bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus
Authors: Laimins L A , Gruss P , Pozzatti R , Khoury G
Published: January 2020

A series of recombinant molecules were constructed which direct the expression of the easily assayed gene chloramphenicol acetyltransferase. We have used these recombinants to show that the 73/72-base-pair tandem repeat unit from the Moloney murine sarcoma virus long terminal repeat shares a number of properties with the prototypic enhancer element, the simian virus 40 72-base-pair repeat. Specifically, the Moloney murine sarcoma virus sequence significantly enhances the level of gene expression at both 5' and 3' locations and in either orientation relative to the test gene. It is able to enhance gene activity both from its own promoter and from a heterologous (simian virus 40) promoter. The 73/72-base-pair subunits of the Moloney murine sarcoma virus enhancer differ in sequence by four nucleotides and also in the strength of their enhancer function. The promoter distal A repeat is at least three times as active as the promoter proximal B repeat in enhancing chloramphenicol acetyltransferase expression. Results of these studies also show that the enhancer sequence alone is unable to induce gene activity but requires other promoter elements, including a proximal GC-rich sequence and the Goldberg-Hogness box.
Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus
Authors: Coen D M , Aschman D P , Gelep P T , Retondo M J , Weller S K , Schaffer P A
Published: January 2020

Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less. DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli. These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434). In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427). The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420). tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity. The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448). The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase.
Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies
Authors: van Wyke K L , Yewdell J W , Reck L J , Murphy B R
Published: January 2020

Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. We found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of the three epitopes are located in nonoverlapping domains on the matrix protein molecule.
Dissection of HA20, a double mutant of bacteriophage SPO1
Authors: Stewart C R
Published: January 2020

HA20, a mutant of Bacillus subtilis phage SPO1, is deficient in both DNA replication and late transcription. HA20 contains mutations in two different genes, which suggested that the two effects might be caused independently. However, single-mutation derivatives, affected only in gene 27, were deficient for both activities. Thus, a single mutation apparently affects both DNA replication and late transcription.
Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas
Authors: Dudley J , Risser R
Published: January 2020

Endogenous mouse mammary tumor virus genomes are amplified and located in novel cell DNA sequences in many mouse T-cell lymphomas. Transplanted tumors recovered from the same mouse strain and shown to be of independent origin by chromosomal analysis, by the presence of JH immunoglobulin gene rearrangements, or by the integration patterns of exogenous Moloney MuLV genomes frequently showed similar patterns of novel mouse mammary tumor virus-containing cell DNA fragments. This process of amplification and relocation can occur within a limited number of cell generations and in C57BL/6 mice does not lead to the synthesis of mature virus-encoded proteins. In some instances, amplified mouse mammary tumor virus genomes contained novel restriction cleavage sites in the gag-pol region. The restricted time course of occurrence, lack of synthesis of mature virion proteins, and apparent site specificity indicate that this process of retrovirus amplification differs significantly from virus replication after exogenous infection.
Unstable heterozygosity in a diploid region of herpes simplex virus DNA
Authors: Varmuza S L , Smiley J R
Published: January 2020

We have examined the behavior of a herpes simplex virus strain KOS isolate in which the two inverted repeats flanking the short segment of viral DNA differ in length by approximately 60 base pairs. We find that individual viral DNA molecules exist which contain the two distinguishable repeats, demonstrating that heterology between the repeats is tolerated. However, viruses heterozygous for the two different repeats are unstable, segregating both classes of homozygotes at a high frequency. We propose that this segregation is a consequence of the high-frequency recombination events which also result in genome segment inversion.
Organization of six early transcripts synthesized from a vaccinia virus EcoRI DNA fragment
Authors: Mahr A , Roberts B E
Published: January 2020

Four early transcripts and the polypeptides they encode have been mapped to the vaccinia virus EcoRI F DNA fragment, which spans the vaccinia HindIII J and H fragments. In addition, two transcripts for which no encoded polypeptides have been identified have also been mapped. Elucidation of the organization of these six transcripts by hybrid selection, S1 nuclease mapping, translation of size-fractionated RNA, and by filter hybridization demonstrates that approximately 90% of this DNA fragment is transcribed during early infection. All but one of these RNAs (1.35 kilobases [kb]) are transcribed in a rightward direction. The leftmost transcript of 0.6 to 0.7 kb encodes a 19,000-dalton (19K) polypeptide that has been determined to be thymidine kinase (D.E. Hruby and L.A. Ball, J. Virol. 43:403-409, 1982; G. Bajszar et al., J. Virol. 45:62-72, 1983). Immediately following the 3' end of this RNA is the 1.7-kb transcript encoding a 36K polypeptide. The 5' end of a 1.25-kb RNA encoding a 22K polypeptide is downstream of the 5' end of the 1.7-kb RNA, and its 3' end may be coterminal with the 3' end of the 1.7-kb RNA. The 2.45-kb transcript is coterminal at its 5' end with the message encoding thymidine kinase and is coterminal at its 3' end with the adjacent 1.7-kb mRNA. This RNA was not demonstrated to encode a polypeptide. Approximately 300 nucleotides from the 3' end of the 1.7-kb RNA is a 3.6-kb transcript encoding a 110K polypeptide. The 3' end of this large RNA lies several hundred nucleotides from the 3' end of the 1.35-kb RNA which is transcribed in the opposite direction. No splicing of RNA has been detected with the S1 nuclease mapping technique of Berk and Sharp. The organization of three late messages, which overlap these early transcripts, has been determined, and these results are discussed in the accompanying paper.
Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase
Authors: Champoux J J , Gilboa E , Baltimore D
Published: January 2020

