bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete Nucleotide Sequences of Two Adjacent Early Vaccinia Virus Genes Located Within the Inverted Terminal Repetition
Authors: Venkatesan Sundararajan , Gershowitz Alan , Moss Bernard
Published: January 2020

The proximal part of the 10,000-base pair (bp) inverted terminal repetition of vaccinia virus DNA encodes at least three early mRNAs. A 2,236-bp segment of the repetition was sequenced to characterize two of the genes. This task was facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotide linkers with synthetic restriction sites provided points for radioactive labeling before sequencing by the chemical degradation method of Maxam and Gilbert (Methods Enzymol.
65
:499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease S1-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide sequences for at least 60 bp upstream of both transcriptional initiation sites are more than 80% adenine · thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus sequences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single open reading frame of 420 nucleotides. Preceding the first AUG, there is a heptanucleotide that can hybridize to the 3′ end of 18S rRNA with only one mismatch. The derived amino acid sequence of the protein indicated a molecular weight of 15,500. The gene transcribed in the leftward direction encodes an RNA 1,000 to 1,100 nucleotides long with an open reading frame of 996 nucleotides and a leader sequence of only 5 to 6 nucleotides. The derived amino acid sequence of this protein indicated a molecular weight of 38,500. The 3′ ends of the two transcripts were located within 100 bp of each other. Although there are adenine · thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences are absent.
Lambdoid Coliphage HK139 Integrates Between
his
and
supD
Authors: Dhillon T. S. , Poon Alice P. W. , Hui Yin Wah , Dhillon Elvera K. S.
Published: January 2020

Phage HK139 is UV inducible and λ homoimmune and has the host range of φ80. It can recombine with λ as well as with φ80, and in the prophage form it is found integrated between the loci
his
and
supD.
Chromosomal localization of the Moloney sarcoma virus mouse cellular (c-mos) sequence
Authors: Swan D , Oskarsson M , Keithley D , Ruddle F H , D'Eustachio P , Vande Woude G F
Published: January 2020

The Moloney sarcoma virus-specific onc gene, referred to as v-mos, was used as probe to hybridize to restricted DNAs from various mouse-Chinese hamster hybrid cell lines. These hybrid cells contain, in addition to all of the Chinese hamster chromosomes, various numbers (less than a full complement) of mouse chromosomes. Comparison of the presence or absence of the mouse cellular mos gene with the known karyotype in each of the hybrid cell lines allows us to conclude that the mos gene is on mouse chromosome 4.
Adenovirus type 12-transformed rat embryo brain and rat liver epithelial cell lines: adenovirus type 12 genome content and viral protein expression
Authors: Paraskeva C , Brown K W , Dunn A R , Gallimore P H
Published: January 2020

By Southern blotting analysis, six adenovirus type 12 (Ad-12)-transformed rat embryo brain cell lines and one Ad-12-transformed rat liver epithelial line were shown to contain all or nearly all the Ad-12 genome. Another Ad-12 rat liver epithelial cell line contained a repeating structure consisting of only the left-hand 16% of the Ad-12 genome. Three Ad-12-specified proteins (molecular weights, 52,000, 41,000, and 18,000) were found by immunoprecipitation to be common to all of these cell lines. Two rat liver epithelial lines, produced from an Ad-12-infected culture and previously shown to be T-antigen negative by immunofluorescence, contained no detectable Ad-12 genome or Ad-12-specified proteins. Although some of the rat embryo brain transformants had been shown previously to express "late" Ad-12 mRNA, no Ad-12 structural proteins were found to be produced by these cell lines.
Abelson murine leukemia virus P120: identification and characterization of tyrosine phosphorylation sites
Authors: Reynolds F H , Oroszlan S , Stephenson J R
Published: January 2020

Tryptic peptides containing two major in vivo P120gag-abl tyrosine phosphorylation acceptor sites were identified, phosphorylated in vitro, and purified to homogeneity. The tyrosine site in peptide a is localized at a position six residues distal to its trypsin cleavage site, whereas the tyrosine acceptor site in peptide b is at residue seven. A third peptide, c, contains an amino-terminal phosphotyrosine residue: phosphorylation of this latter peptide only occurs to a significant extent in vivo.
Separation and Properties of Two Kinds of Simian Virus 40 Late Transcription Complexes
Authors: Llopis Rafael , Stark George R.
Published: January 2020

