bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA
Authors: Pourcel C , Louise A , Gervais M , Chenciner N , Dubois M F , Tiollais P
Published: January 2020

Mouse L cells transformed with recombinant plasmids carrying hepatitis B virus (HBV) DNA fragments were used to study the transcription of the viral surface antigen gene (gene S). An HBV-specific, polyadenylated, 2.3-kilobase RNA was mapped on the HBV genome. This RNA hybridized with approximately 75% of the genome and excluded the region of the HBV core antigen gene (gene C). The 2.3-kilobase RNA species was present only in cell lines that produced hepatitis B surface antigen. An HBV-specific 2.3-kilobase RNA was also detected in human hepatoma cell line PLC/PRF/5 which produced hepatitis B surface antigen. A study of gene S expression in the transformed mouse L cells allowed us to localize the regions of initiation and termination of gene S transcription. Our results strongly suggest that the 2.3-kilobase RNA molecule is the mRNA of the major polypeptide of the envelope, which carries the viral surface antigen determinants.
Mechanism of replication of bacteriophage phi X174 XX. Sensitivity of nascent DNA to single-strand-specific nucleases
Authors: Matthes M , Denhardt D T
Published: January 2020

We reported earlier that dephosphorylated nascent phi X174 viral strand DNA molecules were less extensively degraded from the 5' end by spleen exonuclease than were non-nascent molecules. Experiments described here revealed that the insensitivity to the 5'-OH end-specific nuclease was more evident among the longer molecules in the population than among the shorter, all of the molecules being less than unit length in size. The smallest molecules in the population were about as sensitive to the enzyme as the control molecules and hence must possess unblocked 5'-terminal nucleotides. Degradation of the nascent DNA with the 3' end-specific snake venom phosphodiesterase revealed only a small enrichment for [3H]thymidine near the 3' end, seemingly insufficient to account completely for the apparent insensitivity of the longer molecules to spleen exonuclease. When the nascent molecules were isolated without the use of proteolytic enzymes, some pronase-sensitive material was found associated with the DNA, particularly the longer molecules. We suggest that the resistance of the longer nascent (pronase-treated) molecules to spleen exonuclease occurs because they have remnants of the viral gene A or A* protein covalently bound to the 5' end.
Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity
Authors: Volk W A , Synder R M , Benjamin D C , Wagner R R
Published: January 2020

Nineteen independently isolated hybridomas producing monoclonal antibodies to the glycoprotein of vesicular stomatitis virus were isolated and studied for their capacity to neutralize viral infectivity. By measuring competitive binding of 125I-labeled monoclonal antibodies in a radioimmunoassay. 11 different, non-cross-reacting antigenic determinants were identified on the vesicular stomatitis virus G protein. All monoclonal antibodies reacting with determinants 1, 2, 3, and 4 resulted in viral neutralization, whereas those binding to the other seven determinants did not neutralize infectivity. The mixture of two monoclonal antibodies binding to different determinants resulted in a more rapid neutralization than either antibody alone, suggesting that different antibodies can exert a synergistic effect on viral neutralization. Kinetic experiments revealed biphasic neutralization curves similar to those expected for heterologous antibody. No evidence could be obtained to relate biphasic kinetics of viral neutralization to heterogeneous populations either of antibody molecules or of virus. The possible significance of the kinetic data with monoclonal antibodies is discussed.
New way to isolate simian virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent
Authors: Boyce F M , Sundin O , Barsoum J , Varshavsky A
Published: January 2020

A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.
Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form
Authors: Sakai K , Narita H , Adachi A , Tsuruta S , Yorifuji T , Ishimoto A
Published: January 2020

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.
Herpesvirus glycoprotein synthesis and insertion into plasma membranes
Authors: Peake M L , Nystrom P , Pizer L I
Published: January 2020

In the presence of the antibiotic tunicamycin (TM), glycosylation of herpes simplex virus glycoproteins is inhibited and non-glycosylated polypeptides analogous to the glycoproteins are synthesized (Pizer et al., J. Virol. 34:142-153, 1980). The synthesis of viral proteins and DNA occurs in TM-treated cells. By electron microscopy, nucleocapsids can be observed both in the nucleus and the cytoplasm of TM-treated cells; a small number of enveloped virions were observed on the cell surface. Analyses of the proteins in partially purified virus readily detects viral glycoproteins in the control cells, but neither glycoproteins nor nonglycosylated polypeptide analogs were observed in the virus prepared from TM-treated cells. By labeling the surface of infected cells with 125I, viral glycoproteins were detected as soon as 90 min after infection even when protein synthesis was inhibited with cycloheximide and glycosylation was blocked with TM. Labeling the proteins synthesized in infected cells with [35S]methionine showed that the surface glycoproteins detected in the cycloheximide- and TM-treated cells were not synthesized de novo after infection, but were placed on the cell surface by the infecting virus. Studies with metabolic inhibitors and a temperature-sensitive mutant blocked early in the infectious cycle showed that glycoproteins gA/gB and gD were synthesized soon after infection, but that the synthesis of gC was delayed. Under conditions of infection, in which gC and its precursor pgC are not produced, we have been able to observe the relationships between the glycosylated polypeptides that correspond to pgA/pgB and the nonglycosylated analog made in the presence of TM.
Emergence of simian virus 40 variants during serial passage of plaque isolates
Authors: Norkin L C , Tirrell S M
Published: January 2020

