bluuebunny/crossref_metadata_2025 huggingface dataset:
Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion
Authors: Lemaster S , Roizman B
Published: January 2020

The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component.
Characterization of tau antigens isolated from uninfected and simian virus 40-infected monkey cells and papovavirus-transformed cells
Authors: Simmons D T
Published: January 2020

Tau antigens (also known as cellular or nonviral tumor antigens) were detected in uninfected and simian virus 40-infected monkey cells after immunoprecipitation with serum from hamsters bearing simian virus 40-induced tumours (anti-T serum). These two proteins (56,000 daltons) were digested to similarly sized peptides with various amounts of Staphylococcus aureus V8 protease. The Tau antigen isolated from infected monkey cells was closely related but was not identical to the corresponding protein from human cells transformed by simian virus 40, as determined by two-dimensional mapping of their methionine-labeled tryptic peptides. Hamster cells transformed by various primate papovaviruses (simian virus 40, BK virus, and JC virus) synthesized indistinguishable Tau antigens, as determined by two-dimensional peptide mapping. When tested by the same procedure, these proteins and the ones made in monkey and human cells were found to be related to the Tau antigens isolated from simian virus 40-transformed mouse and rat cells. Based on these results, an "evolutionary tree" was constructed to show the relationship among the methionine-containing tryptic peptides of all of these proteins.
Abrogation of radiation leukemia virus-induced lymphomagenesis by antisera to thymotropic but not to ecotropic or dual-tropic viruses
Authors: Haas M , Jongstra J
Published: January 2020

Leukemia induction by culture-grown thymotropic radiation leukemia virus or by tumor-derived virus present in cell-free tumor extracts was abrogated by incubation of either virus with anti-thymotropoc virus serum, but not by antiserum raised against ecotropic or dual-tropic (mink cell focus-inducing type) viruses that were isolated from radiation leukemia virus-induced thymic leukemias. Thus, virus similar or identical to the cultured thymotropic leukemogenic species may also be the major biologically active principle in tumor-derived extracts, even though the latter also contain viruses of the dual-tropic, mink cell focus-inducing type class.
Evidence for heterogeneity in populations of T5 bacteriophage. II. Some particles are unable to inject their second-step-transfer DNA
Authors: Hulen C , Labedan B , Legault-Demare J
Published: January 2020

A new class of bacteriophage was characterized in purified T5 stocks. Regardless of the host cell, these phages were irreversibly blocked at the first-step-transfer stage under conditions in which whole DNA injection normally takes place. However, they expressed their first-step-transfer functions. These observations confirmed the previously established heterogeneity of T5 bacteriophage populations and provided a new way to define a phage function necessary to release the blocking of T5 DNA injection at the first-step-transfer stage.
Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells
Authors: Krzyzek R A , Mitchell R L , Lau A F , Faras A J
Published: January 2020

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts. The distribution of src protein kinase activity in the cytosol and particulate cell fractions was identical to that of pp60src, indicating no detectable differences in the activity of cytosol- and particulate-associated pp60src. When subcellular components of the cell were fractionated by discontinuous sucrose gradient centrifugation, similar amounts of both pp60src and src protein kinase activity cosedimented with the plasma membrane fractions from both transformed and revertant vole cells, as well as from ASV-infected chicken embryo fibroblasts. src protein kinase activity associated with plasma membrane fractions prepared from vole cells and ASV-infected chicken embryo fibroblasts was resistant to extraction with high salt concentrations, but partial elution was achieved with nonionic detergent. Thus, in both transformed and morphologically reverted vole cells, pp60src is intimately associated with the plasma membrane. Since transforming virus can be rescued from revertant vole cells by fusion to chicken embryo fibroblasts, revertant vole cell pp60src is capable of inducing morphological transformation. Thus, although the data presented herein suggest that transformation requires the association of pp60src with the plasma membrane, the binding of pp60src to the plasma membrane per se is insufficient to induce morphological transformation and requires the additional interaction with a specific target membrane protein which appears to be defective in revertant vole cells.
Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus
Authors: Kung H J , Fung Y K , Majors J E , Bishop J M , Varmus H E
Published: January 2020

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.
Simian virus 40 transcriptional complexes incorporate mercurated nucleotides into RNA in vitro
Authors: Chikaraishi D M , Danna K J
Published: January 2020

