bluuebunny/crossref_metadata_2025 huggingface dataset:
Polar encapsidation of adenovirus DNA: cloning and DNA sequence of the left end of adenovirus type 3
Authors: Kosturko L D , Sharnick S V , Tibbetts C
Published: January 2020

The left-end adenovirus type 3 DNA sequence is very similar to those of other subgroup B adenoviruses, especially in the area between the HinfI site (320 base pairs) and the early-region Ia gene. This segment of the genome has been implicated as necessary for the left-end polarity of adenovirus DNA encapsidation. This segment and the sequences flanking it are compared with the corresponding sequences of adenovirus type 5 and adenovirus type 12, and the extent and pattern of intersubgroup homologies are discussed.
Immunization against the polyoma virus-induced tumor-specific transplantation antigen by early region mutants of the virus
Authors: Dalianis T , Magnusson G , Ito Y , Klein G
Published: January 2020

To investigate the relation between the polyoma tumor-specific transplantation antigen and the virus-coded proteins, mice were immunized by inoculation of a variety of viable polyoma virus mutants and then challenged with polyoma virus-induced tumors. Two classes of early region mutants were used. One class produces a normal small T-antigen and truncated middle and large T-antigens. The second class (hr-t mutants) forms a normal large T-antigen together with N-terminal fragments of small and middle T-antigens. All mutants, transforming as well as nontransforming, induced protection against polyoma virus tumors. However, there were quantitive differences between the mutants. The finding that an hr-t mutant could induce tumor rejection suggests that full-length middle and small T-antigens are not necessary for the induction of this response. Since intact middle T-antigen is the only virus-coded protein known to associate with the plasma membrane, the possibility must be considered that the polyoma virus tumor-specific transplantation antigen consists of cellular components.
Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies
Authors: Emini E A , Jameson B A , Lewis A J , Larsen G R , Wimmer E
Published: January 2020

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.
Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1
Authors: Allen G P , Turtinen L W
Published: January 2020

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.
Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells
Authors: Kaschka-Dierich C , Werner F J , Bauer I , Fleckenstein B
Published: January 2020

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.
Conserved Sequences at the Origin of Adenovirus DNA Replication
Authors: Stillman Bruce W. , Topp William C. , Engler Jeffrey A.
Published: January 2020

The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis.
Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells
Authors: Christensen J B , Brockman W W
Published: January 2020

The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.
Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation
Authors: Bryant D , Parsons J T
Published: January 2020

Transformation of cells by Rous sarcoma virus results from the expression of the viral src gene product, pp60src. Site-directed mutagenesis techniques have been used to construct defined deletion mutations within the src gene of Prague A strain of Rous sarcoma virus. The deletion of DNA sequences at the Bg/II restriction site in the src gene yielded both transformation-defective mutants (tdCH4, 64, and 146) and a mutant temperature sensitive for morphological transformation (tsCH119). The genome of tsCH119 contains an in-phase deletion of approximately 160 base pairs, which mapped to the immediate 3' side of the Bg/II restriction site. Upon infection of chicken cells, tsCH119 encoded a structurally altered src protein, pp53src, containing a deletion of amino acid residues 202 to 255. Immune complexes containing pp53src isolated from tsCH119-infected cells grown at 41 degrees C exhibited only 50% less tyrosine-specific kinase activity than immune complexes isolated from cells grown at 35 degrees C. pp53src immunoprecipitated from tsCH119-infected cells grown at either 35 or 41 degrees C contained phosphoserine and phosphotyrosine. We suggest that tsCH119 represents a class of mutants containing mutations mapping within a functionally important domain of the src protein, distinct from the domain specifying the protein kinase activity.
Characterization of two conformational forms of the major DNA-binding protein encoded by herpes simplex virus 1
Authors: Knipe D M , Quinlan M P , Spang A E
Published: January 2020

