bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Kinase Activity in Hepatitis B Virus
Authors: Albin Catherine , Robinson William S.
Published: January 2020

Protein kinase activity was found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B virus-infected patients, in virion cores, and in hepatitis B core antigen particles purified from hepatitis B virus-infected hepatic tissue and was not found in purified hepatitis B surface antigen particle preparations free of Dane particles. Only a fraction of the major polypeptide (apparent size, 19,700 daltons) in Dane particle cores and hepatitis B core antigen particles from infected liver appeared to be phosphorylated, and phosphorylation changed the electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to that expected for a polypeptide of 20,600 daltons. Five minor polypeptides with apparent sizes between 38,000 and 63,000 daltons were phosphorylated in Dane particles and Dane particle core preparations but were not detected in hepatitis B core antigen particles from infected liver. None of these had electrophoretic mobilities corresponding to those of known hepatitis B surface antigen polypeptides. Prolonged storage of purified hepatitis B core antigen particles or incubation with human immunoglobulin G preparations containing antibody to the hepatitis B core antigen with or without antibody to the hepatitis B e antigen resulted in the conversion of the polypeptide with an apparent size of 20,600 daltons to ones with apparent sizes of 14,700 and approximately 6,000 daltons, suggesting proteolytic cleavage of the 20,600-dalton polypeptide under these conditions.
Genetic Analysis of Orbiviruses by Using RNase T
1
Oligonucleotide Fingerprints
Authors: Walker Peter J. , Mansbridge Jonathan N. , Gorman Barry M.
Published: January 2020

Corresponding double-stranded RNA segments of the related orbiviruses Wallal and Mudjinbarry produced distinctly different RNase T
1
fingerprint patterns. No extensive sequence reiteration was observed between segments of Mudjinbarry virus. Fingerprint analysis of the genome of recombinant orbiviruses confirmed segment reassortment as a mechanism of interchange of genetic information. When temperature-sensitive mutants of each virus were crossed in mixed infection, a consistent pattern of segment reassortment was correlated with generation of the wild-type phenotype. Thus, the temperature-sensitive lesion of group II Wallal serogroup mutants was mapped to segment 1. The group I mutant lesion appears to be located in segment 2.
Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens
Authors: Gurney E G , Harrison R O , Fenno J
Published: January 2020

We have isolated three clones of hybrid cells which synthesize antibodies specific for determinants on simian virus 40 (SV40) T antigens. Mouse myeloma NS1 cells were fused with spleen cells from mice that had been immunized with SV40-transformed mouse cells. Hybrid cells were selected in HAT medium and cloned in soft agar. We used an enzyme-linked immunosorbent assay for detection and quantification of mouse antibodies against SV40 T antigens. Monoclonal antibodies from 3 of the 24 clones that scored as positive in the enzyme-linked immunosorbent assay were verified by immunoprecipitation to be specific for SV40 T antigens. Two clones (7 and 412) produced antibodies that recognized denaturation-sensitive antigenic determinants unique to large T antigen. Antibodies from clone 7 appeared to have a low affinity for large T antigen. Antibodies from clone 412 had a higher affinity for large T antigen but did not recognize a subclass of large T antigen that was recognized by tumor serum. Antibodies of the third clone, clone 122, recognized a denaturation-stable antigenic determinant of the 53,000-dalton mouse nonviral T antigen in SV40-transformed cells. Antibodies from clone 122 also recognized similar (51,000- to 56,000-dalton) nonviral T antigens in SV40-transormed or lytically infected cells from five mammalian species and in four uninfected mouse lines. From these observations, we have concluded that (i) the 94,000-dalton SV40 large T antigen may exist as immunologically distinguishable subclasses, and (ii) the nonviral T antigens of five mammalian species share at least one antigenic determinant.
Persistence of herpes simplex virus genes in cells of neuronal origin
Authors: Levine M , Goldin A L , Glorioso J C
Published: January 2020

The growth characteristics of the KOS strain of herpes simplex virus type 1 (HSV-1) in cell lines of nervous tissues origin were examined in an attempt to develop a tissue culture system mimicking the in vivo state of HSV-1 latency. We have previously reported that the B103 rat brain neuroma cell line is nonpermissive for growth of the KOS strain. In this report, we show that this nonpermissiveness is a temperature- and multiplicity-dependent phenomenon, with minimum virus yields at an elevated temperature and a low multiplicity of infection. Under these conditions, B103 cells survived infection with active wild-type or mutant HSV-1, whereas similarly treated Vero cells were killed. Six independent cultures of B103 cells surviving HSV-1 infection have been established. The surviving cells ceased production of any HSV-1 virus by 14 days postinfection and resumed growth and division at rates comparable to those of uninfected B103 cells. Survivor cells continued to express HSV-1-specific antigens, however, as detected by indirect immunofluorescence and by surface iodination followed by immunoprecipitation and polyacrylamide gel electrophoresis. The survivor cells did not express all of the surface proteins seen on productively infected B103 cells, and they were not susceptible to complement-mediated immune cytolysis with anti-HSV-1 antiserum. These results demonstrate that at least a portion of the HSV-1 genome is being harbored in these survivor cells.
Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies
Authors: van Wyke K L , Hinshaw V S , Bean W J , Webster R G
Published: January 2020