Simian virus 40 Sarkosyl transcriptional complexes incorporated mercurated nucleotide precursors into preinitiated RNA chains. Synthesis with mercurated precursors was three- to fivefold slower than with nonmercurated nucleotides (12 to 20 pmol per 10(6) cells per h at 25 degrees C), and 50 to 70% of the RNA product bound specifically to sulfhydryl-agarose. The amount of mercurated CMP incorporated into RNA was measured by specific binding of [35S]cysteine to the mercury residues. More than 90% of the mercurated RNA hybridized to simian virus 40 DNA.
Lipopolysaccharide induces retroviral antigen expression in 129/J mouse lymphocytes: evidence for assembly of a defective viral particle
Authors: Jongstra J , Moroni C
Published: January 2020

In contrast to those of many other mouse strains, spleen cell cultures of 129/J mice do not release reverse transcriptase activity into the supernatant upon stimulation with bacterial lipopolysaccharide. We report here that lipopolysaccharide induced the expression of intracellular viral proteins in 129/J spleen cells. Furthermore, we found that stimulated spleen cells released retroviral particles. We conclude that 129/J mice are inducible with lipopolysaccharide but that the virus produced is a defective particle deficient in reverse transcriptase activity.
Comparative analysis: intracellular precursor polyproteins of baboon endogenous retroviruses and human viral isolate HL23V
Authors: Whiteley S A , Naso R B
Published: January 2020

Intracellular precursor polyproteins of three baboon endogenous retrovirus (BaEV) isolates, m7, 455K, and BILN, were compared with the intracellular proteins of the type C human isolated HL23V by radioimmunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide analysis. Human and canine cells infected with m7-BaEV and canine thymus cells infected with BILN-BaEV were characterized by identical precursor polyproteins Pr85gag, Pr70-71gag, Pr65gag, and gPr85env. Canine cells infected with 455K-BaEV consistently showed a slightly different pattern of precursor polyproteins. These included Pr85gag, Pr70gag, Pr67gag, and gPR85env. By tryptic digest mapping of peptides containing [3H]leucine, m7-BaEV and 455K-BaEV were shown to be highly related. By comparison, mapping studies showed that BILN-BaEV was less highly related to m7-BaEV than ws 455K-BaEV. Differences in these related BaEV isolates presumably reflected virus-specific differential cleavage of core protein precursors or alterations in polyprotein primary structure or both. Chase-incubated cells infected with BaEV also contained a stable, p28-related polyprotein termed P72gag. This polyprotein migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than the major core protein precursor Pr70-71gag and appeared to arise by posttranslational modification of Pr70-71gag. Immunoprecipitation of extracts of HL23V-infected cells with antisera to simian sarcoma-simian-associated virus proteins and BaEV proteins confirmed that these cells contained two unrelated viral components, one that was similar to m7-BaEV or BILN-BaEV and a second that was related to simian sarcoma-simian-associated virus. Tryptic digest mapping of BaEV and HL23V prcursor polyproteins suggested that the BaEV-like component of HL23V weas more closely related to m7-BaEV than to 455K-BaEV or BILN-BaEV.
Nucleotide sequence changes in polyoma ts-a mutants: correlation with protein structure
Authors: Deininger P L , LaPorte P , Friedmann T
Published: January 2020

The mutations in three polyoma ts-a mutants have been determined. Two mutants, ts-25 and ts-52, have different single-base changes at the same position (2883) in the early region corresponding to a conserved glycine residue very near the C-terminus of the polyoma large T antigen. Mutant ts-48 has a single-base change at position 2341, as well as a second change at position 1228, in the region of large T antigen shared with medium T antigen.
Cloning of herpes simplex virus type 1 sequences representing the whole genome
Authors: Goldin A L , Sandri-Goldin R M , Levine M , Glorioso J C
Published: January 2020

Sequences representative of the whole genome of herpes simplex virus type 1 (HSV-1) strain KOS were cloned in the plasmid vector pBR325 in the form of EcoRI-generated DNA fragments. The cloned fragments were identified by digestion of the chimeric plasmid DNA with restriction enzymes EcoRI or EcoRI and BglII followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested HSV-1 virion DNA. The cloned fragments showed the same migration patterns as the corresponding fragments from restricted virion DNA, indicating that no major insertions or deletions were present. The presence of HSV-1 sequences in the chimeric plasmids was confirmed by hybridization of plasmid DNA to HSV-1 virion DNA. Additionally, some of the cloned fragments were shown to be biologicaly active in that they efficiently rescued three HSV-1 temperature-sensitive mutants in cotransfection marker rescue experiments.
Presence of the cap-binding protein in initiation factor preparations from poliovirus-infected HeLa cells
Authors: Hansen J , Ehrenfeld E
Published: January 2020