We have resolved two electrophoretic species of the major DNA-binding protein, infected cell polypeptide 8 (ICP8), encoded by herpes simplex virus 1. In pulse-chase experiments, we observed the conversion of the ICP8a form, the slower migrating species, to the faster migrating form, ICP8b. Thus, the two species appear to be related as precursor-product. The conversion was not due to proteolytic cleavage, because higher concentrations of reducing agents in the sample buffer shifted the faster moving form to the slower moving species. Also, the two forms have identical peptide patterns as analyzed by partial proteolysis in sodium dodecyl sulfate. Thus, the faster moving species appears to be a conformational isomer containing intramolecular disulfide bonds. The functional significance of the two forms of the protein is discussed.
Genetic studies of coliphage 186. I. Genes associated with phage morphogenesis
Authors: Hocking S M , Egan J B
Published: January 2020

In coliphage 186, 22 essential genes were defined by complementation studies with amber mutants. Eighteen genes were associated with phage morphogenesis: 11 with phage tail formation, and 7 with phage head formation. The remaining four genes are discussed in the accompanying paper (S. M. Hocking and J. B. Egan, J. Virol. 44:1068-1071, 1982).
Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells
Authors: Hill T M , Sinden R R , Sadler J R
Published: January 2020

Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff.
Characterization of herpes simplex virus 2 temperature-sensitive mutants whose lesions map in or near the coding sequences for the major DNA-binding protein
Authors: Spang A E , Godowski P J , Knipe D M
Published: January 2020

By marker rescue with cloned herpes simplex virus 2 DNA fragments, we have mapped the temperature-sensitive mutations of a series of herpes simplex virus 2 mutants to a region of the herpes simplex virus 2 genome that lies within or near the coding sequences for the major DNA-binding protein, ICP8. In cells infected with certain of these mutants at the nonpermissive temperature, the association of the major DNA-binding protein with the cell nucleus was defective. In these cells, the DNA-binding protein accumulated in the cytoplasmic and the crude nuclear detergent wash fractions. At the permissive temperature, the maturation of the mutant ICP8 was similar to that of the wild-type viral protein. With the remainder of the mutants, the nuclear maturation of ICP8 was similar to that encoded by the wild-type virus at the nonpermissive and permissive temperatures as assayed by cell fractionation.
Mutants deleted in the agnogene of simian virus 40 define a new complementation group
Authors: Mertz J E , Murphy A , Barkan A
Published: January 2020

Analysis of the DNA sequence of the late leader region of simian virus 40 indicates that it might encode a 61-amino acid, highly basic protein, LP-1. Mutants deleted in this region are viable, but they produce infectious progeny more slowly than wild-type virus in established monkey cells. On the basis of the rates of appearance and the sizes of mixed plaques formed after cotransfections with pairs of mutants, we found that mutants defective in the synthesis of LP-1 complementation was also observed in infections with virions and was bidirectional. Therefore, these mutants define a new complementation group, group G. In addition, a protein of the appropriate molecular weight for LP-1 (approximately 8 X 10(3) ) was synthesized by wild-type virus-infected cells but not by mock-infected or group G gene mutant-infected cells. This protein, whose identity has been established definitively by Jay et al. (Nature (London) 291:346-349, 1981), was synthesized at a high rate at late times after infection, was present predominantly in the cytoplasmic fraction of cells, possessed a fairly short half-life, and was absent from mature virions. Once formed, virions of group G gene mutants behaved biologically and physically like virions of wild-type virus. On the basis of these findings and other known properties of LP-1 and mutants defective in LP-1 synthesis, we hypothesize that LP-1 functions to facilitate virion assembly, possibly by serving as a nonreusable scaffolding protein.
Palindromic adenovirus type 5-simian virus 40 hybrid
Authors: Gluzman Y , Van Doren K
Published: January 2020