Three serial passage series of simian virus 40 (SV40) in CV-1 cells were initiated by infection directly from the same wild-type plaque isolate, three series were initiated by infection with another plaque isolate, and two series were initiated with each of two other plaque isolates. Aberrant SV40 genomes were not detected in any of the passage series until after the fifty undiluted passage, and each series generated a different array of variant genomes. The results show that the variants were not present in the original plaque isolates but, instead, were randomly generated during subsequent high-input multiplicity passages. Although many of the aberrant viral genomes in each passage series contained reiterations of the SV40 origin of replication and some also contained host cell sequences, there was no indication that SV40 is predisposed toward generating any particular variant.
Transformation by avian sarcoma viruses leads to phosphorylation of multiple cellular proteins on tyrosine residues
Authors: Beemon K , Ryden T , McNelly E A
Published: January 2020

Phosphoamino acid compositions were determined for 10 size classes of cellular proteins, separated by electrophoresis through one-dimensional sodium dodecyl sulfate-polyacrylamide gels. Phosphotyrosine-containing proteins were observed in uninfected chicken embryo fibroblasts in every size class analyzed, ranging from approximately 20,000 to greater than 200,000 daltons. Transformation of chicken embryo fibroblasts by Rous sarcoma virus or PRC II avian sarcoma virus led to increases in phosphorylation of proteins at tyrosine residues in all of these size classes. A large fraction of the phosphotyrosine-containing protein molecules observed in Rous sarcoma virus-transformed cells was larger than 100,000 daltons with a second broad peak in the 35,000- to 60,000-dalton range. This study suggests that there are a number of substrates of viral or cellular tyrosine-specific protein kinases, which have not yet been identified by other methods.
Antibody to a host protein prevents initiation by the poliovirus replicase
Authors: Dasgupta A , Hollingshead P , Baltimore D
Published: January 2020

In vitro transcription of poliovirus RNA was catalyzed by the combination of a virus-coded polymerase and a host cell protein (host factor). Antibody to host factor inhibited template-dependent synthesis of complementary RNA where presumably RNA chain initiation occurred. On the contrary, elongation of already initiated RNA chains catalyzed by the replicase-template complex was not inhibited by anti-host factor antibody. These results strongly favored our previous notion that the host factor was needed for the initiation step of viral complementary RNA synthesis.
Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells
Authors: Stringer J R
Published: January 2020

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.
Characterization of a Temperature-Sensitive Fiber Mutant of Type 5 Adenovirus and Effect of the Mutation on Virion Assembly
Authors: Chee-Sheung Cecilia Cheng , Ginsberg Harold S.
Published: January 2020

A temperature-sensitive, fiber-minus mutant of type 5 adenovirus, H5
ts
142, was biochemically and genetically characterized. Genetic studies revealed that H5
ts
142 was a member of one of the three apparent fiber complementation groups which were detected owing to intracistronic complementation. Recombination analyses showed that it occupied a unique locus at the right end of the adenovirus genetic map. At the nonpermissive temperature, the mutant made stable polypeptides, but they were not glycosylated like wild-type fiber polypeptides. Sedimentation studies of extracts of H5
ts
142-infected cells cultured and labeled at 39.5°C indicated that a limited number of the fiber polypeptides made at the nonpermissive temperature could assemble into a form having a sedimentation value of 6S (i.e., similar to the trimeric wild-type fiber), but that this 6S structure was not immunologically reactive. When H5
ts
142-infected cells were shifted to the permissive temperature, 32°C, fiber polypeptides synthesized at 39.5°C were as capable of being assembled into virions as fibers synthesized in wild type-infected cells; de novo protein synthesis was not required to allow this virion assembly. In H5
ts
142-infected cells incubated at 39.5°C, viral proteins accumulated and aggregated into particles having physical characteristics of empty capsids. These particles did not contain DNA or its associated core proteins. However, when the infected culture was shifted to 32°C, DNA appeared to enter the empty particles and complete virions developed. The intermediate particles obtained had the morphology of adenoviruses, but they contained less than unit-length viral genomes as measured by their buoyant density in a CsCl density gradient and the size of their DNA as determined in both neutral and alkaline sucrose gradients. The reduced size of the intermediate particle DNA was demonstrated to be the result of incompletely packaged DNA molecules being fragmented during the preparative procedures. Hybridization of labeled DNA extracted from the intermediate particles to filters containing restriction fragments of the adenovirus genome indicated that the molecular left end of the viral genome preferentially entered these particles.
Restriction enzyme mapping of vaccinia virus DNA
Authors: DeFilippes F M
Published: January 2020