Crude preparations of initiation factors from mock-infected and poliovirus-infected HeLa cells were analyzed for the presence of proteins which could be cross-linked to the 5' cap group of mRNA. A protein having an apparent molecular weight of 26,000, similar to the cap-binding protein in rabbit reticulocytes described by Sonenberg and Shatkin (Proc. Natl. Acad. Sci. U.S.A. 75:4843-4847, 1978), was found in the ribosomal salt wash from both uninfected and infected cells. Cross-linking of this polypeptide was inhibited by the cap analog m7GMP. In addition, cross-linking of a protein having an approximate molecular weight of 60,000 was similarly inhibited by cap analog. The smaller cap-binding protein fractionated in a 0 to 40% ammonium sulfate precipitate of ribosomal salt wash; the larger protein was found in the 40 to 70% ammonium sulfate fraction. Although the cap-binding proteins were present in both mock-infected and poliovirus-infected ribosomal salt wash, only preparations from uninfected HeLa cells were able to restore translation of capped vesicular stomatitis virus mRNA by extracts prepared from poliovirus-infected cells.
Virus-coded DNA endonuclease from avian retrovirus
Authors: Golomb M , Grandgenett D P , Mason W
Published: January 2020

Reverse transcriptase from avian retrovirus has a physically associated DNA endonuclease with novel substrate and cofactor requirements. A similar endonuclease activity copurifies with pp32, a protein from viral cores that has been identified with the non-alpha region of the beta subunit of reverse transcriptase. Several temperature-sensitive mutants of avian retrovirus with thermolabile DNA polymerase were tested for thermal sensitivity of their DNA endonuclease activity. Two pol mutants of Rous sarcoma virus, ts335 and ts337, had thermolabile DNA endonuclease; a temperature-resistant revertant of ts335 had a heat-stable DNA endonuclease. DNA endonuclease is therefore a product of the pol gene and an integral part of the reverse transcriptase. A second class of pol mutants, typified by ts568 and ts553, had thermolabile DNA polymerase, but heat-stable DNA endonuclease.
Immunological Cross-Reactivity Between Simian Virus 40 Large T Antigen and D2 Hybrid T Antigen
Authors: Baumann Ernst A. , Stedman Deborah , Raptis Leda , Fuks Abraham , Hand Roger
Published: January 2020

A specific antiserum was raised in rabbits against D2 hybrid T antigen that had been purified from HeLa cells infected with the adenovirus/simian virus 40 hybrid, Ad2
+
D2. The specificity of this serum was compared with that of a conventional hamster antiserum against simian virus 40-induced tumors by immunoprecipitation and by a new radioimmune assay that can detect nanogram quantities of D2 hybrid T antigen.
Virus-associated RNAs of naturally occurring strains and variants of group C adenoviruses
Authors: Mathews M B , Grodzicker T
Published: January 2020

We compared the sequences of the virus-associated (VA) RNAs of group C adenoviruses, serotypes 1, 2, 5, and 6, and of three variants of adenovirus type 2 (Ad2) selected for loss of the BamHI restriction site in the VA RNAI gene. In the naturally occurring strains. VA RNAI exists in two forms which differ by two nucleotides: one form is found in Ad2 and Ad6, and the other is found in Ad1 and Ad5. There are three sites of variation in Va RNAII, the Ad1, Ad2, and Ad5 forms each differing from Ad6 VA RNAII at one of the positions. One of the selected variants has a four-base duplication within the BamHI cleavage site, whereas the two others have acquired a VA RNAI sequence indistinguishable from that of Ad5. The findings are interpreted in terms of the secondary structures of the VA RNAs and the interrelationships among the viruses.
Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions
Authors: Newcomb W W , Brown J C
Published: January 2020