A family of novel adenovirus type 5-simian virus 40 (Ad5-SV40) recombinants (Ad5++D1) whose genomes consist of symmetrically inverted structures was isolated. Particles of Ad5++D1 could contain one of several recombinant genomes that differed incrementally from one another by a full-length copy of linear SV40 DNA. The members of the Ad5++D1 family appeared to be in genetic equilibrium with one another. In all probability this equilibrium was maintained by homologous recombination, resulting in the loss or gain of one or two unit length copies of the SV40 genome. The genome of the most abundant recombinant from consisted of a giant inverted repeat which was some 35,000 nucleotide pairs in length. Beginning from one end, the recombinant genome consisted of 3,534 nucleotides derived from the left end of the adenovirus type 5 genome; these nucleotides were joined to 2.7 copies of SV40 DNA arranged as head-to-tail tandems. This entire structure was then repeated in the opposite orientation, thereby forming a large inverted repeat whose structure was Ad5-SV40-SV40-04VS-04VS-5dA. The population of hybrid genomes was stable and was maintained through serial rounds of infection.
Porcine parvovirus: virus purification and structural and antigenic properties of virion polypeptides
Authors: Molitor T W , Joo H S , Collett M S
Published: January 2020

Porcine parvovirus (PPV) was extensively purified from infected swine fetal homogenates by CaCl2 precipitation followed by CsCl density centrifugation. Two species of particles possessing PPV-specific hemagglutinating activity were observed banding at densities of 1.39 and 1.30 g/ml, representing full and empty 20-nm virion particles, respectively. Both classes of particles contained three major polypeptides. A, B, and C, with respective molecular weights of 83,000, 64,000, and 60,000. The amount of polypeptide A was similar in both species (approximately 10%); however, the B protein was most abundant in the 1.30-g/ml particles, whereas the C protein was the major polypeptide found in the 1.39-g/ml particles. Antisera generated to each sodium dodecyl sulfate-polyacrylamide gel-purified virion structural protein had reactivities qualitatively similar to those of conventional antisera raised against intact PPV in a variety of standard serological assays. The antisera generated against the individual sodium dodecyl sulfate-denatured PPV polypeptides were able to react with native, intact PPV virions and were capable of neutralizing virus infectivity.
Persistence of Interferon Action in Mouse Cells Permeabilized with Lysolecithin
Authors: Lee Elsbeth J. , Sreevalsan Thazepadath
Published: January 2020

DNA synthesis, as measured by incorporation of [
3
H]TTP, was inhibited in Swiss mouse 3T3 cells treated with interferon and subsequently permeabilized with lysolecithin. The degree of inhibition observed was similar in intact or permeabilized cells. The interferon-induced antiviral state was retained in permeabilized cells.
Molecular cloning of infectious viral DNA from ecotropic neurotropic wild mouse retrovirus
Authors: Jolicoeur P , Nicolaiew N , DesGroseillers L , Rassart E
Published: January 2020

Among a mixture of amphotropic and ecotropic murine leukemia viruses (MuLVs) isolated from paralyzed wild mice, only N-tropic ecotropic MuLV, cloned by cell culture techniques, has been shown to induce paralysis after reinjection into susceptible mice (M. B. Gardner, Curr. Top. Microbiol. Immunol. 79:215-239, 1978). The viral DNA genome of one of these neurotropic MuLVs (Cas-Br-E) has been cloned in Charon 21A at the SalI site. One clone, designated NE-8, was studied in more detail. A restriction endonuclease map of this cloned DNA was derived. Cloned viral DNA microinjected into NIH 3T3 cells produced infectious MuLV which was characterized as XC+, ecotropic, and N-tropic. The virus that was recovered after the microinjection of NE-8 DNA was also injected into susceptible SIM.S and NIH Swiss mice and was found to induce lower limb paralysis in these animals. These results make it highly unlikely that other agents (which might have escaped detection and separation from ecotropic MuLV by the techniques previously used) play a role in the etiology of this disease and clearly indicate that the ecotropic MuLV genome harbors sequences responsible for this paralysis. The availability of this clone DNA would now allow us to map these sequences on the genome.
Activation of latent Epstein-Barr virus genomes: selective stimulation of synthesis of chromosomal proteins by a tumor promoter
Authors: Lin J C , Smith M C , Pagano J S
Published: January 2020