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.
Transformation phenotype of polyoma virus-transformed rat fibroblasts: plasminogen activator production is modulated by the growth state of the cells and regulated by the expression of an early viral gene function
Authors: Perbal B
Published: January 2020

The expression of two transformation parameters, namely, ability to grow in agar and plasminogen activator production, was studied in several rat fibroblasts transformed by either wild-type or thermo-sensitive (tsa and ts25) polyoma viruses. The production of plasminogen activator was found to be dependent upon the growth state of the infected cells during a period of several days after infection. The analysis of the transformed phenotype of 25 tsa transformants and of 19 ts25 transformants independently isolated under various growth conditions led to the conclusion that there is no correlation between the regulation processes involved in plasminogen activator production and ability to grow without anchorage. The results obtained also suggested that the production of plasminogen activator is under the control of a functional large T antigen.
Comparative structural analysis of glycoprotein gD of herpes simplex virus types 1 and 2
Authors: Eisenberg R J , Ponce de Leon M , Cohen G H
Published: January 2020

We studied the synthesis and processing of the type-common glycoprotein gD in herpes simplex virus type 2 (HSV-2) and compared it structurally to glycoprotein gD of herpes simplex virus type 1 (HSV-1). We demonstrated that in HSV-2, gD undergoes posttranslational processing from a lower-molecular-weight precursor (pgD51) to a higher-molecular-weight product (gD56). Tryptic peptide analysis by cation-exchange chromatography indicated that this processing step altered neither the methionine nor the arginine tryptic peptide profile of gD of HSV-2. Comparative tryptic peptide analysis of gD of HSV-1 and HSV-2 showed that the methionine and arginine tryptic peptide profiles of these two proteins were very similar, but not identical. Some of the resolved peptides coeluted from the cation-exchange column, suggesting that some amino acid sequences of the two proteins might be very similar. However, each protein also appeared to possess several type-specific tryptic peptides. The structural similarity of these two glycoproteins correlates well with their antigenic cross-reactivity since monoprecipitin antibody to gD of HSV-1 also immunoprecipitates gD of HSV-2 and neutralizes the infectivity of both viruses to approximately the same extent.
Bacteriophage T4-related macromolecular synthesis under restriction of plasmid Rts1
Authors: Raghavan N , Ishaq M , Kaji A
Published: January 2020

Rts1 is a plasmid which confers upon the host bacteria the capacity to restrict T4 bacteriophage growth at 32 degrees C but not at 42 degrees C. Pulse-labeling of phage-infected cells showed that Rts1 restricts the synthesis of T1 DNA. Despite efficient restriction of T4 phage growth and DNA synthesis, infected Escherichia coli 20SO harboring Rts1 synthesized both early and late T4 phage RNA. Synthesis of early T4 phage RNA under restrictive conditions (32 degrees C) was almost equal to that found under nonrestrictive conditions, and a lesser, but significant, amount of late T4 phage RNA was made in almost complete absence of T4 DNA synthesis. Moreover, very little, if any, T4 phage-coded lysozyme was detected in the infected E. coli 20SO/Rts1 at 32 degrees C, whereas normal amounts of lysozyme were present at 42 degrees C.
Comparison of Epstein-Barr virus strains of different origin by analysis of the viral DNAs
Authors: Bornkamm G W , Delius H , Zimber U , Hudewentz J , Epstein M A
Published: January 2020

Epstein-Barr virus (EBV) originating from Burkitt's lymphoma (P3HR-1 and CC34-5), nasopharyngeal carcinoma (M-ABA), transfusion mononucleosis (B95-8), and a patient with acute myeloblastic leukemia (QIMR-WIL) was isolated from virus-carrying lymphoid cell lines after induction with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Viral DNA was analyzed by partial denaturation mapping and by use of the restriction endonucleases EcoRI, HindIII, and SalI and separation of fragments in 0.4% agarose. By using the restriction enzyme data of B95-8 (EBV) and W91 (EBV) obtained by Given and Kieff (D. Given and E. Kieff, J. Virol. 28:524-542, 1978), maps were established for the other virus strains. Comigrating fragments were assumed to be identical or closely related among the different strains. Fragments of different strains migrating differently were isolated, purified, radioactively labeled, and mapped by hybridization against blots of separated viral fragments. The results were as follows. (i) All strains studied were closely related. (ii) The number of internal repeats was variable among and within viral strains. (iii) B95-8 (EBV) was the only strain with a large deletion of about 12,000 base pairs at the right-hand side of the molecule. At the same site, small deletions of about 400 to 500 base pairs were observed in P3HR-1 (EBV) and M-ABA (EBV) DNA. (iv) P3HR-1 (EBV), the only nontransforming EBV strain, had a deletion of about 3,000 to 4,000 base pairs in the long unique region adjacent to the internal repeats carrying a HindIII site. (v) Small inserted sequences of 150 to 400 base pairs were observed in M-ABA (EBV) and B95-8 (EBV) at identical sites in the middle of the long unique region. (vi) Near this site, an insertion of about 1,000 base pairs was found in P3HR-1 (EBV) DNA. (vii) The cleavage patterns of P3HR-1 virus DNA and the results of blot hybridizations with P3HR-1 virus fragments are not conclusive and point to the possibility that in addition to the normal cleavage pattern some viral sequences may be arranged differently. Even though it is possible that small differences in the genome organization may have significant biological effects, the great similarity among different EBV strains does not favor the hypothesis that disease-specific subtypes exist.
Characterization of Aleutian disease virus as a parvovirus
Authors: Bloom M E , Race R E , Wolfinbarger J B
Published: January 2020