The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.
Antigenic analysis of woodchuck hepatitis virus surface antigen with site-specific radioimmunoassays
Authors: Cote P J , Engle R E , Langer C A , Ponzetto A , Gerin J L
Published: January 2020

Monoclonal antibodies to five nonoverlapping antigenic domains of woodchuck hepatitis virus surface antigen (WHsAg) were used to develop site-specific radioimmunoassays. The assays were based on the solid-phase sandwich principle in which different combinations of individual domain-specific antibodies were used as immunoadsorbents and radioiodinated probes. Over 85% of the combinations tested were able to detect serum WHsAg, including those using the same antibody as immunoadsorbent and probe. The limits for antigen detection in one site-specific system ranged between 16 and 80 ng of WHsAg per ml. The antigenic similarity of serum WHsAg from 13 colony woodchucks was shown with several combination assay systems. WHsAg was equally immunoreactive in these assay systems whether obtained by immunoaffinity chromatography or standard rate zone centrifugation methods. Further site-specific analysis demonstrated that Formalin treatment of purified antigen did not affect the immunoreactivity of these WHsAg sites.
Mapping of a herpes simplex virus type 2-encoded function that affects the susceptibility of herpes simplex virus-infected target cells to lysis by herpes simplex virus-specific cytotoxic T lymphocytes
Authors: Carter V C , Jennings S R , Rice P L , Tevethia S S
Published: January 2020

A function(s) involved in the altered susceptibility of herpes simplex virus type 2 (HSV-2)-infected cells to specific lysis by cytotoxic T lymphocytes was mapped in the S component of HSV-2 DNA by using HSV-1 X HSV-2 intertypic recombinants (RH1G44, RS1G25, R50BG10, A7D, and C4D) and HSV-1 MP. Target cells infected with R50BG10, A7D, and C4D exhibited reduced levels of cytolysis, as did HSV-2-infected cells, whereas RH1G44 and RS1G25 recombinant-infected and HSV-1 MP-infected cells showed levels of lysis equal to that of HSV-1 KOS-infected cells. The intertypic recombinants R50BG10, RS1G25, RH1G44, and HSV-1 MP induced cross-reactive cytotoxic T lymphocytes. Coinfection of cells with HSV-1 KOS and either HSV-2 186 or R50BG10 recombinant also resulted in a decrease in the level of specific lysis by anti-HSV cytotoxic T lymphocytes.
Transcriptional and genetic analyses of the herpes simplex virus type 1 genome: coordinates 0.29 to 0.45
Authors: Holland L E , Sandri-Goldin R M , Goldin A L , Glorioso J C , Levine M
Published: January 2020

We have constructed a map of the genes encoded by a 23,000-nucleotide-pair region of herpes simplex virus type 1. This region, defined by the three adjacent EcoRI fragments N (map coordinates 0.298 to 0.315), F (0.315 to 0.421), and M (0.421 to 0.448), has previously been shown by genetic analysis to contain the genes for thymidine kinase, nucleocapsid protein p40, glycoprotein B, DNA-binding protein, and DNA polymerase. We report the identification and mapping of RNAs defining 13 viral genes encoded by the region 0.298 to 0.448. The transcriptional pattern shows families of overlapping messages, similar to those observed in other regions of the viral genome. We also isolated mutants representing four distinct complementation groups and physically mapped several of the mutations to regions within EcoRI fragment F by marker rescue. Mutations representing complementation groups 1-9 (glycoprotein B), 1-1 (DNA-binding protein), and 1-3 (DNA polymerase) were mapped to coordinates 0.361 to 0.368 to 0.411, and 0.411 to 0.421, respectively. A fourth previously undefined complementation group was mapped to the region between glycoprotein B and DNA-binding protein. Comparing the transcription mapping with marker rescue data suggests that the genes for glycoprotein B, DNA-binding protein, DNA polymerase, and nucleocapsid protein p40 are expressed as 3.3-, 4.2-, 4.3- or 4.2- or both, and 2.4-kilobase mRNAs, respectively.
Type 1 and Type 2 Herpes Simplex Viruses: Serological and Biological Differences
Authors: Plummer G. , Waner J. L. , Phuangsab A. , Goodheart C. R.
Published: January 2020