Simian virus 40 (SV40) transcription complexes were labeled in cells with 3-min pulses of [
3
H]uridine 48 h after infection and were extracted from nuclei in isotonic buffer or in a buffer containing Sarkosyl. In sucrose gradients, the labeled complexes sedimented faster than both free RNA and most SV40 nucleoproteins. Most of the pulse-labeled nascent RNA hybridized to the entire late region of SV40, remained bound to viral DNA in Cs
2
SO
4
gradients, and ranged in size from a few nucleotides to about 5,000 nucleotides, with a peak at about 700. In contrast, the SV40-associated RNA polymerase activity in the same preparations sedimented near the major peak of SV40 nucleoproteins and was clearly separated from the transcription complexes bearing pulse-labeled nascent RNA. The two kinds of transcription complexes were released from isolated nuclei at different rates. Complexes bearing pulse-labeled RNA were released immediately when the nuclei were agitated in a Dounce homogenizer in isotonic buffer, whereas most of the complexes bearing RNA polymerase active in vitro were released more slowly, during subsequent incubation of the nuclei at 0°C. Since the complexes bearing pulse-labeled nascent RNA were virtually inactive in vitro, the blocked complexes described by Laub et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3297-3301, 1980) probably account for almost all the SV40-associated RNA polymerase activity studied previously by many investigators. New procedures must be developed to preserve the activity of the pulse-labeled complexes if the many advantages of the SV40 system for studying transcription by nucleoprotein complexes in vitro are to be realized fully.
Structure and synthesis of a simian virus 40 super T-antigen
Authors: Lovett M , Clayton C E , Murphy D , Rigby P W , Smith A E , Chaudry F
Published: January 2020

Mouse cells transformed by simian virus 40 often contain virus-coded tumor antigens distinct from those synthesized in productively infected permissive cells. The SV3T3 C120 cell line produces no large T-antigen of apparent molecular weight 94,000 but instead a super T-antigen of apparent molecular weight 145,000. We used recombinant DNA techniques to isolate the template for this super T-antigen and determined its structure by DNA sequencing. The integrated viral early transcription unit contains an in-phase, perfect tandem duplication of 1,212 base pairs. Transfer hybridization and endonuclease S1 mapping experiments were performed to elucidate the structures of the stable, cytoplasmic mRNAs of SV3T3 C120 cells, mRNAs of 3.9 and 3.6 kilobases, containing the small t- and large T-antigen splices, respectively, were transcribed from the internally duplicated early transcription unit. We showed by in vitro translation that these mRNAs encode small t-antigen and the super T-antigen of molecular weight 145,000. Peptide mapping studies of the SV3T3 C120 super T-antigen were consistent with its being derived from an internally duplicated template, since the protein has methionine and cysteine tryptic fingerprints virtually identical to those of normal large T-antigen, with certain methionine peptides present in greater than one molar yield.
Functional analysis of a simian virus 40 super T-antigen
Authors: Clayton C E , Lovett M , Rigby P W
Published: January 2020

The SV3T3 C120 line of simian virus 40-transformed mouse cells synthesizes no large T-antigen of molecular weight 94,000 but instead a super T-antigen of molecular weight 145,000. In the accompanying paper (Lovett et al., J. Virol. 44:963-973, 1982), we showed that the integrated viral DNA segment SV3T3-20-K contains a perfect, in-phase, tandem duplication of 1.212 kilobases within the large T-antigen coding sequences. Our data suggested that this integrated template encodes mRNAs of 3.9 and 3.6 kilobases, the smaller of which directs the synthesis of the super T-antigen of molecular weight 145,000. We transfected the DNA segment SV3T3-20-K into nonpermissive rat cells and into TK- mouse L cells and analyzed the T-antigens and viral mRNAs in the transfectants; these data prove directly the coding assignments suggested previously. The super T-antigen retained the ability to induce morphological transformation, and may even transform better than the wild-type protein. It also retained the ability to bind to the cell-coded p53 protein. Transfection into permissive CV-1 cells showed that the super T-antigen encoded by SV3T3-20-K was incapable of initiating DNA replication at the viral origin. The duplication in SV3T3-20-K thus defines a mutation which separates the transformation and DNA replication functions of large T-antigen. We discuss why such mutations may be selected in transformed cells.
Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16
Authors: Hilkens J , van der Zeijst B , Buijs F , Kroezen V , Bleumink N , Hilgers J
Published: January 2020

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.
Adenovirus-induced alterations of the cell growth cycle: a requirement for expression of E1A but not of E1B
Authors: Braithwaite A W , Cheetham B F , Li P , Parish C R , Waldron-Stevens L K , Bellett A J
Published: January 2020