We characterized a strain of Aleutian disease virus adapted to growth in Crandall feline kidney cells at 31.8 degrees C. When purified from infected cells, Aleutian disease virus had a density in CsCl of 1.42 to 1.44 g/ml and was 24 to 26 nm in diameter. [3H]thymidine could be incorporated into the viral genome, and the viral DNA was then studied. In alkaline sucrose gradients, Aleutian disease virus DNA was a single species that cosedimented at 15.5S with single-stranded DNA from adeno-associated virus. When the DNA was analyzed on neutral sucrose gradients, a single species was again observed, which sedimented at 21S and was clearly distinct from 16S duplex adeno-associated virus DNA. A similar result was obtained even after incubation under annealing conditions, implying that the bulk of Aleutian disease virus virions contained a single non-complementary strand with a molecular weight of about 1.4 X 10(6). In addition, two major virus-associated polypeptides with molecular weights of 89,100 and 77,600 were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virus purified from infected cultures labeled with [35S]methionine. These data suggest that Aleutian disease virus is a nondefective parvovirus.
DNA nucleotide sequence heterogeneity between the Towne and AD169 strains of cytomegalovirus
Authors: Pritchett R F
Published: January 2020

The results of reciprocal DNA-DNA reassociation kinetics indicated that although the DNAs of human cytomegalovirus (CMV) strains Towne and AD169 shared approximately 90% of their nucleotide sequences, about 10% heterogeneity did exist. The implication was that, with respect to one another, the DNAs of CMV Towne and CMV AD169 contained unique nucleotide sequences. To obtain more direct evidence, 32P-labeled DNA of one virus strain was reassociated in the presence of excess unlabeled DNA of the heterologous virus strain. Those 32P-labeled DNA sequences remaining single stranded were separated from double-stranded DNA on hydroxyapatite columns and incubated with Southern blots containing XbaI restriction enzyme fragments of the homologous virus DNA. This approach not only enriched for nucleotide sequences unique to each strain of virus, but also provided for the identification of the restriction enzyme fragments in which the unique sequences were contained. The CMV Towne unique sequences were found in XbaI fragments A, C, G, L, N, and Q of CMV Towne DNA. The CMV AD169 unique sequences were found in XbaI fragments A, C, G, and J of CMV AD169 DNA. The possible significance of these data with respect to variation among other CMV isolates is discussed.
Localization of Membrane-Associated Proteins in Vesicular Stomatitis Virus by Use of Hydrophobic Membrane Probes and Cross-Linking Reagents
Authors: Zakowski Jack J. , Wagner Robert R.
Published: January 2020

The location of membrane-associated proteins of vesicular stomatitis virus was investigated by using two monofunctional and three bifunctional probes that differ in the degree to which they partition into membranes and in their specific group reactivity. Two hydrophobic aryl azide probes, [
125
I]5-iodonaphthyl-1-azide and [
3
H]pyrenesulfonylazide, readily partitioned into virion membrane and, when activated to nitrenes by UV irradiation, formed stable covalent adducts to membrane constituents. Both of these monofunctional probes labeled the glyco-protein G and matrix M proteins, but [
125
I]5-iodonaphthyl-1-azide also labeled the nucleocapsid N protein and an unidentified low-molecular-weight component. Protein labeling of intact virions was unaffected by the presence of cytochrome
c
or glutathione, but disruption of membrane by sodium dodecyl sulfate greatly enhanced the labeling of all viral proteins except G. Labeling of G protein was essentially restricted to the membrane-embedded, thermolysin-resistant tail fragment. Three bifunctional reagents, tartryl diazide, dimethylsuberimidate, and 4,4′-dithiobisphenylazide, were tested for their capacity to cross-link proteins to membrane phospholipids of virions grown in the presence of [
3
H]palmitate. Only G and M proteins of intact virions were labeled with
3
H-phospholipid by these cross-linkers; the reactions were not affected by cytochrome
c
but were abolished by disruption of virus with sodium dodecyl sulfate. Dimethylsuberimidate, which reacts with free amino groups, cross-linked
3
H-phospholipid to both G and M protein. In contrast, the hydrophilic tartryl diazide cross-linked phospholipid primarily to the M protein, whereas the hydrophobic 4,4′-dithiobisphenylazide cross-linked phospholipid primarily to the intrinsic G protein. These data support the hypothesis that the G protein traverses the virion membrane and that the M protein is membrane associated but does not penetrate very deeply, if at all.
Incorporation of influenza virus M-protein into liposomes
Authors: Bucher D J , Kharitonenkov I G , Zakomirdin J A , Grigoriev V B , Klimenko S M , Davis J F
Published: January 2020