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a potent inducer of Epstein-Barr virus (EBV) gene expression. The optimal conditions for maximum activation of latent EBV genomes by TPA were determined. Although TPA is able to induce replication of EBV genomes in P3HR-1 cells in all phases of growth, the greatest increase in viral genome copies per cell (15-fold above the control level) occurred in nonproliferating cells as opposed to cells growing exponentially (6-fold above the control level). The synthesis of chromosomal proteins in nonproliferating cells under the conditions that induce maximum activation of latent virus genomes by TPA was studied. Selective stimulation in chromosomal protein synthesis accompanied the increase in EBV genomes in P3HR-1 cells despite an overall reduction in total cellular protein synthesis. Comparison of the chromosomal proteins from TPA-induced P3HR-1 cells and from superinfected Raji cells revealed comigrating chromosomal polypeptides of 145K, 140K, 135K, 110K, 85K, and 55K that are presumably EBV associated. The selective stimulation of synthesis of these chromosomal proteins in TPA-treated P3HR-1 cells was closely associated with the activation of latent EBV genomes.
Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatitis virus
Authors: Gallione C J , Rose J K
Published: January 2020

The nucleotide sequence of the mRNA encoding the glycoprotein from the New Jersey serotype of vesicular stomatitis virus (VSV) was determined from a cDNA clone containing the entire coding region. The sequence of 12 5'-terminal noncoding nucleotides present in the mRNA but not in the cDNA clone was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1,573 nucleotides long (excluding polyadenylic acid) and encodes a protein of 517 amino acids. Only six nucleotides occur between the translation termination codon and the polyadenylic acid. Short homologies between the untranslated termini of this mRNA and the mRNAs of the Indiana serotype were found. The predicted protein sequence was compared with that of the glycoprotein of the Indiana serotype of VSV and with the glycoprotein of rabies virus, using a computer program which determines optimal alignment. An amino acid identity of 50.9% was found for the two VSV serotypes. Approximately 20% identity was found between the rabies virus and VSV New Jersey glycoproteins. The positions and sizes of the transmembrane domains, the signal sequences, and the glycosylation sites are identical in both VSV serotypes. Two of five serine residues which were possible esterification sites for palmitate in the glycoprotein from the Indiana serotype are changed to glycine residues in the glycoprotein from the New Jersey serotype. Because the glycoprotein of the New Jersey serotype does not contain esterified palmitate, we suggest that one or both of these residues are the probable esterification sites in the glycoprotein from the Indiana serotype.
Bacteriophage SPO1 structure and morphogenesis. I. Tail structure and length regulation
Authors: Parker M L , Eiserling F A
Published: January 2020

Bacteriophage SPO1, a structually complex phage with hydroxymethyl uracil replacing thymine, has been studied by structural and chemical methods with the aim of defining the virion organization. The contractile tail of SPO1 consists of a complex baseplate, a tail tube, and a 140-nm-long sheath composed of stacked disks (4.1 nm repeat), each containing six subunits of molecular weight 60,300. The subunits are arranged in six parallel helices, each with a helical screw angle (omega 0) of 22.5 degrees. The baseplate was shown to undergo a structural rearrangement during tail contraction into a hexameric pinwheel. A mutation in gene 8 which produced unattached heads and tails also produced tails of different lengths. The tail length distribution suggests that the smallest integral length increment is a single disk of subunits. The structural arrangement of subunits in long tails is identical to that of normal tails, and the tails can contract. Many of the long tails showed partial stain penetration within the tail tube to a point which coincides with the top of a unit-length tail. The implications of these findings with respect to tail length regulation are discussed.
Glycosylation of herpes simplex virus type 1 gC in the presence of tunicamycin
Authors: Wenske E A , Courtney R J
Published: January 2020