Mutants dl312, dl314, hr1, and hr3 with mutations in region E1A of adenovirus type 5 were defective for the induction of cell cycle abnormalities detectable by flow cytometry, cell DNA replication, thymidine kinase production, and chromosome aberrations and did not synthesize the viral DNA-binding protein (E2A) in rat cells. dl311, a leaky E1A mutant, induced cell cycle effects at high multiplicity in only one of three experiments, and synthesized the DNA-binding protein. hr7 (E1B) gave a wild-type response in all tests. dl313 was also positive in all tests, although it induced fewer polyploid cells than did wild-type virus, probably because of the leftward extension of the dl313 E1B deletion into E1A. sub315 and sub316, with mutations which also span the E1A-E1B border, synthesized DNA-binding protein, but caused no cell cycle alterations detectable by flow cytometry in rat or mouse cells. Although the participation of other viral early regions cannot be completely excluded, our results suggest that alteration of cell cycle progression is a direct effect of E1A unrelated to its control of other viral early regions, and may be the function of E1A in transformation.
Comparison of the oligosaccharide moieties of the major envelope glycoproteins of the subgroup A and subgroup B avian myeloblastosis-associated viruses
Authors: Hunt L A , Wright S E
Published: January 2020

The nature of the oligosaccharide chains of the major envelope glycoprotein, gp85, from avian myeloblastosis-associated viruses has been examined for the subgroup A and subgroup B viruses replicated in fibroblasts from the same chicken embryos. Pronase-digested glycopeptides from [3H]mannose- or [3H]glucosamine-labeled viruses were analyzed by the combined techniques of gel filtration, endo-beta-N-acetylglucosaminidase digestion, and concanavalin A affinity chromatography. The gp85 protein from these two viruses, and also from another subgroup A avian leukosis virus replicated in the same cells, contained a diverse array of asparagine-linked oligosaccharides of the acidic type [(sialic acid +/- galactose-N-acetylglucosamine)2-4-(mannose)3-N-acetylglucosamine2(+/- fucose)-asparagine], hybrid type (sialic acid +/- galactose-N-acetylglucosamine-(mannose)5,4-N-acetylglucosamine2-asparagine), and neutral type [(mannose)5-9-N-acetylglucosamine2-asparagine], with the more highly branched (tri or tetraantennary or both) acidic-type structures representing the predominant class of oligosaccharide. Minor differences were observed between the gp85 of the subgroup B versus subgroup A viruses.
Metabolic turnover of phosphorylation sites in simian virus 40 large T antigen
Authors: Van Roy F , Fransen L , Fiers W
Published: January 2020

Four (groups of) phosphorylation sites exist in the large T antigen of simian virus 40, and they involve at least two serine and two threonine residues (Van Roy et al. J. Virol. 45:315-331, 1983). All the phosphorylation sites were found to be modified and again dephosphorylated at discrete rates, with phosphoserine residues having the highest turnover rate. The measured half-lives ranged between 3 h (for the carboxy-terminal phosphoserine site) and 5.5 h (for the amino-terminal phosphothreonine site). The influence of four temperature-sensitive A mutations on phosphorylation of large T antigen was also examined. At restrictive temperature, phosphorylation of the carboxy-terminal phosphoserine in mutated large T antigen was found to be particularly impaired. These data emphasize the physiological importance of the latter phosphorylation site.
Epstein-Barr HR-1 Virion DNA Is Very Highly Methylated
Authors: Diala Edward S. , Hoffman Robert M.
Published: January 2020

The virion DNA of Epstein-Barr virus strain HR-1 was found to be methylated to a very large extent, approaching 15%. This extent of methylation is in great contrast to other classes of tumor viruses, and it is the highest that we know of in any mammalian system.
Characterization of the soluble glycoprotein released from vesicular stomatitis virus-infected cells
Authors: Chatis P A , Morrison T G
Published: January 2020