Forty isolates of herpes simplex virus were compared by means of cross-neutralization curves. The 11 oral isolates were serotype 1, and all 29 genital/anal isolates were serotype 2. The cytopathic effects of the two serotypes were consistently different. Passage of strains of type 1 and type 2 in mice and in rabbits yielded two variants, although the majority of the strains remained unchanged serologically and in their cytopathic effects. The two variants were derived from type 1 strains and differed from the parent strains in their cytopathic effects, each of them producing syncytia and enlarged plaques. They had, however, retained the serotypic properties and the deoxyribonucleic acid (DNA) densities of their parent strains. The Roizman syncytial/macroplaque strain of herpes simplex virus was also included in the study; the density of its DNA (1.727 g/ml) was typical of type 1 strains, and serologically it seemed to be basically a type 1 strain, although it was neutralized by type 2 antiserum slightly better than were other type 1 strains. Growth curves were performed of the two serotypes in rabbit kidney, human fibroblast, and mouse embryo tissue cultures. The type 2 strains attained lower titers of infectivity in these three cell systems; the levels of infectivity of type 2 virus in the culture fluid decreased much more rapidly after the maximum had been attained than did the levels of infectivity of the type 1 strains, due to the greater instability of the type 2 virus. Parallel titrations of different strains in tissue cultures and intracerebrally in mice indicated that the latter assay system was usually more sensitive for type 2 strains than it was for type 1 strains. The paralytic sequelae and inflammatory changes of lumbar ganglia and spinal cord in young rabbits inoculated extraneurally with strains of the two serotypes also indicate that the type 2 virus is more virulent in laboratory animals than is type 1 virus.
Properties of the Deoxyribonucleic Acid Contained in the Defective Particle Coliphage 15
Authors: Frampton E. W. , Mandel M.
Published: January 2020

Escherichia coli
strain 15 TAU, which requires thymine, arginine, and uracil for growth and harbors an apparently defective prophage, was induced by exposure to ultraviolet light (580 ergs/mm
2
) or to mitomycin C (5 μg/ml). Phage particles (coliphage 15) were recovered from the resulting lysate by treatment with deoxyribonuclease, filtration, and several cycles of differential centrifugation. Analysis of the phage particles obtained by using cesium chloride density gradient centrifugation in a preparative ultracentrifuge resulted in the resolution of three components. The major component had a peak density of 1.52 to 1.53 g/cm
3
followed by components with densities of 1.5 and 1.49 g/cm
3
. The guanine plus cytosine content of coliphage 15 deoxyribonucleic acid (DNA) was determined by both analytical ultracentrifugation in cesium chloride and by thermal denaturation in standard saline citrate buffer. Respective values of 46.4 ± 1% and 46.6 ± 1% guanine plus cytosine content were obtained. Coliphage 15 DNA formed molecular hybrids with messenger ribonucleic acid (RNA) from both uninduced and ultraviolet-induced cultures of
E. coli
15 TAU, but did not hybridize with
E. coli
ribosomal RNA. The molecular weight of coliphage 15 DNA was determined by constant velocity sucrose density gradient centrifugation to be about 33 × 10
6
daltons.
Immunodiffusion Analysis of Yaba Poxvirus Structural and Associated Antigens
Authors: Olsen Richard G. , Yohn David S.
Published: January 2020

Yaba poxvirus virions were extracted and purified from Rhesus monkey tumors. A saline-soluble virion fraction (Y-xp), obtained by mechanical fractionation of purified virions with an X-press, contained seven components in acrylamide gel electrophoresis; five of these components were reactive in immunodiffusion with whole virion and Y-xp antisera produced in rabbits and monkeys. The saline-insoluble residue remaining after X-press treatment was hydrolyzed with sodium dodecyl sulfate, urea, and 2-mercaptoethanol (SUM). This fraction, Y-sum, contained five components, four of which were demonstrable by immunodiffusion. There was no evidence of antigenic relationships between Y-xp and Y-sum antigens in immunodiffusion. In acrylamide gel electrophoresis, one Y-xp and one Y-sum component had similar mobilities. Y-xp but not Y-sum antisera contained viral-neutralizing antibodies. Virus-free saline extracts of Yaba tumor prepared with Genetron (YS) were essentially devoid of virion structural antigens. They failed to induce precipitating antibodies for virion antigens, were nonreactive in immunodiffusion with virion antisera, and gave low complement-fixation titers with virion antisera. Yaba virion antigens were recovered from the Genetron tumor sediment by SUM and alkaline hydrolysis. Antisera prepared to YS extracts gave a maximum of 17 precipitin lines in immunodiffusion with YS extracts; none was identified as a virion structural antigen. Saline extracts of tumor prepared without Genetron contained immunogenic amounts of 5 virion antigens and 12 to 14 associated antigens. Animals immunized with infected cell culture extracts (virus-free) formed antibodies to six to seven virion antigens. The implications of using extracts of Yaba poxvirus-infected tissues in complement-fixation tests to measure virion antibodies were discussed.
Irreversible Effects of Cycloheximide During the Early Period of Vaccinia Virus Replication
Authors: Moss Bernard , Filler Ronald
Published: January 2020