The presence of O-glycosidic linkages on herpes simplex virus type 1 (HSV-1) glycoproteins was indicated by the synthesis and glycosylation of HSV-1 glycoproteins in the presence of tunicamycin. Monospecific antiserum to HSV-1 gC immunoprecipitated a 92,000-molecular-weight protein synthesized in the presence of tunicamycin and isotopically labeled with glucosamine or galactose. Anti-gAB did not immunoprecipitate a carbohydrate-labeled HSV-1 protein synthesized in the presence of tunicamycin. The purified glucosamine-labeled 92,000-molecular-weight protein synthesized in the presence of tunicamycin and the fully glycosylated forms of gAB and gC were tested for their sensitivity to mild alkaline hydrolysis. Purified gAB was resistant to mild alkaline hydrolysis, whereas gC and the 92,000-molecular-weight protein were both sensitive to mild alkaline hydrolysis. These results suggest that O-glycosidic linkages are associated with the HSV-1 gC glycoprotein.
Role of antibodies and host cells in plaque enhancement of Murray Valley encephalitis virus
Authors: Kliks S C , Halstead S B
Published: January 2020

Chicken antisera to Murray Valley encephalitis (MVE) virus, when incubated with virus and assayed for plaques on chicken embryo (CE) monolayers, neutralized MVE virus at high concentrations of antibody, but caused increases in plaque counts at low concentrations of antibody. Plaque enhancement did not occur when the same virus-antibody mixtures were assayed on a continuous line of rhesus monkey kidney cells (LLC-MK2), nor when the anti-MVE antibody was of mammalian origin and the assay system was CE monolayers. Correspondingly, chicken anti-MVE did not enhance the plaque formation of MVE virus in a stable line of mouse macrophages, P-388D1, whereas rabbit and mouse anti-MVE did enhance plaque formation. This enhancing activity was associated with noncytophilic immunoglobulin G (IgG). The Fc terminus of the IgG molecule was required, as no plaque enhancement occurred with chicken anti-MVE Fab. These data indicate that there is a requirement for taxonomic complementarity between Fc termini and Fc receptors in the above systems. CE cell monolayers were found to contain approximately 2% of Fc receptor-bearing cells among the fibroblast-like cells. Fc receptor-bearing cells in CE monolayers were isolated and found to be of the mononuclear phagocytic lineage. These mononuclear phagocytes, which originate in lymphoid tissues and blood associated with CE tissue fragments, are integrated into primary CE monolayers and form infectious centers in the presence of virus and low dilutions of antibody.
Structural proteins and cell-free translation products of total RNA and hybrid-selected RNA from two DNA variants of vaccinia virus
Authors: Bedard D L
Published: January 2020

Two major variants of vaccinia virus, large (L) and small (S), differ by a deletion of 9.7 kilobase pairs. The structural proteins and the translation products of RNA transcribed in vitro from each of these variants were analyzed by gel electrophoresis. Recombinant plasmids were used to select RNA transcribed from the L variant sequences corresponding to the deletion. This RNA yielded translation products indicating that a minimum of 11 polypeptides, including two structural proteins, map within the deletion.
Structure of Erwinia carotovora temperate bacteriophage 59 and its DNA
Authors: Kishko Y G , Ruban V I , Tovkach F I , Murashchyk I G , Danileychenko V V
Published: January 2020

The temperate phage 59 from Erwinia carotovora and its DNA were studied. The phage particles have an icosahedral head and a long noncontractile tail with a base plate. The virus DNA makes up 50% of the total virus and exists as a linear molecule (molecular weight, 2.6 X 10(7)). A model of virus structural organization is presented.
Regulation of endogenous murine mammary tumor virus expression in C57BL mouse lactating mammary glands: transcription of functional mRNA with a block at the translational level
Authors: Vaidya A B , Taraschi N E , Tancin S L , Long C A
Published: January 2020