M-protein from influenza virus vaccine was purified by sodium dodecyl sulfate-gel chromatography and incorporated into liposomes by solubilization with octylglucoside and subsequent dialysis. Liposomes containing M-protein formed a distinct population with a density of 1.22 g/ml on sucrose-gradient centrifugation, regardless of the net charge on the liposomes. Treatment of the liposomes by freeze-fracture followed by electron microscopic examination showed multilamellar structures in those liposomes without M-protein; liposomes containing M-protein were mulberry-like structures which appeared unilamellar. These studies show incorporation of M-protein into the lipid bilayer.
Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells
Authors: Pignatti P F , Cassai E
Published: January 2020

HEp-2 cells were infected with herpes simplex virus type 1 and labeled with [3H]thymidine and 14C-amino acids. Infected cells or nuclei prepared from them were extracted with Triton X-100 and NaCl, utilizing a method recently described, and the low-speed supernatant (extract) was partially purified by sedimentation on sucrose gradients. A nucleoprotein complex which sedimented as a wide peak around 200S was identified. The nucleoprotein complex contained viral DNA, which banded at the expected density in CsCl isopycnic gradients and was intact after measurements taken on electron microscopic photographic enlargements. The autoradiographic pattern of 14C-labeled proteins after electrophoresis showed that only a few of the virus-specific polypeptides were present in the nucleoprotein complexes, in particular, VP5, VP12, VP15.2, VP19, and VP24. Cellular histones were absent. The extracts and the nucleoprotein complexes were centrifuged to equilibrium in metrizamide density gradients without prefixation. Electron microscopic direct visualization of the nucleoprotein complexes after sucrose or metrizamide purification revealed that the proteins were preferentially associated with one end of the DNA molecule and formed large irregular terminal thickenings or capsid-like transparent shells enclosing polyglobular cores. No nucleosomes were observed on herpes simplex virus nucleoprotein complexes. The same type of complex was detected after phosphonoacetic acid addition, and grossly altered nucleocapsids were formed.
Characterization of vesicular stomatitis virus mutants by partial proteolysis
Authors: Metzel P S , Reichmann M E
Published: January 2020

Structural proteins of temperature-sensitive (ts) mutants of vesicular stomatitis virus, Indiana serotype, were compared with those of wild-type and revertant virions by electrophoresis on polyacrylamide gels of partial digests with Staphylococcus aureus V8 protease. Mutants of complementation groups III (tsG31 and tsG33), II (tsG22), and IV (tsG41) differed from the wild-type virion in peptide profiles of their M, NS, and N proteins, respectively. The differences were only detectable over a narrow range of enzyme-substrate ratios and were due to peptides transiently generated during incomplete digestion. Proteins of revertants to tsG31, tsG22, and tsG41 exhibited the wild-type virion peptide pattern, indicating that reversion had restored their original conformation. However, in the case of tsG22, the NS peptide profile reverted to the wild-type phenotype only partially, suggesting that a silent mutation might have taken place during either the original chemical mutagenesis or the following repeated laboratory passages. The apparent alteration in protein conformation and its restoration upon reversion of the mutants indicated that the lesions of groups III and IV were located in the M and N proteins, respectively. Moreover, for the first time, the site of mutation of group II could be positively identified as the NS protein cistron.
Pichinde virus L and S RNAs contain unique sequences
Authors: Leung W C , Ramsingh A , Dimock K , Rawls W E , Petrovich J , Leung M
Published: January 2020

Using oligodeoxyribonucleotides produced by limited DNase I digestion of calf thymus DNA as a primer, we synthesized complementary DNA (cDNA) from the L and the S RNAs of Pichinde virus. The reaction conditions for in vitro cDNA synthesis were optimized to allow transcription of about 90% of either L or S RNA. No significant hybridization was observed when the L cDNA was hybridized to the S RNA, or when the S cDNA was hybridized to the L RNA. The results indicate that the L and S RNAs of Pichinde virus contain unique nucleotide sequences.
In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA
Authors: Jochemsen H , Hertoghs J J , Lupker J H , Davis A , van der Eb A J
Published: January 2020