The presence of cycloheximide, an inhibitor of protein synthesis, during the period 30 to 60 min after vaccinia infection produced an irreversible block in virus replication. In contrast (i) cycloheximide given at earlier or later times, even for prolonged periods, did not prevent continuation of the infectious cycle after removal of the drug, and (ii) treatment with cycloheximide during the first 2 hr did not prevent virus growth when the early stages of replication proceeded more slowly due to infection with a low multiplicity of virus. These findings were interpreted as an indication that protein synthesis is required at a critical time in the virus growth cycle. Under the conditions in which brief cycloheximide treatment prevented virus growth, ribonucleic acid (RNA) synthesis continued at an undiminished rate for at least 2 hr after removal of the drug. Although this RNA appeared identical by polyacrylamide gel electrophoresis to “early” viral messenger RNA, it was not found associated with ribosomes or polyribosomes. Failure to observe viral protein synthesis was consistent with the latter finding. It appeared unlikely that the translational block resulted from inadequate removal of cycloheximide, since the effects of the drug were shown to be reversible at earlier or later times in infection or even at the same time when a lower multiplicity of virus was used. Interference with the normal synthesis of specific viral protein factors required for translation was postulated to explain the results.
Immunological Activity Associated with the Nucleocapsid and Envelope Components of an Arbovirus
Authors: Bose Henry R. , Sagik Bernard P.
Published: January 2020

Complement-fixing antigens were detected in the nucleocapsid and envelope of Sindbis virus. Neutralizing antibodies are directed against antigens in the viral envelope.
Transfer Ribonucleic Acid in KB Cells Infected with Adenovirus Type 2
Authors: Raška Karel , Frohwirth Daniel H. , Schlesinger R. Walter
Published: January 2020

Populations of transfer ribonucleic acid (tRNA) extracted from control and type 2 adenovirus (Ad2)-infected KB cells were compared. No consistent differences in acceptor activity for 11 amino acids were observed. Comparison of methylated albumin-kieselguhr (MAK) elution profiles of arginyl-tRNA from control and infected cells revealed a minor modification in that the proportion of arginyl-tRNA eluting at high salt concentration was somewhat greater in infected cells. No similar differences were observed in MAK elution profiles of aspartyl-, isoleucyl-, leucyl-, phenylalanyl-, seryl-, tyrosyl-, and valyl-tRNA. Hybridization of 4
S
RNA from infected cells labeled by incorporation of
3
H-uridine with Ad2 deoxyribonucleic acid revealed the presence of a complementary species of RNA in this preparation. Hybridization of
3
H-arginyl-tRNA and of
3
H-aminoacyl-tRNA labeled by charging with
3
H-arginine or a
3
H-mixture of amino acids, respectively, failed to detect the presence of virus-specific tRNA in Ad2-infected cells.
Early Intracellular Events in the Replication of T4 Bacteriophage Deoxyribonucleic Acid
Authors: Miller Robert C.
Published: January 2020

The relationship between
32
P suicide stabilization and deoxyribonucleic acid (DNA) replication during infection of
Escherichia coli
by bacteriophage T4 was reinvestigated. Replication of the parental phage DNA was detected at early stages of stabilization.
Double-Strand Scissions in Bacteriophage T4 Deoxyribonucleic Acid as a Result of
32
P Decay
Authors: Miller Robert C.
Published: January 2020

Double-strand scissions produced by decay of
32
P incorporated into T4 deoxyribonucleic acid (DNA) were detected in cross-linked DNA and DNA containing
32
P in only one strand of the double helix.
Initial Site of Synthesis of Virus During Rescue of Simian Virus 40 from Heterokaryons of Simian Virus 40-Transformed and Susceptible Cells
Authors: Wever Grace H. , Kit Saul , Dubbs D. R.
Published: January 2020