The cleavage sites for the restriction enzymes Bg/I, HindIII, KpnI, SalI, SmaI, and XhoI were located, from primary data, on the DNA isolated from the WR strain of vaccinia virus. Bg/I and SmaI divide the DNA into five segments which can be isolated by sucrose gradient centrifugation. These large segments provide a convenient means to group segments produced by other enzymes. The construction of physical maps was initiated by identifying the segments at each end of the DNA and then finding segments which were adjacent to these terminal sections. This was accomplished by isolating large shear fragments which contained the covalently linked termini of the DNA. Most of the data needed to derive the maps were obtained by isolating segments produce by one enzyme and then cleaving these individual segments with a second enzyme.
Studies with aphidicolin on the Fv-1 host restriction of Friend murine leukemia virus
Authors: Chinsky J , Soeiro R
Published: January 2020

The murine gene Fv-1 exerts a major control over the replication of Friend murine leukemia virus (F-MuLV). An effect of the gene product has been determined to be at the level of accumulation and integration of viral DNA. Aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, was studied in murine cells infected either permissively or nonpermissively with regard to the Fv-1 genotype. Results indicated that inhibition of DNA polymerase alpha did not affect the accumulation of form III viral DNA in either permissive or nonpermissive cells. However, the normal accumulation of circular form I DNA in permissive cells was inhibited. The block in the accumulation of form I DNA resembled that occurring in some F-MuLV Fv-1-nonpermissive infections. Additionally, aphidicolin treatment resulted in the accumulation of novel low-molecular-weight viral DNA species, normally detectable in a nonpermissive infection of NIH cells with B-tropic F-MuLV. These data suggest that the Fv-1 gene product may interact with host DNA polymerase alpha to prevent viral replication.
Synthesis of virus-specific polypeptides and genomic RNA during the replicative cycle of Pichinde virus
Authors: Dimock K , Harnish D G , Sisson G , Leung W C , Rawls W E
Published: January 2020

A stock of plaque-purified Pichinde virus, prepared under conditions designed to limit the amounts of defective interfering virus, was used to infect BHK cells. At daily intervals after infection, cells were examined for infectious and radiolabeled virus particle production and for the synthesis of virus-specific polypeptides. Quantitative comparisons were also made of the concentrations of genomic Pichinde virus L and S RNAs in the cytoplasm of infected cells on different days after infection. Our results showed that virus particle production, rates of protein synthesis, and the intracellular levels of viral genomic RNAs all increased and decreased with similar kinetics, and that this regulation was independent of the cell growth cycle. We were unable to relate these changes in viral macromolecule and virus production to the appearance of readily identifiable defective interfering particles. Our findings suggest that regulation of virus replication early during the replicative cycle of Pichinde virus may not be dependent upon the generation of defective interfering virus.
Location of the structural genes for glycoproteins gD and gE and for other polypeptides in the S component of herpes simplex virus type 1 DNA
Authors: Lee G T , Para M F , Spear P G
Published: January 2020

To map the structural genes for the gD and gE polypeptides and for other viral products encoded in the S component of herpes simplex virus type 1 DNA, we selected mRNAs capable of hybridizing to cloned viral DNA fragments and translated the mRNAs in vitro to determine which polypeptides were encoded therein. The gD and gE polypeptides were identified by immunoprecipitation with appropriate monoclonal and monospecific antibodies, whereas the other polypeptides were characterized only by their electrophoretic mobilities in polyacrylamide gels. We found that gD mRNA hybridized to a single SacI subfragment of BamHI fragment J, whereas gE mRNA hybridized to an adjacent SacI subfragment of BamHI fragment J and also to BamHI fragment X. These and other results permit the conclusion that the structural gene for gD is located between map coordinates 0.911 and 0.924, and the gene for gE is between map coordinates 0.924 and 0.951. We also found that mRNAs for polypeptides of 55,000, 42,000, 33,000, and 22,000 molecular weight hybridized to DNA fragments spanning the regions from map coordinates 0.911 to 0.924, 0.897 to 0.911, 0.939 to 0.965, and 0.939 to 0.965, respectively. Finally, in accord with the results of others, we found that mRNA for a 68,000-molecular-weight polypeptide hybridized to the two noncontiguous BamHI fragments N and Z, which share a reiterated DNA sequence.
Genetic alterations of RNA leukemia viruses associated with the development of spontaneous thymic leukemia in AKR/J mice
Authors: Thomas C Y , Coffin J M
Published: January 2020