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transformed by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.5%). In addition, the adenovirus type 5-transformed human embryonic kidney line 293.C31 was investigated. The products were immunoprecipitated with serum from tumor-bearing hamsters and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The results show that all transformed cells investigated contain early region 1a-specific RNAs which can be translated into proteins with molecular weights of 34,000 (34K), 36K, 40K, and 42K. Transformed cells that also contain an intact early region 1b synthesized RNA which can be translated into proteins with molecular weights of 19K and 65K. Minor proteins of 15K, 16K, 17.5K, 18K, 25K, and 29K were also observed, but these proteins could not be mapped unambiguously. Cells transformed by the 8% HindIII-G apparently lack RNA encoding the 65K protein, but they do contain RNA coding for the 19K protein.
Subgenomic viral DNA species synthesized in simian cells by human and simian adenoviruses
Authors: Daniell E
Published: January 2020

DNA synthesized after infection of simian tissue culture cells (BSC-1 or CV-1) with human adenovirus type 2 or 5 or with simian adenovirus 7 was characterized. It was demonstrated that as much as 40% of the virus-specific DNA in nuclei of infected monkey cells consists of subgenomic pieces. No subgenomic viral DNA species were detected in the nuclei of human (HeLa) cells infected with these adenovirus types. Restriction analysis showed that these short viral DNA molecules contain normal amounts of the sequences from the ends of the viral genome, whereas internal regions are underrepresented. The production of subgenomic DNAs is not correlated with semipermissive infection. Although adenovirus types 2 and 5 are restricted in monkey cells, these cells are fully permissive for simian adenovirus 7. HR404, an adenovirus type 5 mutant which is not restricted in monkey cells, produced the same percentage of subgenomic DNAs as did its wild type (restricted) parent, and coinfection of monkey cells with adenovirus type 5 DNAs. The array of predominant size classes among the heterogeneously sized short DNAs is serotype specific. Extensive plaque purification and comparison of wild-type adenovirus type 5 with several viral mutants indicated that the distribution of aberrant sizes of DNA is characteristic of the virus and not a result of random replicative errors and then enrichment of particular species.
Herpesvirus papio DNA is similar in organization to Epstein-Barr virus DNA
Authors: Heller M , Gerber P , Kieff E
Published: January 2020

EcoRI, HindII, SalI, nd XbaI restriction endonuclease maps of herpesvirus papio (HVPapio) DNA were derived by determining the fragment sizes and the linkage relationships between fragments generated by the different enzymes. The data indicate that HVPapio DNA has a single molecular arrangement which is similar to that of Epstein-Barr virus DNA. The size of the DNA was 110 X 10(6) to 114 X 10(6) daltons. Restriction fragments from both ends varied in the number of repeats of a 4 X 10(5)-dalton sequence, TR, and hybridized to each other. This suggests that there is an identical repeating unit, TR, at both ends of the DNA. There were usually six tandem repetitions (range, 1 to 11) of a 2 X 10(6)-dalton sequence, IR, within the DNA. IR separated the DNA into two domains of largely unique sequence complexity, a 9 X 10(6)-dalton segment, Us, and an 88 X 10(6)-dalton segment, UL. There was homology between DNA fragments which mapped at 25 X 10(6) to 29 X 10(6) to 91 X 10(6) to 95 X 10(6) daltons in UL.
Characterization of supercoiled nucleoprotein complexes released from detergent-treated vaccinia virions
Authors: Soloski M J , Holowczak J A
Published: January 2020

Treatment of vaccinia virions with 1% sodium dodecyl sulfate in the absence of reducing agents resulted in the release of subviral particles termed "subnucleoids," which contained viral DNA in combination with four polypeptides with molecular weights of 90,000, 68,000, 58,000 and 10,000. Biochemical and electron microscopic studies showed that viral DNA in combination with these polypeptides was maintained in a superhelical configuration. When subnucleoids were "fixed" with glutaraldehyde and formaldehyde and then examined by electron microscopy, spherical particles were observed, in which the supercoiled DNA was folded into globular structures that were 20 to 60 nm in diameter and were interconnected by DNA-protein fibers resembling the nucleosome structures described for eucaryotic chromatin.
Structure and restriction enzyme maps of the circularly permuted DNA of staphylococcal bacteriophage phi 11
Authors: Löfdahl S , Zabielski J , Philipson L
Published: January 2020

One restriction enzyme map of Staphylococcus aureus bacteriophage phi 11 DNA was established by reciprocal double digestions with the enzymes EcoRI, HaeII, and KpnI. The sequential order of the EcoRI fragments was thereafter established by a novel approach involving blotting of DNA partially cleaved with EcoRI and the probing the blots with nick-translated terminal fragments. A circular map of the phi 11 DNA was established, and the phage genome was circularly permuted based on the failure to end label mature viral DNA, restriction maps of replicating DNA, and finally, homoduplex analysis in the electron microscope. A restriction enzyme map of the prophage form of phi 11 DNA was obtained by analysis of chromosomal DNA from a lysogenic strain.
Characterization of RNA polymerase products of Nebraska calf diarrhea virus and SA11 rotavirus
Authors: Bernstein J M , Hruska J F
Published: January 2020