Expression of endogenous murine mammary tumor viruses (MuMTVs) in various mouse strains is regulated in different ways, and in the absence of exogenous MuMTV, this regulation influences the incidence of spontaneous mammary tumors. Two mouse strains with low mammary tumor incidence, BALB/c and C57BL, control endogenous MuMTV expression at different stages. Neither of the strains had any detectable MuMTV polypeptides in its lactating mammary glands (LMG). However, in C57BL LMG, substantial amounts of MuMTV RNA were present, whereas very little viral RNA was detected in BALB/c LMG. By determining MuMTV RNA levels in LMG of hybrids and backcrosses of BALB/c and C57BL mice, we found that there are three unlinked, independently segregating genetic loci in C57BL mice that are responsible for the presence of moderately high amounts of MuMTV RNA in LMG. The viral RNA in C57BL LMG was processed and transported to the cytoplasm where it was found to cosediment with EDTA-sensitive polysomes. No viral proteins were detected in run-off reactions that permit completion of nascent polypeptide synthesis with polysomes from C57BL LMG, and sensitive radioimmunoassays failed to detect any MuMTV proteins in these tissues. In contrast, MuMTV mRNA purified from C57BL LMG did direct the synthesis of both gag and env MuMTV polypeptides when added to a heterologous rabbit reticulocyte lysate cell-free translation system. We propose that MuMTV mRNA in C57BL LMG, for unknown reasons, is blocked at the translational level.
Characterization of am404, an amber mutation in the simian virus 40 T antigen gene
Authors: Rawlins D R , Collis P , Muzyczka N
Published: January 2020

We analyzed the biological activity of an amber mutation, am404, at map position 0.27 in the T antigen gene of simian virus 40. Immunoprecipitation of extracts from am404-infected cells demonstrated the presence of an amber protein fragment (am T antigen) of the expected molecular weight (67,000). Differential immunoprecipitation with monoclonal antibody demonstrated that am T antigen was missing the carboxy-terminal antigenic determinants. The amber mutant was shown to be defective for most of the functions associated with wild-type T antigen. The mutant did not replicate autonomously, but this defect could be complemented by a helper virus (D. R. Rawlins and N. Muzyczka, J. Virol. 36:611-616, 1980). The mutant failed to transform nonpermissive rodent cells and did not relieve the host range restriction of adenovirus 2 in monkey cells. However, stimulation of host cell DNA, whose functional region domain has been mapped within that portion of the protein synthesized by the mutant, could be demonstrated in am404-infected cells. A number of unexpected observations were made. First, the am T antigen was produced in unusually large amounts in a simian virus 40-transformed monkey cell line (COS-1), but overproduction was not seen in nontransformed monkey cells regardless of whether or not a helper virus was present. This feature of the mutant was presumably the result of the inability of am T antigen to autoregulate, the level of wild-type T antigen in COS-1 cells, and the unusually short half-life of am T antigen in vivo. Pulse-chase experiments indicated that am T antigen had an intracellular half-life of approximately 10 min. In addition, although the am T antigen retained the major phosphorylation site found in simian virus 40 T antigen, it was not phosphorylated. Thus, phosphorylation of simian virus 40 T antigen is not required for the stimulation of host cell DNA synthesis. Finally, fusion of am404-infected monkey cells with Escherichia coli protoplasts containing appropriate procaryotic suppressor tRNAs showed that am404 is a suppressible nonsense mutation.
Sequence repeats in a polyoma virus DNA region important for gene expression
Authors: Ruley H E , Fried M
Published: January 2020

The sequenced prototype strains (A2 and A3) of polyoma virus lack sequence duplications characteristic of other papovaviruses. However, we found that five polyoma virus strains (P16, Toronto large plaque, MV, Ts 48, and NG59R) contain tandemly duplicated sequences in a region near the late RNA leader. Although the duplications vary in size (31 to 84 base pairs) and location (between nucleotide [nt] 5068 and nt 5185), the sequence between nt 5114 and nt 5137 is contained within all five duplicated segments. This region is known to be important in polyoma virus early gene expression, and it contains sequences capable of enhancing the expression of nonviral genes. Inspection of the sequences at and around the ends of the repeats indicated that the duplications do not arise by homologous recombination, and there was no indication that a sequence-specific mechanism results in their formation. However, the variation in the structure of the repeats among different polyoma virus strains suggests that these sequence duplications are a recent evolutionary occurrence. The potential biological significance of this variation is discussed.
The adenovirus type 2-simian virus 40 hybrid virus Ad2+ND4 requires deletion variants to grow in monkey cells
Authors: Lewis A M , Westphal H
Published: January 2020