Simian virus 40 (SV40) can be rescued from certain SV40-transformed hamster cells by fusion with susceptible African green monkey kidney (CV-1) cells, in the presence of ultraviolet-irradiated Sendai virus. We have determined the sites in which SV40 is produced during rescue in these heterokaryons. To determine the sequence, nuclei were isolated from fused cells at various times after fusion, separated on sucrose-density gradients, and assayed for infectious center formation and virus content on CV-1 monolayers. Virus was first detected in the transformed nucleus (40 hr postfusion), and later associated with both transformed and susceptible nuclei (68 to 72 hr). Viral rescue apparently does not depend upon the transfer of SV40 deoxyribonucleic acid to a susceptible CV-1 nucleus, since the transformed nucleus is the primary site of virus production. The time course of certain cytological events in the rescue process and in productive infection was found to be similar.
Carrier Cultures of Simian Foamy Virus
Authors: Clarke J. K. , Samuels Janis , Dermott Evelyn , Gay F. W.
Published: January 2020

The production of cultures of HEp-2 and BHK-21 cells persistently infected with a type 1 simian foamy virus is described. After infection, HEp-2 cells showed no structural changes, whereas BHK-21 cells lost their normal spindle shape and showed mitochondrial damage, and some cells contained many lysosomes. Thin sections also showed that a few BHK-21 cells contained virus particles in low concentration, and infectious virus could be isolated from both the cells and the supernatant fluid. No virus was seen in thin sections of HEp-2 cells, although infectious virus in low titer could be recovered intermittently from lysed cells. Both carrier cultures were immune to challenge with homologous virus and antigen could be detected in over 90% of the cells even after growth for 9 weeks in the presence of virus-neutralizing serum. The distribution of antigen in carrier cultures of both cell types is described and compared with that seen in cytocidal infections.
Intracellular Inactivation of Bacteriophage T4 Early Deoxyribonucleic Acid
Authors: Mathews Christopher K.
Published: January 2020

As previously shown, a small amount of polynucleotide material is added to parental T4 deoxyribonucleic acid (DNA) molecules within the first 5 min of infection. I have asked whether this process is essential for phage replication. Two approaches—one involving decay of
32
P incorporated into this “early DNA” and the other involving photoinactivation of bromodeoxyuridine-containing early DNA—indicate that it is.
Bacteriophage Tail Components
Authors: Kozloff L. M. , Verses C. , Lute M. , Crosby L. K.
Published: January 2020

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants,
wh
1 and
wh
11, have been examined. Various lines of evidence support the view that the product of the
wh
locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced “early” enzyme is a component of the phage coat.
Stalked Bacteria: Properties of Deoxriybonucleic Acid Bacteriophage φCbK
Authors: Agabian-Keshishian Nina , Shapiro Lucille
Published: January 2020

The
Caulobacter crescentus
bacteriophage φCbK was studied with respect to the physical and chemical properties of both the phage and its deoxyribonucleic acid (DNA). Electron micrographs reveal the phage to be among the largest DNA bacteriophages reported, with head dimensions of 64 by 195 nm and a flexible tail 275 nm in length. The phage is composed of 57% DNA. This DNA is double-stranded as indicated by (i) the sharp increase in extinction coefficient over a narrow range of temperature increase, (ii) an increase in density in CsCl upon thermal denaturation, and (iii) the equivalence of guanine and cytosine as well as adenine and thymine, determined by chemical analysis. φCbK DNA cosediments with
Escherichia coli
phage T2 DNA and has therefore been assigned an
S
20,w
value of 63.5
S
. The size of the phage and its DNA and the percentage of DNA indicate that the φCbK phage head is relatively loosely packed. The properties of the DNA from bacteriophage φCbK are similar to those of host
C. crescentus
DNA with respect to buoyant density, thermal transition point, and guanine plus cytosine content.
Structure and genetic complexity of the genomes of herpesvirus defective-interfering particles associated with oncogenic transformation and persistent infection
Authors: Baumann R P , Dauenhauer S A , Caughman G B , Staczek J , O'Callaghan D J
Published: January 2020