Vesicular stomatitis virus-infected Chinese hamster ovary cells release into the extracellular medium a soluble form of the vesicular stomatitis virus glycoprotein (G protein) termed Gs (Kang and Prevec, Virology 46:678-680, 1971). The properties of this molecule and the cellular site at which it is generated were characterized. By comparing the sizes and the peptide maps of the unglycosylated forms of G and Gs, we found that between 5,000 and 6,000 daltons of the carboxy-terminal end of the G protein is cleaved to generate the Gs molecule. This truncated molecule contains no fatty acid. Gs released from cells grown at 39 degrees C migrated on polyacrylamide gels slightly slower than Gs released at 30 degrees C. The unglycosylated form of Gs also showed this size difference. Furthermore, unglycosylated Gs was resolved into two species upon isoelectric focusing: the relative amounts of the two species depended upon the temperature at which infected cells were incubated. Full-sized unglycosylated virus-associated G also was resolved into two species, but the more basic form predominated at both 30 and 39 degrees C. The appearance of Gs in the extracellular medium depended upon the presence of stable, full-sized G at the cell surface. The amount of Gs released was quantitated in seven different situations in which the migration of G to the cell surface was inhibited. In all cases, the amount of Gs released was also decreased. In addition, incubation of cells surface labeled with 125I resulted in the release of 125I-labeled Gs protein, as well as full-sized G protein. These results suggest that Gs is generated primarily by proteolytic cleavage of plasma membrane-associated G at a site in the molecule just amino terminal to the membrane-spanning region of the molecule.
New Class of Leukemogenic Ecotropic Recombinant Murine Leukemia Virus Isolated from Radiation-Induced Thymomas of C57BL/6 Mice
Authors: Rassart E. , Sankar-Mistry P. , Lemay G. , DesGroseillers L. , Jolicoeur P.
Published: January 2020

We previously reported the establishment of several lymphoid cell lines from X-ray-induced thymomas of C57BL/Ka mice, and all, except one, produce retroviruses (P. Sankar-Mistry and P. Jolicoeur, J. Virol.
35
:270-275, 1980). Biological characterization of five of these new primary radiation leukemia viruses (RadLVs) indicated that they had a B-tropic, fibrotropic, and ecotropic host range and were leukemogenic when reinjected into C57BL/Ka newborn mice. The leukemogenic potential of one isolate (G
6
T
2
) was further assessed and shown to be retained after prolonged passaging on fibroblasts in vitro. Restriction endonuclease analysis of the DNA of four of our new RadLV isolates (G
6
T
2
, Ti-7, Ti-8, and Ti-9) revealed that G
6
T
2
and Ti-7 murine leukemia virus (MuLV) genomes had identical restriction maps, whereas Ti-8 and Ti-9 genomes were different from each other and from the G
6
T
2
and Ti-7 genomes. The physical maps of these genomes were similar to that of known ecotropic MuLV genomes (including the C57BL/Ka endogenous ecotropic MuLV) within their long terminal repeats,
env
, the right portion of
pol
, and the left portion of
gag
. However, a region covering the end of
gag
and the beginning of
pol
was different and showed several similarities with xenotropic MuLV genomes of BALB/c, AKR, and C58 mice previously mapped. Our results suggest that these primary RadLV genomes are recombinants between the parental ecotropic MuLV genome and a nonecotropic (xenotropic) sequence. This nonecotropic
gag-pol
region might be important in conferring the leukemogenic potential to these isolates. Therefore, these RadLVs appear to form a new class of leukemogenic recombinant MuLVs recovered from leukemic tissues of mice. They appear to be distinct from the recombinant AKR mink cell focus-inducing MuLVs which have a dual-tropic host range and harbor xenotropic
env
sequences. To further study the leukemogenic potential of these RadLVs, the genome of one of them (G
6
T
2
) was cloned in Charon 21A as an infectious molecule.
Polyoma virus minichromosomes: characterization of the products of in vitro DNA synthesis
Authors: Gourlie B B , Pigiet V P
Published: January 2020

A two-dimensional (neutral-alkali) agarose gel electrophoretic system was used to separate three families of replicative intermediate (RI) polyoma virus DNA molecules (form I, form II, and form III RIs). Two of these families, form II and III RIs, are the result of artifactual nicking of one of the parental strands of supercoiled RIs (form I RIs) during in vitro replication of soluble minichromosomes. Kinetic studies in vitro showed that the nicked RIs serve as templates for limited DNA synthesis. The nicked species are not converted into normal products, however. The nicking reaction, which appears to be specific for the parental strands, is dependent on magnesium ions and occurs concurrently with the in vitro synthesis of DNA.
Far upstream initiation sites for adenovirus early region 1A transcription are utilized after the onset of viral DNA replication
Authors: Osborne T F , Berk A J
Published: January 2020