The Ad2+ND4 virus is an adenovirus type 2 (Ad2)-simian virus 40 (SV40) recombination. The Ad2 genome of this recombinant has a rearrangement within early region 3; Ad2 DNA sequences between map positions 81.3 and 85.5 have been deleted, and the SV40 DNA sequences between map positions 0.11 and 0.626 have been inserted into the deletion in an 81.3-0.626 orientation. Nonhybrid Ad2 is defective in monkey cells; however, the Ad2+ND4 virus can replicate in monkey cells due to the expression of the SV40-enhancing function encoded by the DNA insert. Stocks of the Ad2+ND4 hybrid were produced in primary monkey cells by using the progeny of a three-step plaque purification procedure and were considered to be homogeneous populations of Ad2+ND4 virions because they induced plaques in primary monkey cells by first-order kinetics. By studying the kinetics of plaque induction in continuous lines (BSC-1 and CV-1) of monkey cells, we have found that stocks (prepared with virions before and after plaque purification) of Ad2+ND4 are actually heterogeneous populations of Ad2+ND4 virions and Ad2+ND4 deletion variants that lack SV40 and frequently Ad2 DNA sequences at the left Ad2-SV40 junction. Due to the defectiveness of the Ad2+ND4 virus, the production of progeny in BSC-1 and CV-1 cells requires complementation between the Ad2+ND4 genome and the genome of an Ad2+ND4 deletion variant. Since the deletion variants that have been obtained from Ad2+ND4 stocks do not express the SV40-enhancing function in that they cannot produce progeny in monkey cells, we conclude that they are providing an Ad2 component that is essential for the production of Ad2+ND4 progeny. These data imply that the Ad2+ND4 virus is incapable of replicating in singly infected primary monkey cells without generating deletion variants that are missing various amounts of DNA around the left Ad2-SV40 junction in the hybrid genome. As the deletion variants that arise from the Ad2+ND4 virus are created by nonhomologous DNA recombination, the generation of deletion variants in monkey cells infected with Ad2+ND4 may be a useful model for studying this process.
Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene
Authors: Majors J E , Varmus H E
Published: January 2020

To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein.
Involvement of Escherichia coli K-12 DNA polymerase I in the growth of bacteriophage Mu
Authors: McBeth D L , Taylor A L
Published: January 2020

We examined several aspects of bacteriophage Mu development in Escherichia coli strains that carry mutations in the polA structural gene for DNA polymerase I (PolI). We found that polA mutants were markedly less efficient than PolI wild-type (PolI+) strains in their capacity to form stable Mu lysogens and to support normal lytic growth of phage Mu. The frequency of lysogenization was determined for polA mutants and their isogenic PolI+ derivatives, with the result that mutants were lysogenized 3 to 8 times less frequently than were PolI+ cells. In one-step growth experiments, we found that phage Mu grew less efficiently in polA cells than in PolI+ cells, as evidenced by a 50 to 100% increase in the latent period and a 20 to 40% decrease in mean burst size in mutant cells. A further difference noted in infected polA strains was a 10-fold reduction in the frequency of Mu-mediated transposition of chromosomal genes to an F plasmid. Pulse labeling and DNA-DNA hybridization assays to measure the rate of phage Mu DNA synthesis after the induction of thermosensitive prophages indicated that phage Mu replication began at about the same time in both polA and PolI+ strains, but proceeded at a slower rate in polA cells. We conclude that PolI is normally involved in the replication and integration of phage Mu. However, since phage Mu does not exhibit an absolute requirement for normal levels of PolI, it appears that residual PolI activity in the mutant strains, other cellular enzymes, or both can partially compensate for the absence of normal PolI activity.
Enzymatic construction and selection of bacteriophage G4 mutants with modifications of a DNA secondary structure in the J-F intercistronic region
Authors: Müller U R
Published: January 2020

The J-F intercistronic region of bacteriophage G4 has the potential to form a perfectly base-paired hairpin structure, thought to act as a terminator of transcription. To investigate this proposed structure-function relationship, viable mutants were constructed by site-specific mutagenesis with small deletions of 2 to 4 base pairs in the center of the corresponding palindromic sequence. These sequence modifications had a small positive effect on the growth efficiency of the phage. The approach of biochemical rather than biological selection of these mutant phages is generally applicable to the construction of virus and plasmid vectors.
In vivo cleavage of cytosine-containing bacteriophage T4 DNA to genetically distinct, discretely sized fragments
Authors: Carlson K , Wiberg J S
Published: January 2020