The complexity and structural organization of defective-interfering (DI) particle DNA of equine herpesvirus type 1 (EHV-1) have been elucidated by using restriction enzyme and Southern blot hybridization analyses. DI particles were generated by serial high-multiplicity passage of EHV-1 in L-M cells, and total viral DNA was extracted from virus purified from supernatants of these serial passages. EHV-1 DI particle DNA was quantitatively separated from standard (STD) DNA by several cycles of CsCl isopycnic banding in a vertical rotor. Restriction endonuclease digestion profiles of pure DI DNA were completely different from the mapped patterns observed for EHV-1 STD DNA. Digestion of pure defective DNA with restriction enzymes (Bg/II, EcoRI, and XbaI), for which there are few or no cleavage sites within the S (short) region of the EHV-1 STD genome, yielded high-molecular-weight supermolar DNA bands, suggesting that a large subgenomic repeat unit was present in defective DNA. DNA blot hybridization analysis with the Bg/II supermolar fragment of defective DNA, intact DI particle genomic DNA, and EHV-1 STD DNA restriction enzyme fragments as 32P-labeled probes indicated that the EHV-1 DI particle genome originates predominately from the STD DNA S region (0.77 to 1.00 map units) and to a lesser extent from the left terminus of the unique long (UL) region (0.00 to 0.05 map units). None of the EHV-1 DNA sequences associated to date with EHV-1 oncogenesis (0.32 to 0.38 map units; O'Callaghan et al. in B. Roizman [ed.], Herpesviruses, in press; Robinson et al., Cell 32:204-219, 1983, and Proc. Natl. Acad. Sci., U.S.A., 78:6684-6688, 1981) were detected in the DI particle DNA. The importance of these data with regard to DNA replication of DI particles and the role of DI particles in one model system of EHV-1 oncogenic transformation are discussed.
Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis
Authors: Babiss L E , Ginsberg H S
Published: January 2020

To determine the role adenovirus 5 early region 1b-encoded 21- and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (0 to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region 1b functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dl118, H5dl110, H5in127, and H5dl163) indicates that all of the viruses contain mutations which affect the 55-kilodalton protein, whereas dl118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the mutants which is phenotypically identical to wild-type adenovirus 5). When compared with characteristics of sub309, the early and late transcription and DNA replication of the mutants were similar, but synthesis of late polypeptides and late cytoplasmic mRNAs was greatly reduced. Quantitation of mutant virus-specific late mRNAs associated with polysomes revealed a threefold reduction when compared with that of sub309. Analysis of infected cell extracts further revealed that these mutants were incapable of efficiently shutting off host cell protein synthesis, suggesting that the 55-kilodalton protein plays a role in this process. These data suggest that early region 1b products may function by interacting with additional viral or host cell macromolecules to modulate host cell shutoff or that some late viral mRNA or polypeptide may potentiate this reaction.
Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis
Authors: Gardella T , Medveczky P , Sairenji T , Mulder C
Published: January 2020

A simple gel technique is described for the detection of large, covalently closed, circular DNA molecules in eucaryotic cells. The procedure is based on the electrophoretic technique of Eckhardt (T. Eckhardt, Plasmid 1:584-588, 1978) for detecting bacterial plasmids and has been modified for the detection of circular and linear extrachromosomal herpesvirus genomes in mammalian cells. Gentle lysis of suspended cells in the well of an agarose gel followed by high-voltage electrophoresis allows separation of extrachromosomal DNA from the bulk of cellular DNA. Circular viral DNA from cells which carry the genomes of Epstein-Barr virus, Herpesvirus saimiri, and Herpesvirus ateles can be detected in these gels as sharp bands which comigrate with bacterial plasmid DNA of 208 kilobases. Epstein-Barr virus producer cell lines also show a sharp band of linear 160-kilobase DNA. The kinetics of the appearance of this linear band after induction of viral replication after temperature shift parallels the known kinetics of Epstein-Barr virus production in these cell lines. Hybridization of DNA after transfer to filters shows that the circular and linear DNA bands are virus specific and that as little as 0.25 Epstein-Barr virus genome per cell can be detected. The technique is simple, rapid, and sensitive and requires relatively low amounts of cells (0.5 X 10(6) to 2.5 X 10(6)).
Neoplastic transformation by E26 leukemia virus is mediated by a single protein containing domains of gag and myb genes
Authors: Klempnauer K H , Bishop J M
Published: January 2020

The genome of avian leukemia virus E26 shares homology with v-myb, the oncogene of avian myeloblastosis virus, and encodes a protein with an Mr of 135,000. Analyses of tryptic oligopeptides show that this protein is related to the proteins encoded by gag (Pr76gag) as well as v-myb (p45v-myb[AMV] ) and c-myb (p75c-myb). We found no evidence for the existence of additional myb-related proteins or subgenomic species of myb-related RNA in myeloblasts transformed by strain E26.
Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein
Authors: Satake M , Venkatesan S
Published: January 2020

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.
Murine resistance to street rabies virus: genetic analysis by testing second-backcross progeny and verification of allelic resistance genes in SJL/J and CBA/J mice
Authors: Lodmell D L , Chesebro B
Published: January 2020