Vesicular stomatitis virus was extracted with 60 mM octylglucoside in the absence of salts and in the presence of 0.5 M NaCl. The resulting extracted virus particles were examined by electron microscopy, and the proteins present were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extraction in the absence of salts yielded subviral structures which we cell "skeletons" as originally suggested by Cartwright et al. (J. Gen. Virol. 7:19-32, 1970). The skeletons contained the viral N, M, and L proteins, but they lacked the glycoprotein (G) entirely. Morphologically, the skeletons resembled intact vesicular stomatitis virus but they were slightly longer and smaller in diameter. Like native vesicular stomatitis virus, skeletons were found to have lateral striations spaced 5.0 to 6.0 nm apart along the length of the structure. In contrast to extraction in the absence of NaCl, extraction of vesicular stomatitis virus with 60 mM octylglucoside in the presence of 0.5 M NaCl yielded highly extended viral nucleocapsids in which N was the predominant protein; no M or G proteins could be detected. These results support the view that the M protein is involved in maintaining the nucleocapsid in the compact form found in native virions.
Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum
Authors: Ozer H L , Slater M L , Dermody J J , Mandel M
Published: January 2020

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.
Growth and Persistence of Polyoma Early Region Deletion Mutants in Mice
Authors: McCance D. J.
Published: January 2020

Replication of early region deletion mutations in polyoma in the kidneys of mice was in most cases reduced by 10- to 10,000-fold, as compared with wild-type polyoma, and the mutants failed to produce the persistent infection observed with wild-type polyoma.
Biochemical Studies on Adenovirus Multiplication
Authors: Warocquier Rita , Samaille Jean , Green Maurice
Published: January 2020

The synthesis of infectious virus is reduced by 99% in adenovirus type 2-infected KB cells maintained at 42 C in suspension. Studies to delineate the steps in virus biosynthesis blocked at 42 C revealed the following. (i) The inhibition of ribosomal ribonucleic acid (RNA) synthesis and the block in the conversion of ribosomal RNA precursors to ribosomal RNA which normally occur in infected cells at 37 C was accentuated at 42 C. (ii) The same species of early and late viral messenger RNA were synthesized at 37 and 42 C. (iii) The shift from host cell deoxyribonucleic acid (DNA) synthesis to viral DNA replication occurred earlier at 42 C than at 37 C. These findings indicate that the thermosensitive block(s) in virion formation does not occur at the level of viral DNA replication or transcription of viral mRNA but probably involves the synthesis of late viral proteins or the maturation of the virion.
Morphogenesis of Venezuelan Equine Encephalomyelitis Virus
Authors: Bykovsky A. F. , Yershov F. I. , Zhdanov V. M.
Published: January 2020

Morphogenesis of Venezuelan equine encephalomyelitis virus was studied by means of electron microscopy. Virus-specific structures (factories, viroplasts) were found at early stages of infection; these structures were composed of fibrillar and cylindrical formations, aggregates of ribosomes, and viral nucleoids. The latter emerged from fibrillar and cylindrical structures. Aggregates of viral nucleoids were found in the cytoplasm and occasionally in the nuclei of virus-infected cells. Viral envelopes and mature virions were formed on the cell membranes and on the membranes of intracellular vacuoles. Anomalous forms of virions (both polygenomic and oligogenomic) were observed.
Capsid Mosaics of Intermediate Strains of Human Adenoviruses
Authors: Norrby Erling
Published: January 2020

Antisera against isolated capsid components of intermediate adenovirus strains, types 3-16 (the San Carlos agent) and 15-9 and of “parental” prototype strains were compared in neutralization tests, hemagglutination-inhibition (HI) tests employing soluble and virion-associated hemagglutinin as antigens, and by electron microscopy. Hexons of the intermediate strains were found to be similar to, but not identical to, those of the prototype strains with which a cross-reaction occurred in neutralization tests (types 3 and 15). In contrast, fibers of intermediate strains displayed characteristics relating them to the corresponding components of prototype strains to which a relationship has been found in HI tests. Fibers (and possibly even pentons) of types 9 and 15-9 appeared to be identical, whereas fibers of types 3-16 and 16 displayed antigen specificities of both common and unique nature.
Analysis of the DNA-terminal protein from different serotypes of human adenovirus
Authors: Rekosh D
Published: January 2020

The DNA-terminal proteins from adenovirus type 12, type 3, and type 5 were analyzed after labeling in vitro with 125I by the chloramine T method, and were shown to be serotype specific. We also studied the kinetics of cleavage by alkali of the terminal protein-DNA linkage and showed that the half-time of cleavage with either 0.1 M NaOH or 0.5 M piperidine at 37 degree C is about 15 to 30 min. The substitution of the volatile base piperidine for NaOH in this procedure provided a useful tool for rapid analysis of the labeled protein.
Integration of Rous sarcoma virus DNA into chicken embryo fibroblasts: no preferred proviral acceptor site in the DNA of clones of singly infected transformed chicken cells
Authors: Lerner T L , Skalka A M , Hanafusa H
Published: January 2020