T1-oligonucleotide fingerprinting and mapping were used to study the expression of RNA leukemia viruses in leukemic and preleukemic AKR/J mice, with techniques designed to minimize the loss or inadvertent selection of viruses in vitro before biochemical analysis. In leukemic animals, complex mixtures of ecotropic and mink-tropic viruses were expressed. Unique but similar polytropic virus-like genomes were present in each tumor isolate. In preleukemic mice, viral isolates from the thymus that were grown on NIH3T3 fibroblasts contained genomes with non-Akv polytropic virus-related oligonucleotides. This phenomenon was not evident in fingerprints of viruses from the spleen and bone marrow of the same animals. Remarkably, the non-Akv oligonucleotides located in the 3' portion of the P15E gene, the U3 noncoding region, and the 5' part of the gp70 gene were often expressed independently. Our results suggest the following. (i) Recombinant viruses can be detected in the thymuses of young preleukemic AKR mice and increase in relative abundance with age. (ii) During in vivo generation of the recombinant leukemogenic viruses, the selection of polytropic virus-related sequences in the 3' part of p15E and the U3 region and the 5' portion of gp70 occurs independently. (iii) Independent biological properties encoded in the gp70 and p15E regions of env of the recombinant viruses may mediate viral selection or leukemogenicity. (iv) The leukemogenic polytropic viruses of AKR/J mice arise via genetic recombination involving at least three endogenous viral sequences.
Construction and Characterization of Viable Deletion Mutants of Simian Virus 40 Lacking Sequences near the 3′ End of the Early Region
Authors: Polvino-Bodnar Maryellen , Cole Charles N.
Published: January 2020

Five viable deletion mutants of simian virus 40 (SV40) were prepared and characterized. These mutants lack 15 to 60 base pairs between map positions 0.198 and 0.218, near the 3′ end of the early region of SV40 and extend further into the body of the A gene, encoding the large T antigen, than previously described deletion mutants. These mutants were isolated after transfection of monkey kidney CV-1p cells with full-sized linear DNA prepared by partial digestion of form I SV40 DNA with restriction endonucleases
Hin
fI or
Mbo
II, followed by removal of approximately 25 base pairs of DNA from the 5′ termini using λ-5′-exonuclease and purification of the DNA in agarose gels. Based on camparisons of the DNA sequence of SV40 and polyoma virus, these mutations map in the 19% of the SV40 A gene that shares no homology with the A gene of polyoma virus. The mutations exist in two different genetic backgrounds: the original set of mutants (
dl
2401 through
dl
2405) was prepared, using as a parent SV40 mutant
dl
862, which has a deletion at the single
Hpa
II site (0.725 map unit). A second set (
dl
2491 through
dl
2495) contains the same deletions in a wild-type SV40 (strain SV-S) background. Relative to wild-type SV40, the original mutants showed reduced rates of growth, lower yields of progeny virus and viral DNA, and smaller plaque size; in these properties the mutants resembled parental
dl
862, although mutant progeny yields were usually lower than yields of
dl
862, suggesting a possible interaction between the two deletions. The second set of mutants had growth properties and progeny yields similar to those of wild-type SV40; however, Southern blotting experiments indicated that viral DNA replication proceeds at a slightly reduced rate. All of the mutants transformed mouse NIH/3T3 cells and mouse embryo fibroblasts at the same frequency as wild-type SV40. Mutants
dl
2402,
dl
2492, and
dl
2405 consistently produced denser and larger foci in both types of cells. All mutants directed the synthesis of shortened large T antigens. Adenovirus helper function was retained by all mutants.
Products of reverse transcription in avian retrovirus analyzed by electron microscopy
Authors: Junghans R P , Boone L R , Skalka A M
Published: January 2020

DNA products synthesized in avian retroviral particles permeabilized with melittin have been analyzed in an electron microscope. These studies have provided further insight and subsequent refinement in the melittin activation techniques. Our electron microscope analyses verify the existence of the plus-strand single-stranded DNA branches, presumed to originate by strand-displacement synthesis (L. R. Boone and A. M. Skalka, J. Virol. 37:117-126, 1981). The branches occur at many locations along the DNA molecules and are observed at very early times, even before the minus-strand copies of the RNA genome are completed. Circular forms of different derivations are observed at early and at late times, which are possible intermediates in viral replication. Novel forms termed H structures are also described. In addition to the identification of possible intermediates, these analyses have provided further information on the sequence of events in retroviral reverse transcription. These new data are combined with previous results to generate a model of reverse transcription which incorporates strand-displacement synthesis as an essential feature.
Genetic studies on capsid-length determination in bacteriophage T4. I. Isolation and partial characterization of second-site revertants of a gene 23 mutation affecting capsid length
Authors: Doherty D H
Published: January 2020