Intraperitoneal challenge with street rabies virus of second-backcross offspring produced from susceptible females mated with either randomly selected or rabies-resistant first-backcross males indicated that murine resistance is under the influence of the concurrent presence of each of two segregating genes. Furthermore, the greater than or equal to 96% resistance of offspring produced from (SJL X CBA)F1 and (CBA X SJL)F1 hybrids crossed to susceptible A.SW or A/WySn mice demonstrated that resistance genes of SJL/J and CBA/J mice are allelic.
Localization of the simian virus 40 small t antigen in the nucleus and cytoplasm of monkey and mouse cells
Authors: Ellman M , Bikel I , Figge J , Roberts T , Schlossman R , Livingston D M
Published: January 2020

Monkey and mouse cells producing simian virus 40 small t antigen in the absence of clearly detectable intact or truncated large T antigens were subjected to indirect immunofluorescence and biochemical cell compartment analyses. Results revealed specific immunofluorescence and small t polypeptide in both the nucleus and cytoplasm of these cells.
Differential phosphorylation of cytoplasmic and nuclear variants of simian virus 40 large T antigen encoded by simian virus 40-adenovirus 7 hybrid viruses
Authors: Scheidtmann K H , Schickedanz J , Walter G , Lanford R E , Butel J S
Published: January 2020

The phosphorylation patterns of cytoplasmic and nuclear forms of simian virus 40 large T antigen encoded by simian virus 40-adenovirus 7 hybrid viruses were analyzed by two-dimensional peptide mapping. The PARA(cT) mutant which encodes a large T antigen defective for nuclear transport was used as source for cytoplasmic large T antigen. The data suggest that the large T antigen is phosphorylated in a sequential manner at a subset of sites in the cytoplasm and at additional sites in the nucleus.
Time Course Analysis and Mapping of
Autographa californica
Nuclear Polyhedrosis Virus Transcripts
Authors: Rohel Dennis Z. , Faulkner Peter
Published: January 2020

To study the expression of the
Autographa californica
nuclear polyhedrosis virus (AcNPV) genome, intracellular virus-specific proteins and mRNAs were pulsed-labeled, extracted, and analyzed at 6-h intervals during the replicative cycle. Most RNAs were detected between 12 and 24 h postinfection (p.i.), but many continued to be synthesized until late in infection. Polyhedrin and p8 mRNAs were the two most abundant late viral RNA transcripts, and they were synthesized at high rates until late in the infection cycle (60 h p.i.). The abundancy control of polyhedrin and p8 polypeptides was considered to be at the level of transcription. Two other major mRNAs in infected cells were 0.6-kilobase RNA, which was synthesized at its highest rate 12 to 18 h p.i., and 2.8-kilobase RNA, which was synthesized from 12 h p.i. until 48 h p.i. Cytoplasmic polyadenylic acid-containing RNA was isolated at 6-h intervals and was analyzed by Northern blot hybridization. At least 50 virus RNA transcripts were recognized, sized, and mapped onto the genome. Six RNAs hybridized to
Eco
RI-H, -I, and -J, and
Hin
dIII-Q AcNPV DNA restriction fragments, seven RNAs hybridized to
Eco
RI-B and -D DNA fragments, five RNAs hybridized to
Eco
RI-A and -E regions of the genome, four RNAs hybridized to
Eco
RI-C and -N DNA fragments, and one RNA species hybridized to
Eco
RI-O AcNPV DNA. A transcription map of the AcNPV genome was constructed, and the data were correlated with previously published translation maps.
Positive identification of a measles virus cDNA clone encoding a region of the phosphoprotein
Authors: Bellini W J , Englund G , Richardson C D , Rozenblatt S
Published: January 2020

A measles virus-specific cDNA clone, C1-G, that was previously assigned as a hemagglutinin-specific clone has been reassigned as a phosphoprotein-specific clone. The nucleotide sequence of C1-G was used to deduce the amino acid sequence. A synthetic peptide was constructed from a portion of the deduced sequence, and antisera were prepared. The antibodies directed against this synthetic peptide specifically precipitated the phosphoprotein of measles virus and not the hemagglutinin.
Analysis of T antigen and viral DNA in mouse cells transformed by K virus, a nononcogenic murine papovavirus
Authors: Kanda T , Takemoto K K
Published: January 2020

A tumorigenic line of mouse cells transformed by the nononcogenic murine K papovavirus was established and characterized. Tumor-bearing animals produced antibody specific for K virus-transformed cells; the K virus T antiserum did not react with polyomavirus-transformed cells. Immunoprecipitation of the T antigen in the transformed cells revealed large T antigen (molecular weight 84,000). About 10 to 15 copies of K virus DNA were found to be integrated in the cellular DNA.
Phosphorylation of the ribosomal protein S6 is elevated in cells transformed by a variety of tumor viruses
Authors: Blenis J , Erikson R L
Published: January 2020