The endogenous RNA polymerase of the calf rotavirus was shown to synthesize single-stranded RNA transcripts of one polarity which were identical in size to the denatured parental double-stranded RNA segments. The transcripts were notable in their absence of polyadenylate sequences. The polymerase activity associated with the cores of calf rotavirus was minimally altered by the reaction, since calf rotavirus cores could be reused after purification, whereas the RNA polymerase products appeared to be released from the virion.
Association of cellular 56,000- and 32,000-molecular-weight protein with BK virus and polyoma virus t-antigens
Authors: Rundell K , Major E O , Lampert M
Published: January 2020

Two cellular proteins (molecular weights: 56,000 and 32,000) were specifically co-immunoprecipitated by simian virus 40 and BK virus t-antigens and by polyoma virus non-T early proteins.
Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase
Authors: Goff S , Traktman P , Baltimore D
Published: January 2020

A rapid assay for retroviral reverse transcriptase activity released into the culture medium by infected cells was developed. With the assay, 4,000 clonally infected cell lines could be tested in a few hours. We have adapted the assay for use as a screen for the detection of spontaneous viral mutants. Mutants of Moloney murine leukemia virus have been isolated which (i) produce a thermolabile reverse transcriptase, (ii) are temperature sensitive for release of enzyme activity, or (iii) can only productively infect cells already producing gag-related polypeptides. The assay has also been useful for the isolation of nonproducer cells infected with various replication-defective transforming viruses.
Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents
Authors: Rotter V , Boss M A , Baltimore D
Published: January 2020

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.
Phosphorylation of threonine in the proline-rich carboxy-terminal region of simian virus 40 large T antigen
Authors: Scheidtmann K H , Kaiser A , Carbone A , Walter G
Published: January 2020

The position of phosphothreonine in the predicted primary structure of simian virus 40 large T antigen was determined by different methods. After digestion of large T antigen with trypsin and subsequent two-dimensional peptide mapping, a single peptide containing phosphothreonine could be separated from the bulk of phosphoserine-containing peptides. Its amino acid composition was determined by differential labeling with various amino acids in vivo. The high yield of proline (4.5 mol) within the phosphothreonine peptide indicated that it was derived from the carboxy terminus of large T antigen and had in its unphosphorylated form the sequence Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr-COOH. A phosphopeptide generated by chymotrypsin could be converted into the tryptic phosphothreonine peptide, indicating that the latter was part of the chymotryptic peptide. The origin of the phosphothreonine-containing peptides was independently confirmed by using an antiserum directed against the carboxy terminus of large T antigen. This serum reacted specifically with the proline-rich, phosphothreonine-containing peptides. Further analysis by partial acid hydrolysis indicated that the internal threonine was phosphorylated. The unusual amino acid composition on both sides of the phosphothreonine and the possible function of this phosphorylation site are discussed.
Comparative analysis of RNA genomes of mouse hepatitis viruses
Authors: Lai M M , Stohlman S A
Published: January 2020

The RNA genomes of various murine hepatitis virus (MHV) strains were studied by T1-oligonucleotide fingerprinting analysis with regard to their structure and sequence relationship. It was found that the MHV particles contained only positive-stranded 60S RNA which had a "cap" structure at its 5' end. No negative-stranded RNA was found. It was also shown that most of the MHV strains had diverged quite extensively in their genetic sequences. However, MHV-3, a hepatotropic strain, and A59, a nonpathogenic strain, were found to have very similar oligonucleotide fingerprinting patterns. Yet, each of them contained two to four specific oligonucleotides. The MHV-3-specific oligonucleotides were conserved in almost all of the hepatotropic MHV strains studied. In contrast, two of the A59-specific oligonucleotides were absent from the genomes of all hepatotropic strains. These findings suggest that these unique oligonucleotides might be localized at the genetic region(s) associated with viral pathogenicity or other biological properties of the virus. Comparison of viral structural proteins also suggests that MHV-3 and A59 are more closely related than other MHV strains. The significance of these findings is discussed.
Structure and Functions of the Kirsten Murine Sarcoma Virus Genome: Molecular Cloning of Biologically Active Kirsten Murine Sarcoma Virus DNA
Authors: Tsuchida Nobuo , Uesugi Seiichiro
Published: January 2020

The unintegrated closed circular form of viral DNA prepared from NIH3T3 cells infected with Kirsten murine sarcoma virus was cloned into bacterial plasmid pBR322. The closed circular DNA, which consisted of two different-sized populations, was enriched from the virus-infected cells, linearized with
Bam
HI, and inserted into pBR322 DNA. Four different recombinant DNAs (clones 2, 4, 6, and 7) were obtained, and a physical map of each was constructed by using various restriction enzymes. Clone 4 DNA had the largest insertion, corresponding to a complete copy of the linear DNA. This suggested that this insertion contained two copies of the 0.55-kilobase pair long terminal redundant sequence. Clone 2 and clone 6 insertion DNAs had deletions of 0.2 and 0.5 kilobase pair, respectively, which mapped near the right end (3′ side of viral RNA) of the linear DNA. Clone 7 DNA appeared to have a deletion of a single copy of the large terminal redundant sequence. Transfection of BALB3T3 cells with the clone 4 DNA insertion showed that this DNA had transforming activity. The efficiency of transfection with clone 4 Kirsten murine sarcoma virus DNA was enhanced eightfold by inserting
Eco
RI-cleaved viral DNA into the
Eco
RI site of pBR322. The
Eco
RI-inserted DNA produced foci with single-hit kinetics, suggesting that a single molecule of Kirsten murine sarcoma virus DNA can induce transformation. Results of transfections with
Eco
RI-inserted Kirsten murine sarcoma virus DNA cleaved with various restriction enzymes suggested that the first 3.3-kilobase pair region at the left end of the linear DNA is important for the initiation of transformation or maintenance of transformation or both.
Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2
Authors: Galloway D A , McDougall J K
Published: January 2020