The T4 mutation ptg19-80 affects the mechanism of capsid-length determination. It is located in gene 23, which encodes the major structural protein of the capsid. The mutation results in the production of abnormal-length capsids in high frequencies. This paper describes the isolation and partial characterization of second-site revertants of ptg19-80. In the course of their analysis, it was discovered that ptg19-80 is itself a double mutation consisting of a gene 23 mutation (ptg19-80c), which causes the morphogenetic defect, and a suppressor mutation located near the lysozyme gene. Phenotypic characterization of nine pseudo-wild-type revertants of this double-mutation revealed that these revertants all produced lower frequencies of abnormal capsids than did ptg19-80. Seven of these revertants were shown to contain two suppressor mutations, one mapping in or near gene 22 and done mapping in or near gene 24. Both mutations were required for suppression. These suppressors displayed no discernible phenotype in the absence of ptg19-80c.
Monoclonal antibodies against baboon endogenous virus and against host cell antigens
Authors: Cogniaux J , Olislager R , Sprecher-Goldberger S , Thiry L
Published: January 2020

Monoclonal antibodies were produced by murine hybridomas after immunization with semipurified baboon endogenous virus. In a solid-phase radioimmunoassay, two antibodies (F12-9 and B9-18) reacted with viral antigen only. The antibodies A6-8 and C9-12 also reacted with virus-producing cells but not with control cells, whereas antibodies E4-6 and D12-2 bound to virus-free cells as well. The cytofluorometry technique confirmed these results and showed a competition between antibodies A6-8 and C9-12 for binding to virus-producing cells as well as a competition between antibodies D12-2 and E4-6 for binding to virus-free human cells. An immune precipitation assay with disrupted virions indicated that antibodies A6-8, B9-18, and C9-12 were directed against the gp70 glycoprotein, and that antibody F12-9 reacted with a viral antigen with a molecular weight of 18,000. The syncytia induced in RSa cells by baboon molecular weight of 18,000. The syncytia induced in RSa cells by baboon endogenous virus could be inhibited either when antibody A6-8 or C9-12 was combined to the virus or when the RSa cells were treated with the anticellular antibody D12-2 or E4-6. These two effects were not observed with Mason-Pfizer virus. Thus, of three antibodies with specificities for viral gp70, two (A6-8 and C9-12) were directed at viral sites responsible for syncytium formation. Another antiviral antibody (F12-9) reacted with a protein of unknown function with a molecular weight of 18,000. The two anticellular antibodies were directed at similar or neighboring epitopes, which may be situated within the receptor to the virus.
Core Particles of Hepatitis B Virus and Ground Squirrel Hepatitis Virus I. Relationship Between Hepatitis B Core Antigen- and Ground Squirrel Hepatitis Core Antigen-Associated Polypeptides by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Peptide Mapping
Authors: Feitelson Mark A. , Marion Patricia L. , Robinson William S.
Published: January 2020

The relationships among the core antigen polypeptides of hepatitis B virus (HBV) and ground squirrel hepatitis virus (GSHV) were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. The major core antigen polypeptides of liver-derived HBV (p22) and GSHV (p20.5) shared 56% of the spots in their peptide maps. Comparison of hepatitis B core antigen (HBcAg) p19 or ground squirrel hepatitis core antigen (GSHcAg) p16.5 with their respective major polypeptides indicated that these components probably resulted from cleavage of the major polypeptide of each virus. Other polypeptides smaller than the major component of each virus were often faint on polyacrylamide gels and probably resulted from the cleavage or degradation of components larger than p22 of HBcAg or p20.5 of GSHcAg, since their peptide maps contained spots unique to these high-molecular-weight components. p26 of GSHcAg and p27.5 of HBcAg shared approximately two-thirds of the spots on their peptide maps with those of their respective major core polypeptides. Furthermore, p37.5 of GSHcAg and p40 of HBcAg shared about 60% homology with their respective major polypeptides, and also shared many of the spots that were unique to p26 of GSHcAg or p27.5 of HBcAg but were not found in the peptide map of their respective core antigen polypeptides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis bands larger than 40,000 daltons were variably present, and peptide mapping indicated that these were aggregates of various smaller core antigen-associated polypeptides. The results suggest that p40 of HBcAg and p37.5 of GSHcAg are the largest unique polypeptides in these core particles, and that they are encoded for by the genome of each virus. That a subset of the spots unique to p40 or p37.5 was also found in p27.5 of HBcAg or p26 of GSHcAg, respectively, as compared to the major core polypeptides, also suggests that p27.5 and p26 are unique proteins encoded by the genome of each virus. It is proposed that the core antigen gene of each virus is larger than that which would encode the major polypeptide of each virus, and that the genetic organizations of the core genes of HBV and GSHV are very similar.
Identification and isolation of the main component (gp350-gp220) of Epstein-Barr virus responsible for generating neutralizing antibodies in vivo
Authors: Thorley-Lawson D A , Poodry C A
Published: January 2020