We analyzed retroviral integration into a host genome by using avian sarcoma virus infection of natural target cells under conditions where secondary integration via virus spread was inhibited. This was accomplished by using the noninfectious pol- env- alpha variant of the Bryan high-titer strain of Rous sarcoma virus. A total of 12 independent Bryan high-titer Rous sarcoma virus-transformed chicken embryo fibroblast clones were obtained and mapped by using restriction endonucleases. Provirus-cell junction fragments were identified with appropriate hybridization probes. We found that expression of the viral genes could occur after proviral integration at many sites on the chicken genome and that there was no apparent preference for specific integration sites.
Proteins containing only half of the coding information of early region 1b of adenovirus are functional in human cells transformed with the herpes simplex virus type 1 thymidine kinase gene and adenovirus type 2 DNA
Authors: Klessig D F , Quinlan M P , Grodzicker T
Published: January 2020

We introduced into tk- human 143 cells adenovirus type 2 (Ad2) genes by transformation with a plasmid (p711) containing both Ad2 sequences and the herpes simplex virus type 1 (HSV-1) tk gene. p711 contained approximately the left 8% of the Ad2 genome inserted in the HindIII site of pBR322, whereas the fragment of HSV-1 containing the tk gene was inserted in the BamHI site. Three tk+ cell lines were isolated after selection in HAT medium. The arrangement of viral sequences in the three transformants was analyzed by restriction endonuclease digestion and filter hybridization. All three lines contained a single insertion of Ad2 DNA which was present at approximately one copy per cell. The arrangement of Ad2 sequences in these lines was identical to that found in the linear p711 DNA used in the transformation. S1 analysis of the Ad2-specified RNA from two of these lines indicated that the early region 1a mRNA's were synthesized, though in lower amounts than found in lytic infections. These cell lines contained only the left half of early region 1b (4.6 to 11.2), which encoded the 5' portion of the 1b mRNA's. A complex pattern of 1b RNAs was made in these cell lines. Transcription of most of these RNAs began at or near the 1b promoter and proceeded through the 1b sequences into the flanking pBR322, HSV-1, or host sequences. Since many of the RNAs were terminated or spliced in the HSV-1 (anti-sense strand) or pBR322 sequences, new RNA processing sites must be used in the formation of these mRNA's. All three lines fully complemented the 1a deletion mutant Ad5 dl312. Surprisingly, these lines also permitted the growth of 1b deletion mutants (Ad5 dl313 and Ad5 dl434), although the complementation was not always complete. Presumably the new gene product(s) which contained only part of the 1b gene provided most of the essential function(s) required for viral multiplication. Alternatively, the 1b 19-kilodalton protein which was entirely encoded by the adenovirus sequences present in these cell lines was sufficient for viral growth even in the absence of the 1b 55-kilodalton protein.
Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation
Authors: Templeton D , Eckhart W
Published: January 2020

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.
Continuing germ line integration of AKV proviruses during the breeding of AKR mice and derivative recombinant inbred strains
Authors: Steffen D L , Taylor B A , Weinberg R A
Published: January 2020

The gel electrophoresis-hybridization technique of Southern was used to analyze genetically transmitted proviruses coding for the AKV strain of murine leukemia virus. We were able to identify the restriction endonuclease EcoRI fragments containing two previously unidentified, genetically transmitted AKV proviruses of AKR mice. Comparison of different sublines of AKR mice revealed considerable heterogeneity in their complement of germ line proviruses. This heterogeneity provides evidence that the provirus complement of AKR mice is not stable. Rather, the number of genetically transmitted proviruses increases during inbreeding. Examination of a series of sublines of the C3H strain indicated that this amplification is dependent on viremia. We estimate that, in viremic strains of mice, one new provirus becomes fixed in the germ line every 15 to 30 years.
DNA Synthesis and Gene Expression in
Bacillus subtilis
Infected with Wild-Type and Hypermodification-Defective Bacteriophage SP10
Authors: Witmer Heman , Franks Michael
Published: January 2020