Adenovirus early region 1A (E1A) is the first transcription unit expressed after infection. It encodes a protein which controls the expression of all other early viral genes. The E1A mRNAs have one major capped 5' terminus which maps 31 nucleotides downstream from a T-A-T-A sequence (C. Baker and E. Ziff. J. Mol. Biol. 149:189-221, 1981). In addition, a minor set of E1A mRNAs are observed during the early phase of infection which have 5' termini mapping at approximately -160, -185, and -230 relative to the major cap site (Osborne et al., Cell 29:139-148, 1982). Here we report the occurrence of another set of minor E1A mRNAs which were observed exclusively after the initiation of viral DNA replication. These late specific E1A mRNAs had cap sites which mapped at approximately -300, -325, -360, and -375 relative to the major cap site. The appearance of these minor late E1A mRNAs was blocked by the DNA synthesis inhibitor cytosine arabinoside. These same late specific E1A mRNAs were synthesized from E1A-containing plasmids which replicate in monkey cells. This demonstrated that neither late specific adenovirus proteins nor adenovirus-specific chromatin structure was required for the production of the late specific E1A mRNAs. Adenovirus mutants in which the E1A T-A-T-A box region had been deleted also synthesized the corresponding deleted forms of the late specific mRNAs after initiation of DNA replication. These results indicate that the process of DNA replication alters the specificity of E1A transcription initiation in a promoter region which is at least 375 nucleotides in length.
Recombinational junctions of variants of Moloney murine sarcoma virus: generation and divergence of a mammalian transforming gene
Authors: Donoghue D J , Hunter T
Published: January 2020

Different variants of Moloney murine sarcoma virus (MSV) were examined by nucleotide sequencing to compare the junctions between the acquired cellular sequence, v-mos, and the adjacent virus-derived sequences. These variants included 124-MSV, m1-MSV, and HT1-MSV and also the purportedly independent isolate Gazdar MSV. These four strains have an identical 5' junction between the murine leukemia virus env gene and the v-mos gene. This junction lies within the sixth codon of the chimeric env-mos coding region that encodes the transforming gene product. In contrast, at the 3' junction between the v-mos gene and the murine leukemia virus env gene, the three variants examined here were all different. A small deletion was found in the COOH-terminal portion of the m1-MSV env-mos coding region, indicating that the COOH terminus of this transforming gene product must be different from that of 124-MSV or HT1-MSV. The data presented here are consistent with the thesis that a virus closely related to HT1-MSV was the primordial Moloney MSV, and that all other related strains evolved from it by deletion or rearrangement. The variability observed in the Moloney MSV family is discussed in terms of possible mechanisms for the initial capture of mos sequences by the parental retrovirus and also in comparison with other transforming retrovirus families, such as Abelson murine leukemia virus and Rous sarcoma virus.
Purification and properties of a fifth major viral gag protein from avian sarcoma and leukemia viruses
Authors: Pepinsky R B , Vogt V M
Published: January 2020

We have developed procedures for the purification of a 6,000-dalton protein from avian myeloblastosis virus. This protein is a major component of avian myeloblastosis virus, accounting for over 7% of total protein, and thus is equimolar with the other internal structural proteins in virions. As described in the accompanying paper (Hunter et al., J. Virol. 45:885-888, 1983), the results of N-terminal amino acid sequence analysis identify the protein as a product of the gag gene. We suggest denoting this protein as p10, according to nomenclature that is already in use for a previously identified but poorly defined low-molecular-weight protein or proteins of avian sarcoma and leukemia viruses. In virions p10 appears to be located between the core and the membrane. Several of its properties may explain why p10 has not been characterized previously. Among these are its abnormal amino acid composition, its solubility under conditions where most proteins are fixed into sodium dodecyl sulfate-polyacrylamide gels, and the variability in its electrophoretic migration in different avian sarcoma viruses.
UC-1, a new bacteriophage that uses the tonA polypeptide as its receptor
Authors: Lundrigan M D , Lancaster J H , Earhart C F
Published: January 2020

We characterized UC-1, a previously undescribed Escherichia coli phage. UC-1 was observed to have an icosahedral head and a long, flexible, noncontractile tail: its genome consisted of linear double-stranded DNA having a molecular weight of 34 X 10(6). The product of the tonA gene served as at least part of the receptor for UC-1. E. coli tonA strains neither plated nor adsorbed UC-1 well, tonA mutants were selected on the basis of UC-1 resistance, and ferrichrome, a siderophore which utilizes TonA as its receptor, blocked infection. Restriction analyses, DNA-DNA hybridization experiments, and guanine-plus-cytosine determinations demonstrated that UC-1 DNA was unrelated to that of other phages (T1, T5, and phi 80) which employ TonA as a receptor. Also, mutants specifically resistant to UC-1 were isolated. UC-1 may be useful as a probe for investigating TonA, which functions as a receptor for more ligands than any other membrane protein. Study of the resistant mutants may improve our understanding of how phage DNA penetrates the cell envelope.
Genome structure and virion polypeptides of the primate herpesviruses Herpesvirus aotus types 1 and 3: comparison with human cytomegalovirus
Authors: Ebeling A , Keil G , Nowak B , Fleckenstein B , Berthelot N , Sheldrick P
Published: January 2020