The majority of hybridomas we have characterized against Epstein-Barr virions react with the major glycoproteins gp350 and gp220 (gp350/220). One of these antibodies, ID4C-1, neutralizes virus infection in vitro. The presence of gp350/220 on the viral envelope could be confirmed directly by immunoelectron microscopy. We used lectin affinity (ricin) and immunoaffinity (ID4C-1) to purify gp350/220 and show that this material is able to induce potent virus-neutralizing antibodies. Absorption of four human and one rabbit anti-Epstein-Barr virus sera with purified gp350/220 suggests that this is the primary component responsible for generating neutralizing antibodies in vivo.
Chromosomal clustering of five defined endogenous retrovirus loci in White Leghorn chickens
Authors: Tereba A , Astrin S M
Published: January 2020

By using in situ hybridization, we localized three endogenous chicken retrovirus loci associated with the gs- chf- phenotype to distinct regions on chromosome 1. These three loci, ev4, ev5, and ev8, along with the two other known gs- chf--associated loci in White Leghorn chickens, ev1 and ev13, appear to be evenly distributed along chromosome 1, occurring approximately every 1.8 X 10(7) base pairs. The presence of all five loci on chromosome 1 represents a highly nonrandom distribution and all but eliminates a mechanism of independent random integration events for the generation of these loci.
Regulation of α Genes of Herpes Simplex Virus: The α 27 Gene Promoter-Thymidine Kinase Chimera Is Positively Regulated in Converted L Cells
Authors: Mackem Susan , Roizman Bernard
Published: January 2020

In cells infected with herpes simplex virus 1, the expression of viral genes is coordinately regulated and sequentially ordered; the α genes are expressed first and are followed by β and γ genes in a cascade fashion. Earlier, this laboratory reported (Post et al., Cell
24
:555-565, 1981) that a chimeric gene, constructed by the replacement of 50 base pairs of DNA coding for 5′ nontranslated leader and sequences upstream of the site of transcription initiation of thymidine kinase (a β gene) by corresponding sequences of α gene no. 4, was regulated as an α gene. Of particular interest was the observation that in cells converted to TK
+
phenotype, the chimeric gene was positively regulated by superinfecting virus. In this paper, we report two series of experiments. First, we determined that transcription of α gene no. 27 is initiated at or near a five-nucleotide sequence flanked by an eight-nucleotide perfect inverted repeat situated from 256 to 277 bases to the right of the left terminus of the
Bam
HI B fragment. In the second series of experiments, we constructed a chimeric gene which consisted of the thymidine kinase sequences described above but was fused to a DNA fragment expected to contain the promoter-regulatory region of α gene 27 and stretching from approximately −270 to the +55 nucleotide. The chimeric gene in converted cells was amplified upon superinfection with TK
−
virus only when the promoter-regulatory region was in the correct transcriptional orientation relative to the leader and structural sequences of the thymidine kinase gene. The requirements for amplification of the expression of this chimeric thymidine kinase gene were exactly the same as those previously reported for the α gene no. 4-thymidine kinase chimera and different from those of the standard (β) thymidine kinase. We conclude that the positive regulation of expression of α gene no. 4 deduced in previous studies may be a general property of α genes and that the promoter-regulatory region of α gene no. 27 is within a sequence contained between −270 and +55 nucleotides relative to the transcription initiation site.
Polyoma virus mutant with normal transforming ability but impaired tumorigenic potential
Authors: Gelinas C , Chartrand P , Bastin M
Published: January 2020

Cloned DNA from the P155 mutant of polyoma virus transforms cells in culture as efficiently as wild-type DNA, but has a much lower tumorigenic potential when injected into newborn rats. Like cells transformed by wild-type DNA, cells transformed by the mutant DNA grow in low serum concentrations, form colonies in agar suspension, and grow to high saturation densities compared with untransformed cells. They are, however, much less tumorigenic since they transplant 100- to 2,000-fold less efficiently than cells transformed by wild-type DNA. Substitution of the region between 89.7 and 1.8 map units by the corresponding region of P155 DNA decreased the tumorigenicity of wild-type DNA. When this region was isolated from wild-type DNA and substituted in P155 DNA, the tumorigenicity of the latter increased to values comparable to those of wild-type DNA. This showed that the lesion affecting tumorigenicity occurred between 89.7 and 1.8 map units on the polyoma virus genome. Sequence analysis in this region revealed a 12-base-pair deletion between nucleotides 1,347 and 1,360. This identified P155 as an mlt mutant, i.e., a mutant with a deletion from a region which encodes parts of the large and middle T antigens.
Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells
Authors: Johnson D C , Spear P G
Published: January 2020

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.
Purification and characterization of bovine rotavirus cores
Authors: Bican P , Cohen J , Charpilienne A , Scherrer R
Published: January 2020