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.
Incomplete transformation of rat cells by a deletion mutant of adenovirus type 5
Authors: Shiroki K , Maruyama K , Saito I , Fukui Y , Shimojo H
Published: January 2020

Rat 3Y1 cells were infected with adenovirus type 5 (Ad5) wild type, dl312 (deletion of 902 base pairs between 1.5-4.5 map units), and dl313 (deletion of 2,350 base pairs between 3.5-10.5 map units). After cultivation for 4 weeks, transformed foci appeared in wild type- and dl313-infected cells. No focus was observed in dl312- and mock-infected cells. Foci induced by dl313 were less dense than those induced by wild type. Cell lines (313Y cells) established from dl313-induced foci contained the E1 gene of the dl313 genome (E1a only). Cell lines (5WY cells) established from Ad5 wild type-induced foci contained the E1 gene of wild type (E1a and b). The difference between the transcriptional patterns of the E1 gene in 313Y cells and that in 5WY cells was the same as the difference in dl313- and wild type-infected cells. Colonies were formed in soft agar culture inoculated with 5WY cells, but no colony was formed after inoculation of 313Y cells. The transformed phenotype of 313Y cells was incomplete compared with that in 5WY cells. In nongrowing 3Y1 cells, dl313 and Ad5 wild type induced cellular DNA synthesis but dl312 did not. The above results suggest that the E1a gene is functioning in dl313-infected but not in dl312-infected cells and that such functions as induction of cellular DNA synthesis and transformation of cells are dependent on expression of the Ad5 E1a gene.
Early Syncytium Formation by Bovine Leukemia Virus
Authors: Graves Don C. , Jones Lorraine V.
Published: January 2020

Bovine leukemia virus (BLV) from either persistently infected bat cells or fetal lamb kidney cells induced rapid syncytium formation in F81 indicator cells. Distinct syncytia were seen within 2 h after inoculation of cells with highly concentrated (500-fold) cell-free BLV preparations and within 4 to 8 h when unconcentrated cell-free BLV preparations were used. Indicator cell densities of 1 × 10
5
to 2 × 10
5
were optimal for rapid and maximal syncytium formation. Pretreatment of BLV with reference BLV leukemic serum and antiserum prepared against purified BLV significantly inhibited (95%) syncytium formation. Reference bovine viral diarrhea virus serum, foamy-like bovine syncytial virus serum, and control serum had little effect (17% inhibition). Antiserum to BLV gp51 inhibited syncytium formation by greater than 96%, whereas antiserum to BLV p24 reduced syncytium activity to a much lesser extent (38% inhibition). Treatment of BLV with β-propiolactone (0.005 to 0.05%) had little or no effect upon syncytium-forming activity, whereas UV irradiation (15 ergs/mm
2
per s for 30 min) reduced, but did not completely destroy, the fusion activity. However, both β-propiolactone and UV irradiation drastically reduced the replication potential of BLV, as demonstrated by the lack of p24 expression in the inoculated cells. Concentrations of cycloheximide, cytosine arabinoside, tunicamycin, and 2-deoxy-D-glucose which effectively blocked cellular macromolecular synthesis did not significantly inhibit syncytium formation. These latter results suggested that de novo protein and DNA synthesis as well as protein glycosylation were not required for early syncytium formation. Thus, these experiments demonstrated that replication of BLV by the indicator cells was not essential for cell fusion.
Polyoma virus minichromosomes: associated DNA molecules
Authors: Krauss M R , Benbow R M
Published: January 2020

Electron microscopy was used to identify and quantitate DNA molecules associated with 3H-labeled polyoma minichromosomes which had been fractionated on a sucrose gradient. The percentage of replicating DNA molecules observed in the fractions of the gradient normally designated the replicative intermediate region was up to ninefold higher than in fractions from the mature region. Nevertheless, because of the higher overall concentration of polyoma DNA molecules in the mature region, nearly as many replicating DNA molecules were computed to be in the mature region as in the replicative intermediate region. The replicating molecules in the mature region was predominantly early replicative intermediates. Almost all late replicative intermediates were found in the replicative intermediate region. Under aqueous spreading conditions, a substantial fraction of the replicating DNA structures appeared to be asymmetrical or otherwise unusual, suggesting that extensive single-stranded regions may exist in replicating polyoma minichromosomes.
Phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus
Authors: Kennedy I M , Stevely W S , Leader D P
Published: January 2020