Two serologically distinguishable primate herpesviruses, Herpesvirus aotus type 1 and type 3, were examined with regard to their genomes and structural polypeptides. The duplex DNA genomes of these two viruses were found to be essentially identical in molecular weight (Mr approximately equal to 145 X 10(6)) and guanine plus cytosine composition (55%). Both contained unique and inverted repeat nucleotide sequences of the same size and arrangement, which, as judged by DNA-DNA hybridization and restriction enzyme analyses, were at least 95% homologous. In addition, no differences were observed in electrophoretic profiles of virion polypeptides. Because of their great similarity with respect to these criteria, the two viruses ought to be considered independent isolates (or strains) of a single virus, which should be designated H. aotus type 1. The elevated molecular weight and presence of two sets of inverted repeat sequences closely resemble the structure of the human cytomegalovirus genome. However, no sequence homology (less than 5%) nor similarity in virion polypeptides was detected between H. aotus type 1 and human cytomegalovirus.
Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells
Authors: Erwin C , Brown D T
Published: January 2020

The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).
Defective Interference in the Killer System of
Saccharomyces cerevisiae
Authors: Ridley Susan Porter , Wickner Reed B.
Published: January 2020

The K
1
killer virus (or plasmid) of
Saccharomyces cerevisiae
is a noninfectious double-stranded RNA genome found intracellularly packaged in an icosahedral capsid. This genome codes for a protein toxin and for resistance to that toxin. Defective interfering virus mutants are deletion derivatives of the killer virus double-stranded RNA genome; such mutants are called suppressive. Unlike strains carrying the wild-type genome, strains with these deletion derivatives are neither toxin producers nor toxin resistant. If both the suppressive and the wildtype virus are introduced into the same cell, most progeny become toxin-sensitive nonkillers (J. M. Somers, Genetics
74
:571-579, 1973). Diploids formed by the mating of a killer with a suppressive strain were grown in liquid culture, and RNA was extracted from samples taken up to 41 generations after the mating. The ratio of killer RNA to suppressive RNA decreased with increasing generations; by 41 generations the killer RNA was barely detectable. The copy numbers of the suppressive genome and its parental killer were virtually the same in isogenic strains, as were the growth rates of diploid strains containing either virus alone. Therefore, suppressiveness, not being due to segregation or overgrowth by faster growing segregants, is likely due to preferential replication or maintenance of the suppressive genome. Three suppressive viruses, all derivatives of the same killer virus (T. K. Sweeney et al., Genetics
84:
27-42, 1976), did not coexist stably. The evidence strongly indicates that the largest genome of the three slowly suppressed both of the smaller genomes, showing that larger genomes can suppress smaller ones and that suppression can occur between two suppressives. Of 48 isolates of strains carrying the suppressive viruses, 5 had newly detectable RNA species, all larger than the original suppressive genomes. At least seven genes necessary for maintenance of the wild-type killer virus (
MAK
genes) were needed by a suppressive mutant. No effect of
ski
mutations (affecting regulation of killer virus double-stranded RNA replication) on suppressiveness was observed.
Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants
Authors: Gauntt C J , Trousdale M D , Lee J C , Paque R E
Published: January 2020

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.
Transformation of rat cells by cyt mutants of adenovirus type 12 and mutants of adenovirus type 5
Authors: Mak I , Mak S
Published: January 2020

Several mutants with much reduced oncogenicity (spontaneous mutants H12 cyt 52 and H12 cyt 70 and UV-induced mutants H12 cyt 61, H12 cyt 62, and H12 cyt 68) of the highly oncogenic adenovirus type 12 (Ad12) were studied for their ability to transform primary baby rat kidney cells. Four of the mutants showed much reduced capacity to transform cells in vitro, while H12 cyt 61 transformed cells as efficiently as the wild-type virus. Viral gene expression in several cell lines established from cultures infected by cyt mutants was studied, and it was found that viral sequences belonging to the left 16% of Ad12 were always transcribed. These results suggest that the function of the transformed state is not defective in the cyt mutants studied. Heterotypic complementation studies showed that the defect(s) in a cyt mutant can be corrected by an Ad7 function. Ad5 dl 313, with a deletion between 3.5 and 10.5 map units, transformed rat cells only at high multiplicity. These results suggest that the region E1B of adenoviruses may be required for efficient transformation of rat cells.
Coliphage P1 morphogenesis: analysis of mutants by electron microscopy
Authors: Walker J T , Walker D H
Published: January 2020