Using the chaotropic effect generated by a high concentration of CaCl2, we converted calf rotavirus particles into cores of 40 nm in diameter. These cores were purified by rate zonal centrifugation in sucrose gradients and by isopycnic gradients. They had a sedimentation coefficient of 280S +/- 20S and a density of 1.44 g/ml in CsCl. When analyzed by polyacrylamide gel electrophoresis, they contained three polypeptides (VP125, VP89, and VP78). The major internal polypeptide of the virion (VP39) was recovered in a purified and soluble form in the top fractions of the sucrose gradients. From this stepwise degradation, it appears that VP39 is the most external polypeptide of dense particles. In contrast to reovirus cores, calf rotavirus cores did not exhibit transcriptase activity. Purified VP39 also did not exhibit transcriptase activity when tested after being mixed with purified rotavirus genome RNA as a template. Transcriptase activity was partially recovered when ionic conditions were adjusted to permit the reassociation of VP39 with the cores.
Hypomethylation of Epstein-Barr virus DNA in the nonproducer B-cell line EBR
Authors: Larocca D , Clough W
Published: January 2020

The conversion of the Epstein-Barr virus-negative Ramos cell line has previously been shown to result in an Epstein-Barr virus-positive non-virus-producer cell line, EBR. We report here that Epstein-Barr virus DNA from EBR alone among several cell lines examined was totally unmethylated at three of four sites containing guanine plus cytosine which were tested. This is in direct contrast to reports of high degrees of methylation in the DNAs of other animal viruses, including herpesviruses, isolated from cells in which the viral genome is expressed at a low level.
Mapping the Mutation Site of an
Autographa californica
Nuclear Polyhedrosis Virus Polyhedron Morphology Mutant
Authors: Carstens Eric B.
Published: January 2020

A polyhedron morphology mutant of
Autographa californica
nuclear polyhedrosis virus, designated M5, was compared with wild-type virus by genotypic analysis with
Eco
RI,
Bam
HI,
Hin
dIII,
Sst
I, and
Sma
I restriction endonucleases. M5 DNA revealed several alterations relative to the wild-type pattern: (i)
Eco
RI fragment I was 400 base pairs larger; (ii)
Bam
HI fragment F was missing; (iii)
Hin
dIII fragment F was 400 base pairs larger; (iv) an extra restriction fragment was obtained with both
Hin
dIII and
Sma
I; and (v)
Sst
I fragment G was 400 base pairs larger. M5 virions contained two size classes of circular DNA, one of 100% of the wild type and one of about 58% of the wild-type molecule. A revertant of M5, designated M5R, was isolated on the basis of polyhedron morphology. The genome of M5R contained the insertion of DNA in
Eco
RI fragment I and in
Hin
dIII fragment F, but was similar to the wild type in its other restriction fragment patterns. M5-infected cell cultures synthesized a polyhedrin polypeptide smaller in size than the wild type or M5R.
Epstein-Barr virus DNA XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-1 deletion
Authors: King W , Dambaugh T , Heller M , Dowling J , Kieff E
Published: January 2020

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (Heston et al., Nature (London) 295:160-163, 1982; Miller et al., J. Virol. 18:1071-1080, 1976; Miller et al., Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974; Ragona et al., Virology 101:553-557, 1980). The P3HR-1 virus was known to be deleted for a region which encodes RNA in latently infected, growth-transformed cells (Bornkamm et al., J. Virol. 35:603-618, 1980; Heller et al., J. Virol. 38:632-648, 1981; King et al., J. Virol. 36:506-518, 1980; Raab-Traub et al., J. Virol. 27:388-398, 1978; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1980). This deletion is now more precisely defined. The P3HR-1 genome contains less than 170 base pairs (and possibly none) of the 3,300-base pair U2 region of EBV DNA and is also lacking IR2 (a 123-base pair repeat which is the right boundary of U2). A surprising finding is that EBV isolates vary in part of the U2 region. Two transforming EB viruses, AG876 and Jijoye, are deleted for part of the U2 region including most or all of a fragment, HinfI-c, which encodes part of one of the three more abundant cytoplasmic polyadenylated RNAs of growth-transformed cells (King et al., J. Virol. 36:506-518, 1980; King et al., J. Virol. 38:649-660, 1981; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934).
Adenovirus type 2 fiber mRNA synthesis: no evidence for a cytoplasmic processing pathway
Authors: Lawrence C B , Ramsey W J
Published: January 2020

