restriction mapping and DNA sequencing demonstrated that normal proviral integration events occurred adjacent to c-myc in both tumors, without gross structural alteration of c-myc. The right long terminal repeat of an avian leukosis virus provirus is integrated upstream from the bulk of the c-myc coding sequences and oriented such that transcription can initiate within the long terminal repeat and proceed downstream into c-myc. A comparison of a tumor junction fragment with the v-myc gene showed that there are two regions of v-myc-related sequences (which are probably exons) separated by 1 kilobase of sequences unrelated to v-myc (probably an intron). A DNA sequence analysis of the tumor junction fragments suggested that integration had occurred in exons adjacent to splice donor sites. This suggests that there are additional exons and introns in c-myc. Based on these findings, a model is proposed for the genesis of the tumor-specific RNAs containing viral-5' and c-myc information in avian leukosis virus-induced lymphomas.
In Vitro Translation of
Autographa californica
Nuclear Polyhedrosis Virus Early and Late mRNAs
Authors: Smith Gale E. , Vlak Just M. , Summers Max D.
Published: January 2020

A preliminary translational map of the
Autographa californica
genome was constructed. Eighteen viral DNA restriction fragments were either purified from agarose gels or obtained from pBR322 recombinant DNA plasmids to locate specific gene products. The DNAs were immobilized on nitrocellulose filters and used to select viral mRNAs isolated from RNA obtained from the cytoplasm of infected
Spodoptera frugiperda
cells at 21 h postinfection. The fragment-specific mRNAs were translated in vitro in the presence of
l
-[
3
H]leucine by using a rabbit reticulocyte lysate system and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The approximate locations of 19
A. californica
nuclear polyhedrosis virus (AcMNPV) gene products were mapped. The genes for mRNAs present late in viral infection were mapped to DNA fragments that represent nearly the entire genome. The molecular weights of many of these proteins were similar to those present in purified AcMNPV extracellular virus and to proteins being made in infected cells at 18 to 21 h postinfection. Cytoplasmic RNA was isolated at 4 h postinfection from infected cells, a time early in the viral infection cycle, and hybridized to AcMNPV DNA immobilized on nitrocellulose filters. AcMNPV-specific early RNA was translated in vitro into at least six polypeptides, the most abundant having a molecular weight of 39,000. Viral polypeptides were detected in cells pulse-labeled with
l
-[
3
H]leucine at 3 to 6 h postinfection, with molecular weights similar to those of polypeptides made in vitro from early AcMNPV mRNA.
Molecular cloning of circular unintegrated DNA of two types of the SEATO strain of gibbon ape leukemia virus
Authors: Gelmann E P , Trainor C D , Wong-Staal F , Reitz M S
Published: January 2020

Closed circular unintegrated DNA of the SEATO strain of gibbon ape leukemia virus (GaLV-S) was isolated from canine thymus fibroblasts after cocultivation with chronically infected bat lung fibroblasts. Restriction endonuclease HindIII cleaves GaLV-S DNA once, thus allowing isolation and cloning of HindIII-digested unintegrated DNA in a permitted form. Two clones isolated in the vector, Charon 21A, were nearly identical by restriction enzyme mapping to each of the two types of GaLV-S previously observed. These two types differ at a single SalI site. Unlike previous maps of GaLV-S proviral DNA, however, both clones lack SstI sites in the long-terminal-repeat units. Both the GaLV-S clones and the major species of GaLV-S proviral DNA contain an EcoRI site in the long-terminal-repeat units. The presence of this EcoRI site and the absence of an SstI site in the GaLV-S long-terminal-repeat units differentiate it from all other known GaLV strains and from the closely related nononcogenic simian sarcoma-associated virus. Heteroduplex comparisons of each of the two clones to clones of simian sarcoma-associated virus show no obvious deletion or substitution loops. This suggests that the ability of GaLV-S to induce myeloid leukemia in gibbon apes in not due to an acquired onc gene.
Isolation of a monoclonal antibody viral polypeptide VP2 of simian virus 40
Authors: Kasamatsu H
Published: January 2020

A stable hybridoma cell line, IIG8-203-2, that secretes a monoclonal antibody of the immunoglobulin subclass M was obtained by fusion of mouse myeloma cells with spleen cells of mice that had been immunized with the viral polypeptide VP2 of simian virus 40. The monoclonal antibody recognizes viral polypeptides that migrate with VP2 polypeptides in a sodium dodecyl sulfate-polyacrylamide gel. It also recognizes two intracellular polypeptides (29,000 and 37,000 daltons) in a detergent-insoluble fraction extracted 30 h after virus infection of TC7 cells.
Phosphorylation and dephosphorylation alter the structure of D2 hybrid T antigen
Authors: Baumann E A , Hand R
Published: January 2020