We have analyzed the phosphorylation of the ribosomal protein S6 in several cultures of normal cells and cells transformed with RNA or DNA tumor viruses. Serum deprivation of confluent, normal cells results in low levels of S6 phosphorylation. In contrast, under identical conditions, this protein is highly phosphorylated in virally transformed cells. No differences in the phosphopeptides generated by one-dimensional limited proteolysis were detected in S6 prepared from serum-stimulated or transformed cells. These results suggest that a common event in oncogenic transformation is the phosphorylation of S6.
Protective effect of biological response modifiers on murine cytomegalovirus infection
Authors: Ebihara K , Minamishima Y
Published: January 2020

Pretreatment with two biological response modifiers (BRM), OK-432 and PS-K, protected mice from lethal infection by murine cytomegalovirus (MCMV). This was evidenced by an increase in 50% lethal doses and a decrease in titers of infectious viruses replicated in the liver and spleen. Spleen cells from the BRM-treated mice augmented the natural killer (NK) cell activity and suppressed the replication of MCMV in vitro. During MCMV infection, the NK cell activity of the spleen cells was maintained at a high level in the BRM-treated mice, whereas it was severely impaired in untreated mice. The BRM-induced protection was nullified by concomitant administration of antiasialo GM1 antibody. Interferon was neither induced by BRM treatment nor enhanced in BRM-pretreated and MCMV-infected mice. Thus, the protective effect of OK-432 and PS-K seems to be based on activation of NK cells and prevention of MCMV-induced inhibition of the NK cell activity.
Cloning and physical mapping of enteric adenoviruses (candidate types 40 and 41)
Authors: Takiff H E , Reinhold W , Garon C F , Straus S E
Published: January 2020

We have studied the DNAs of fastidious enteric adenoviruses recovered from the stools of infants with gastroenteritis. By endonuclease analysis, the strains examined represent candidate adenovirus types 40 and 41, which are thought to comprise new adenovirus subgroups F and G. Cloning of DNA from representative enteric adenovirus isolates, together with hybridization and subcleavage analysis, permitted the mapping of restriction enzyme cleavage sites. Although the restriction profiles are different for the two strains, they appear to have several cleavage sites in common. Cross hybridization studies show considerable homology between the subgroup F and G strains but much less homology to adenovirus 2. In addition, regions on both ends of enteric adenovirus genomes (map units, 2.9 to 11.3 and 75 to 100) possess little or no homology to adenovirus 2. Restriction enzyme digests reveal submolar fragments that map to the terminal regions of the genome. Electron micrographic studies of denatured and renatured DNA strands suggest that the submolar fragments may derive from cleavage of defective molecules. Inverted terminal repeat sequences were shown to comprise 0 to 3.2% of the length of complete (greater than or equal to 22 megadaltons) enteric adenovirus DNA molecules but 4 to 69% of incomplete-length (less than 22-megadalton) molecules.
Mapping of the cohesive overlap of duck hepatitis B virus DNA and of the site of initiation of reverse transcription
Authors: Molnar-Kimber K L , Summers J W , Mason W S
Published: January 2020

The hepatitis B-like viruses have a approximately 3.2 kilobase, partially double-stranded DNA genome that is held in a circular conformation by a cohesive overlap between the 5' ends of the two strands. In addition, a protein is covalently bound to the 5' end of the minus strand of virion DNA. The sequence of the cohesive overlap region and its location relative to open reading frames and to the initiation site for minus-strand DNA synthesis, which occurs by reverse transcription of viral RNA, were investigated in duck hepatitis B virus. The 5' ends of virion DNA were mapped by restriction endonuclease analysis of labeled virion DNA, S1 nuclease digestion, and primer extension, using avian myeloblastosis virus DNA polymerase. The cohesive overlap region was shown to be 69 +/- 4 base pairs in length. It contained a 10-base pair inverted repeat in approximately the middle and a 12-base pair direct repeat near each end. The apparent initiation site of reverse transcription was determined by partial sequence analysis of dideoxynucleotide-truncated minus-strand DNA intermediates and comparison of their lengths with the length of a known DNA sequence. It mapped within two to four nucleotides of the 5' end of the minus strand of virion DNA. The results are consistent with the interpretation that the 5' end of the minus strand of virion DNA is the origin of reverse transcription and that the protein covalently bound to virion DNA is the primer of reverse transcription.
A murine model for oral infection with a primate rotavirus (simian SA11)
Authors: Offit P A , Clark H F , Kornstein M J , Plotkin S A
Published: January 2020

Simian rotavirus SA11 was shown to replicate in the gastrointestinal tracts of infant mice after oral inoculation. Clinical symptoms, histopathological changes in the small intestinal mucosa, and the type-specific humoral immune response were all characteristic of rotavirus-induced gastroenteritis. The availability of this small animal model for the study of a primate rotavirus infection should expedite analysis of the immune response necessary for protection against challenge.