We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage.
The
ocr
+
Gene Function of Bacteriophages T3 and T7 Counteracts the
Salmonella typhimurium
DNA Restriction Systems SA and SB
Authors: Krüger Detlev H. , Hansen Sigrid , Reuter Monika
Published: January 2020

In host cells containing the
Salmonella typhimurium
DNA restriction-modification systems SA
+
and SB
+
, replication of the
ocr
+
bacteriophages T3 and T7 is not impaired. However,
ocr
(gene 0.3) mutants of these phages are susceptible to DNA restriction and modification by the SA
+
and SB
+
systems.
Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity
Authors: Yonehara S , Yonehara-Takahashi M , Ishii A
Published: January 2020

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.
Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src
Authors: Parsons S J , McCarley D J , Ely C M , Benjamin D C , Parsons J T
Published: January 2020

Transformation of cells by Rous sarcoma virus is mediated by the product of the viral src gene, pp60src. A hybridoma cell line producing an immunoglobulin G3 antibody to pp60src was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1). Mice were immunized with p60src purified from Escherichia coli cells expressing the src gene product. The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virus-transformed cells and recognized an antigenic determinant located in the amino-terminal third of the pp60src protein.
Further studies on the glycosylated gag gene products of Rauscher murine leukemia virus: identification of an N-terminal 45,000-dalton cleavage product
Authors: Naso R B , Stanker L H , Kopchick J J , Ng V L , Karshin W L , Arlinghaus R B
Published: January 2020

A glycosylated 45,000-Mr protein containing Rauscher murine leukemia virus p15 and p12 antigenic sites and tryptic peptides was identified in Rauscher murine leukemia virus-infected cells. This glycoprotein, termed gP45gag, was also shown to contain a single tryptic peptide also present in gPr80gag and its unglycosylated apoprotein precursor Pr75gag, but lacking in Pr65gag or Pr40gag. The presence of this peptide only in viral precursor proteins containing the so-called leader (L) sequence strongly suggests that gPr45gag is an N-terminal fragment of larger glycosylated gag polyproteins, composed of L sequences in addition to p15 and p12. The kinetics of appearance of radiolabeled gPr45gag and its disappearance during chase-incubation is suggestive of a precursor-like role for this intermediate gene product. An observed 27,000-Mr glycosylated polypeptide, termed gP27gag and containing p15 but not p12, p30, or p10 antigenic determinants, is a candidate cleavage product derived from gPr45gag. These observations suggest that gPr45gag and its putative cleavage product gP27gag represent an authentic pathway for intracellular processing of glycosylated core proteins.
Use of Monoclonal Antibodies Against Two 75,000-Molecular-Weight Glycoproteins Specified by Herpes Simplex Virus Type 2 in Glycoprotein Identification and Gene Mapping
Authors: Para Michael F. , Zezulak Kathleen M. , Conley Anthony J. , Weinberger Michael , Snitzer Karen , Spear Patricia G.
Published: January 2020

We produced two monoclonal antibodies that precipitate different glycoproteins of similar apparent molecular weight (70,000 to 80,000) from extracts of cells infected with herpes simplex virus type 2. Evidence is presented that one of these glycoproteins is the previously characterized glycoprotein gE, whereas the other maps to a region of the herpes simplex virus type 2 genome collinear with the region in herpes simplex virus type 1 DNA that encodes gC.
Marek's disease herpesviruses. IV. Molecular characterization of Marek's disease herpesvirus A antigen
Authors: Glaubiger C , Nazerian K , Velicer L F
Published: January 2020

Marek's disease herpesvirus A antigen (MDHV-A) was identified as a 61,000- to 65,000-dalton glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation from the culture medium of both [35S]methionine- and [14C]glucosamine-labeled infected cells by specific rabbit serum directed against MDHV-A. Rigorous identification was accomplished by selective blocking of this specific immunoprecipitation of the glycoprotein with purified MDHV-A that was isolated at its characteristic isoelectric point. These results identify and characterize MDHV-A in terms of the previously determined physical and chemical properties of the antigen. A molecule of similar size was immunoprecipitated from the culture medium of cells infected with herpesvirus of turkeys, extending previous observations about the identity of a potentially important common antigen shared by MDHV and the nonpathogenic vaccine virus, herpesvirus of turkeys.
New mammalian transforming retrovirus: demonstration of a polyprotein gene product
Authors: Rapp U R , Reynolds F H , Stephenson J R
Published: January 2020