Mutants of bacteriophage T4D that are defective in genes 42 (dCMP hydroxymethylase), 46 (DNA exonuclease), and 56 (dCTPase) produce limited amounts of phage DNA in Escherichia coli B. In this DNA, glucoylated 5-hydroxymethylcytosine is completely replaced by cytosine. We found that this DNA rapidly becomes fragmented in vivo to at least 16 discrete bands as visualized on agarose gels subjected to electrophoresis. The sizes of the fragments ranged from more than 20 to less than 2 kilobase pairs. When DNAs from two of these bands were radioactively labeled in vitro by nick translation and hybridized to XbaI restriction fragments of cytosine-containing T4 DNA, evidence was obtained that the two bands are genetically distinct, i.e., they contain DNA from different parts of the T4 genome. Mutational inactivation of T4 endonuclease II (gene denA) prevented the fragmentation. Three different mutations in T4 endonuclease IV (gene denB) caused the same minor changes in the pattern of fragments. We conclude that T4 endonuclease II is required, and endonuclease IV is involved to a minor extent, in the in vivo production of these cytosine-containing T4 DNA fragments. We view these DNA fragments as "restriction fragments" since they represent degradation products of DNA "foreign" to T4, they are of discrete size, and they are genetically distinct. Thus, this report may represent the first, direct in vivo demonstration of discretely sized genetically distinct DNA restriction fragments.
Chromatin-like structure of adeno-associated virus DNA in infected cells
Authors: Marcus-Sekura C J , Carter B J
Published: January 2020

Adeno-associated virus (AAV) is a single-stranded DNA virus which requires adenovirus as a helper for productive infection. We studied whether intracellular AAV DNA in KB cells was present in a chromatin-like structure by digesting infected cell nuclei with micrococcal nuclease. Virus DNA was detected by agarose gel electrophoresis followed by blotting and hybridization to nick-translated [32P]DNA probes. After coinfection with adenovirus, AAV DNA was present in nucleosome-like structures which were similar to cell nucleosomes and were double stranded as judged by insensitivity to S1 nuclease digestion. In the absence of adenovirus, intracellular AAV DNA also formed similar nucleosome-like structures which were also insensitive to S1 digestion and were formed in both the presence and absence of hydroxyurea. These latter structures probably formed on AAV duplexes created either by reassociation of infecting parental single-stranded DNA or by covalent integration into the cell genome rather than by de novo AAV DNA synthesis. These results have implications for the mechanism of AAV genome replication, transcription, and integration into the cell genome.
Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses
Authors: Lipsich L A , Lewis A J , Brugge J S
Published: January 2020

Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.
Marker rescue of temperature-sensitive mutations of vaccinia virus WR: correlation of genetic and physical maps
Authors: Ensinger M J , Rovinsky M
Published: January 2020

The physical map locations of 62 temperature-sensitive mutations of vaccinia virus WR have been determined by marker rescue experiments, using cloned HindIII fragments of wild-type DNA. Since vaccinia virus DNA is not infectious, marker rescue was performed by infecting monolayers of cells at the nonpermissive temperature with a low multiplicity of the mutant to be rescued and transfecting with calcium phosphate-precipitated recombinant DNA. Wild-type recombinants were measured by using either a direct plaque assay technique or a two-step procedure in which the final yield of virus from the transfected cells was assayed at the permissive and nonpermissive temperatures. Mutants that had been previously assigned to the same complementation-recombination group were rescued by the same HindIII fragment, with the exception of three mutants in one group that were rescued by either one of two adjacent fragments. A comparison between the genetic linkage map of the temperature-sensitive mutations in 30 mutants with their physical locations demonstrated that not only was the order of the genetic map correct but also recombination frequencies generally reflected actual physical distances.