In BHK cells infected with pseudorabies virus, there was a substantial increase in the phosphorylation of ribosomal protein S6. This increase occurred between 2 and 4 h after infection and persisted at least until 9 h. We estimated that in mock-infected cells S6 contained, on an average, one phosphate group per protein chain, whereas in infected cells this rose to between four and five phosphate groups per protein chain. A second ribosomal protein, either S16 or S18, was also phosphorylated after infection. No increase in cyclic AMP was found at the time of phosphorylation. We also found an increased phosphorylation of S6 in herpes simplex virus-infected BHK cells.
Isolation of a replication-efficient mutant of West Nile virus from a persistently infected genetically resistant mouse cell culture
Authors: Brinton M A
Published: January 2020

Flavivirus-resistant mouse embryofibroblasts (C3H/RV) that were infected with West Nile virus, strain E101 (WNV), yielded fewer infectious virions than did cultures of congenic susceptible cells (C3H/HE). Analysis of intracellular viral RNA synthesis indicated that the incorporation of [3H]uridine into 40S genome RNA was markedly reduced in resistant cells, and about 100-fold less labeled 40S RNA was found in pelleted extracellular virions from resistant cultures than in those from susceptible ones. A non-temperature-sensitive mutant of WNV isolated from culture fluid of a persistently infected culture of genetically resistant mouse cells was found to produce higher yields of infectious virus than the parental WNV used to initiate the persistent infection. Analysis of intracellular actinomycin D-resistant RNA indicated that the mutant virus (WNV-RV) was more efficient at incorporating [3H]uridine into 40S RNA in resistant cells than was the parental virus. WNV-RV also synthesized 40S RNA more efficiently than parental virus in congenic susceptible cells and in BHK cells. Analysis of the incorporation of [35S]methionine into viral proteins was likewise enhanced in WNV-RV-infected cells. The WNV-RV mutant provides a tool for studying the regulation of transcription of flavivirus RNA.
Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs
Authors: Cooper J A , Wittek R , Moss B
Published: January 2020

Physical, transcriptional, and translational maps of an EcoRI fragment located between 15,800 and 20,600 base pairs from the left end of the vaccinia virus genome were prepared. Major polypeptides with molecular weights of 14,000 (14K polypeptide), 32,000 and 38,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA made in the presence of cycloheximide and selected by hybridization to lambda recombinant DNA containing the EcoRI fragment. With early RNA made in the presence of cytosine arabinoside, an inhibitor of DNA replication, the polypeptide pattern was similar except for quantitative differences in which less 38K polypeptide was detected as a translation product. With late RNA, isolated 6 h after infection without inhibitors, only traces of the early translation products were found and a new 40K polypeptide was detected. The size of the mRNA's for the 14K, 32K, and 38K polypeptides were determined to be approximately 760,880, and 1,150 nucleotides, respectively, by several independent procedures. Several large early RNAs not shown to code for any additional translation products were also detected. The size of the late message for the 40K polypeptide varied from 920 to 3,100 nucleotides. This heterogeneity appeared to be a general property of vaccinia virus late mRNA's. No evidence of RNA splicing was obtained by analysis of RNA-DNA hybrids after nuclease S1 treatment. Further analyses using separated recombinant DNA strands and restriction fragments indicated that all mRNA's were encoded by the leftward-reading DNA strand and at least two were overlapping. Since early and late mRNA's were encoded by the same DNA strand, the possibility of temporal regulation by transcriptional strand switching was eliminated. In conjunction with previous studies, a transcriptional map of the left 20,600 base pairs of the vaccinia virus genome was derived.
Detection and cloning of murine leukemia virus-related sequences from African green monkey liver DNA
Authors: Martin M A , Bryan T , McCutchan T F , Chan H W
Published: January 2020

By using low-stringency nucleic acid hybridization conditions and specific subgenomic segments of the AKR ecotropic provirus as probes, murine leukemia virus (MuLV)-related sequences were detected in African green monkey (AGM) liver DNA. The MuLV-reactive segments present in restricted AGM DNA ranged from 1.9 kilobases (kb) to greater than 10 kb in size. On the basis of this finding, a 17-kb segment was cloned from a partial EcoRI AGM library in lambda Charon 4A which shared nearly 5 kb of homology with AKR ecotropic MuLV DNA. The MuLV-related sequences detected in restricted preparations of AGM DNA or present in the cloned monkey DNA reacted with probes mapping 2.0 to 7.0 kb from the 5' terminus of the AKR ecotropic provirus. The AGM clone also contained repeated sequences that flanked the MuLV-related segment. Labeled, subgenomic, MuLV-reactive segments of the monkey clone hybridized to multiple restriction fragments of AGM liver DNA, indicating the presence of several copies of the MuLV-related sequences.