A new acute transforming type C retrovirus was isolated from mice inoculated with a virus stock obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/10T1/2 mouse cells. This virus, designated 3611-MSV, transforms embryo fibroblasts and epithelial cells in culture and induces fibrosarcomas in vivo. 3611-MSV is replication defective, requiring a type C helper virus for propagation both in vitro and in vivo. By using endpoint transmission of 3611-MSV to MMCE C17 mouse and FRE 3A rat cells, several nonproductively transformed clonal cell lines have been derived. Pseudotype virus stocks obtained from such clones transform cells in vitro, are highly oncogenic in vivo, and exhibit host range and serological properties that are characteristic of their helper virus component. Analysis of viral antigen expression in 3611-MSV-transformed cells has led to the demonstration of a 90,000-molecular-weight (Mr) polyprotein and a 75,000-Mr probable cleavage product, both containing the amino-terminal murine leukemia virus gag gene proteins p15 and p12. In contrast to gene products of many previously described mammalian transforming viruses, 3611-MSV-encoded polyproteins lack detectable protein kinase activity, and 3611-MSV-transformed cells resemble chemically transformed cell line C3H/MCA-5, from which 3611-MuLV was originally derived, in that they do not exhibit elevated levels of phosphotyrosine. By using molecular hybridization the 3611-MSV transforming gene was found to be distinct from previously described mammalian cellular oncogenic sequences, including c-ras, c-abl, c-fes, c-fms, c-sis, and c-mos.
Activation of Nonexpressed Endogenous Ecotropic Murine Leukemia Virus by Transfection of Genomic DNA into Embryo Cells
Authors: McCubrey James , Risser Rex
Published: January 2020

We studied the infectivity of endogenous ecotropic murine leukemia virus genomes contained in high-molecular-weight DNA prepared from virus-free cells of the AKR-2B line, and from RF, BALB/c, B6, and (BALB/c × B6)F
1
mouse embryo cells. When DNA prepared from virus-free AKR-2B cells was transfected into NIH-3T3 cells, no virus-positive cultures were observed, a result consistent with previous reports. However, when DNAs from virus-free AKR-2B cells or virus-free cells containing the RF/J or BALB/c ecotropic proviruses were transfected into chicken embryo cells that were then cocultivated with SC-1 (mouse) cells, virus-positive cultures were recovered. The specific infectivities of the AKR provirus(es) contained in virus-free cells and the molecularly cloned Akv-1 provirus were similar when chicken embryo cells were used as primary recipients. Virus-positive cultures were also observed when secondary mouse embryo cells were used as recipients for DNA from virus-free AKR-2B and RF/J cells. The transfected chicken embryo-SC-1 cultures produced XC-positive murine leukemia virus that is N-tropic. Virus-positive recipient cultures were observed 10- to 100-fold more frequently when AKR-2B DNA was used than when BALB/c DNA was used as the donor DNA. Our studies indicate that some nonexpressed ecotropic murine leukemia virus proviruses are activated upon transfection into chicken embryo cells. Such studies suggest that there are different factors governing the expression of murine leukemia virus after transfection into established cell lines (NIH-3T3) and into nonestablished secondary cultures (chicken and mouse).
Topography of simian virus 40 A protein-DNA complexes: arrangement of protein bound to the origin of replication
Authors: Tegtmeyer P , Lewton B A , DeLucia A L , Wilson V G , Ryder K
Published: January 2020

DNA binding regions I, II, and III at the origin of replication have different arrangements of A protein (T antigen) recognition pentanucleotides. The A protein also protects each region from DNase in distinctly different patterns. Footprint and fragment assays led to the following conclusions: (i) in some cases a single recognition pentanucleotide is sufficient to direct the binding and accurate alignment of A protein on DNA; (ii) the A protein binds within isolated region I or II in a sequential process leading to multiple overlapping areas of DNase protection within each region; and (iii) the 23-base pair span of recognition sequences in region II allows binding and protection of a longer length of DNA than the 23-base pair span in region I. We propose a model of protein binding that addresses the problem of variations in the arrangement of pentanucleotides in regions I and II and explains the observed DNase protection patterns. The central feature of the model requires each protomer of A protein to bind to a pentanucleotide in a unique direction. The resulting orientation of protein would protect more DNA at the 5' end of the 5'-GAGGC-3' recognition sequence than at the 3' end. The arrangement of multiple protomers at the origin of simian virus 40 replication is discussed.