D2 hybrid T antigen is a protein closely related to simian virus 40 large T antigen and is synthesized in large quantities in cells infected with Ad2+D2, an adenovirus-simian virus 40 hybrid. We have analyzed the effects of phosphorylation on the structure and DNA binding of this protein. On nondenaturing pore-gradient gels, the purified protein migrated with an apparent molecular weight of 135,000, with a minor band at 330,000 molecular weight. In vitro phosphorylation catalyzed by the protein kinase activity associated with the protein resulted in a structural change so that most of it migrated with an apparent molecular weight of 740,000. Treatment of the phosphorylated form of the protein with alkaline phosphatase (which removed 95% of the phosphate) caused the disappearance of the 740,000-molecular-weight form and reappearance of the smaller forms. Partial tryptic digestion showed that D2 T antigen has two major regions of phosphorylation, only one of which was phosphorylated in vitro. The region phosphorylated in vitro was responsible for the aggregation of D2 T antigen and was tentatively assigned to the N-terminal part of the protein. As shown by protein blotting onto nitrocellulose filters, it was mainly the form of 740,000 molecular weight that bound to simian virus 40 DNA. However, sucrose gradient analyses showed that only a fraction of the in vitro-phosphorylated protein bound to DNA, suggesting that aggregation alone is not sufficient for binding.
DNA Binding Properties of Simian Virus 40 Temperature-Sensitive A Proteins
Authors: Wilson Van G. , Tevethia M. J. , Lewton Betsy A. , Tegtmeyer Peter
Published: January 2020

Wild-type simian virus 40 A protein (large T antigen) bound to three tandem regions of simian virus 40 DNA. The binding regions were defined by the ability of A protein to protect simian virus 40 DNA from digestion with limited (footprint assay) or excess (fragment assay) amounts of DNase I. At low concentrations, protein first bound to region I, which maps 30 to 45 base pairs to the early side of the origin of replication. At higher concentrations, A protein also protected region II and then region III. Region II spanned approximately 65 base pairs and corresponded in location to the functional origin of replication that contains a unique
Bgl
I site along with an adjacent adenine-thymine-rich region. Region III was adjacent to the late boundary of region II, but its distal limit was not well defined. Twelve distinct temperature-sensitive (
ts
) A proteins were purified and examined for their ability to bind in regions I to III. Three classes of
ts
A protein were defined on the basis of thermal stability. Class I
ts
A protein displayed wild-type binding either with or without a heat shock. Unheated class II
ts
A protein exhibited wild-type binding, but after a heat shock bound very poorly to the origin of replication. Class III
ts
A protein was defective in its binding even without a heat shock and only protected region I. Classes II and III were coded by mutants mapping in two distinct regions of the genome. For all of the
ts
A proteins examined, there was a positive correlation between the thermolability of origin binding in vitro and the temperature sensitivity of these mutants for DNA replication and transcriptional autoregulation in vivo. This correlation adds support to the essential role of origin binding by A protein in viral DNA replication and early transcription repression.
Time-dependent maturation of the simian virus 40 large T antigen-p53 complex studied by using monoclonal antibodies
Authors: Carroll R B , Gurney E G
Published: January 2020

Newly synthesized simian virus 40 large tumor antigen (T Ag) slowly forms a stable complex with the host tumor antigen, "p53." By the use of immunological and temporal separations and inhibition of aggregation and processing by A locus mutation, we have distinguished specific steps in the reaction sequence leading to formation of the rapidly sedimenting oligomeric complex. The monoclonal antibody PAb101 bound only a fraction of the total soluble pulse-labeled T Ag bound by antitumor serum. After a chase, all T Ag had matured to the form recognized by PAb101. All p53 in the mouse line SVA31E7 was precipitated by the T Ag-specific monoclonal antibody PAb101, even after a short pulse, and is therefore entirely bound to mature T Ag. The p53-specific monoclonal antibody PAb122 precipitates nearly all of the mature T Ag recognized by PAb101, except A locus mutant T Ag, synthesized at the nonpermissive temperature. A locus mutation inhibited entry of newly synthesized T Ag into the oligomeric greater than 28S complex of T Ag and p53.