Adenovirus type 2 fiber mRNA exists in several forms in the cytoplasm which differ in the presence or absence of extra 5'-leader segments (L. T. Chow and T. R. Broker, Cell 15:497-510, 1978). We have investigated the possibility that forms possessing extra leader segments serve as precursors to the mature form in the cytoplasm. Pulse-labeled fiber mRNA became considerably shorter (150 to 250 bases) during a chase; however, most of the pulse-labeled species failed to hybridize to DNA fragments known to encode extra leader segments. Moreover, the entire decrease in size appeared to be due to extensive shortening of the polyadenylic acid tail. Mature-sized fiber mRNA was synthesized normally in cells infected with the nondefective adenovirus type 2-simian virus 40 hybrid virus Ad2+ND5, in which the region encoding the extra leader segments is deleted. These results indicate that the additional 5'-leader segments present in wild-type adenovirus type 2 fiber mRNA are not required for the production of mature fiber mRNA and that species that possess them are not cytoplasmic precursors to the mature form.
Endo-beta-N-acetylglucosaminidase H sensitivity of precursors to herpes simplex virus type 1 glycoproteins gB and gC
Authors: Wenske E A , Bratton M W , Courtney R J
Published: January 2020

The endoglycosidase endo-beta-N-acetylglucominidase H (endo H) was used to examine the nature of the oligosaccharides associated with the herpes simplex virus type 1 glycoproteins gA, gB, and gC. Immunoprecipitates from detergent extracts of infected cells, using monospecific antisera to gAB and gC, were treated with endo H. The low-molecular-weight precursor to gC, pgC(105), was found to be sensitive to endo H. Removal of the endo H-sensitive oligosaccharide chains from pgC(105) resulted in a protein with an apparent molecular weight of 75,000. In contrast, the fully glycosylated gC was not sensitive to endo H treatment. These results suggested that the oligosaccharide chains of pgC(105) were primarily of the simple high-mannose type. Both gA and gB were sensitive to endo H treatment; however, gB appeared to be only partially susceptible, whereas [3H]mannose-labeled gA was not detectable after endo H treatment. These results that gB contained both complex- and simple-type oligosaccharides, and gA contained only simple-type oligosaccharides. An accumulation of the high-mannose glycoproteins pgC(105) and gA was observed in monensin-treated infected cells with a concomitant inhibition of gB and gC. Glycoproteins gA and pgC(105) synthesized in the presence of monensin were also sensitive to endo H treatment.
Long internal direct repeat in Epstein-Barr virus DNA
Authors: Cheung A , Kieff E
Published: January 2020

The nucleotide sequence of the long internal reiteration. IR1, of Epstein-Barr Virus DNA has been determined. The repeat unit is 3,071 base pairs which are 66.8% guanine plus cytosine. There is a CCAAT sequence 39 nucleotides 5' to a TATAA, which could indicate a promotor for transcription. The longest open reading frame is 1,124 base pairs. Also within IR1 is a sequence homologous to the papovavirus origin of DNA replication. The ori-like sequence is within a long palindromic region which is 500 base pairs overall. The palidromic region shares common features with the Alu family members and with eucaryotic transposable elements. The juncture between the short unique region (U1) and IR1 is also sequenced. The transition occurs in BamHI-C at 1,214 base pairs before the BamHI site in the first repeat of IR1. The transition from IR1 to the rightward unique region (U2) has been reported to be at 636 base pairs after the BamHI site in the last repeat of IR1. Thus, relative to the start of IR1 at the juncture with U1, the last copy of IR1 is a partial repeat which contains only the beginning 1,850 base pairs.
Sequence analysis of the polymerase 1 gene and the secondary structure prediction of polymerase 1 protein of human influenza virus A/WSN/33
Authors: Sivasubramanian N , Nayak D P
Published: January 2020

The nucleotide sequence of polymerase 1 (P1) gene of a human influenza virus (A/WSN/33) has been determined by using cDNA clones, except for the last 83 nucleotides, which were obtained by primer extension. The WSN P1 gene contains 2,341 nucleotides and codes for a protein of 757 amino acids (Mr = 86,500). P1 gene possesses a striking tandem repeat of 12 nucleotides (nucleotide position 2,188 to 2,199, 2,200 to 2,211) and a corresponding tandem repeat of tetrapeptide in the P1 protein. The deduced sequence of P1 protein is enriched in basic amino acids, particularly arginine. In addition, it also contains clusters of basic amino acids which may provide sites for the interaction with the template virion RNA capped primer as well as with other proteins involved in viral replication and transcription. A secondary structure prediction, using Chou and Fasman analyses (Annu. Rev. Biochem. 47:251-276, 1978), shows that the P1 protein possesses some unique features, viz., one "four-helical supersecondary structure" and four "polypeptide double helices" (antiparallel beta-pleated sheets) which are considered important in RNA binding.
Adenovirus Type 2 DNA Replicated in Arginine-Starved KB Cells Is Infectious
Authors: Auborn Karen J. , Rouse Harriet
Published: January 2020

Adenovirus type 2 DNA from extracts of KB cells rendered nonpermissive by arginine starvation is infectious.