A hypermodified base (Y-Thy) replaces 20% of the thymine (Thy) in mature DNA of
Bacillus subtilis
phage SP10. Two noncomplementing hypermodification-defective (
hmd
) mutants are described. At 30°C,
hmd
phage carried out a normal program, but at temperatures of ≥37°C, the infection process was nonproductive. When cells were infected at 37°C with
hmd
phage, DNA synthesis started at its usual time (12 min), proceeded at about half the normal rate for 6 to 8 min, and then stopped or declined manyfold. All, or nearly all, of the DNA made under
hmd
conditions consisted of fully hypermodified parental DNA strands H-bonded to unhypermodified nascent strands. The reduced levels of DNA synthesis observed under
hmd
conditions were accompanied by weak expression of late genes. A sucrose gradient analysis of SP10
hmd
+
replicating DNA intermediates was made. Two intermediates, called VG and F, were identified. VF consisted of condensed DNA complexed to protein; VF also contained negatively supercoiled domains covalently joined to relaxed regions. F was composed of linear concatenates from which mature DNA was cleaved. None of those intermediates was evident in cells infected at 37°C with
hmd
phage. Shiftup experiments were performed wherein cells infected with
hmd
phage at 30°C were shifted to 37°C at a time when replication was well under way. DNA synthesis stopped or declined manyfold 10 min after shiftup. The
hmd
DNA made after shiftup was conserved as a form sedimentationally equivalent to the F intermediate, but little mature DNA was evident. It is proposed that Y-Thy is required for replication and DNA maturation because certain key proteins involved with these processes interact preferentially with hypermodified DNA.
Most of the Murine Leukemia Virus Sequences in the DNA of NIH/Swiss Mice Consist of Two Closely Related Proviruses, Each Repeated Several Times
Authors: Steffen David L. , Mural Richard , Cowing Deborah , Mielcarz Jane , Young Janet , Roblin Richard
Published: January 2020

The structure of the endogenous murine leukemia virus (MuLV) sequences of NIH/Swiss mice was analyzed by restriction endonuclease digestion, gel electrophoresis, and hybridization to an MuLV nucleic acid probe. Digestion of mouse DNA with certain restriction endonucleases revealed two classes of fragments. A large number of fragments (about 30) were present at a relatively low concentration, indicating that each derived from a sequence present once in the mouse genome. A smaller number of fragments (one to five) were present at a much higher concentration and must have resulted from sequences present multiple times in the mouse genome. These results indicated that the endogenous MuLV sequences represent a family of dispersed repetitive sequences. Hybridization of these same digested mouse DNAs to nucleic acid probes representing different portions of the MuLV genome allowed construction of a map of the sites where restriction endonucleases cleave the endogenous MuLV sequences. Several independent recombinant DNA clones of endogenous MuLV sequences have been isolated from C3H mice (Roblin et al., J. Virol.
43:
113-126, 1982). Analysis of these sequences shows that they have the structure of MuLV proviruses. The sites at which restriction endonucleases cleave within these proviruses appeared to be similar or identical to the sites at which these nucleases cleaved within the MuLV sequences of NIH/Swiss mice. This identity was confirmed by parallel electrophoresis. We conclude that the apparently complex pattern of endogenous MuLV sequences of NIH/Swiss mice consists largely of only two kinds of provirus, each repeated multiple times at dispersed sites in the mouse genome.
Sequence of the Long Terminal Repeat and Adjacent Segments of the Endogenous Avian Virus Rous-Associated Virus 0
Authors: Hughes Stephen H.
Published: January 2020

Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U
3
region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U
3
region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward
gag
) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the
env
gene and U
3
is approximately 190 bases in length. Essentially this entire segment is present between
env
and
src
in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between
env
and
src
in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to
env
. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between
src
and the LTR. This arrangement has implications for the mechanism by which
src
was acquired. The region which encoded the gp37 portion of
env
appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of
env
imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.
Effect of interferon on the replication of mink cell focus-inducing virus in murine cells: synthesis, processing, assembly, and release of viral proteins
Authors: Bilello J A , Wivel N A , Pitha P M
Published: January 2020

Treatment of mink cell focus-inducing (MCF) virus (isolate AK-13) producing SC-1 cells with mouse fibroblast interferon (150 to 600 U/ml) led to a 100-fold decrease in the release of infectious virus, whereas there was a 2.5- to 10-fold decrease in various parameters of virus particle release. Analysis of labeled virion proteins indicated that a temporal change in virion protein composition occurred after interferon treatment. After a 24-h exposure of chronically infected cells to interferon, the virions produced contained a 85,000-dalton glycoprotein (apparently of nonviral origin) which was in excess of the virus envelope glycoprotein gp70. Particles produced from cells treated with interferon for 32 to 48 h were nearly devoid of gp70 and contained substantially lower quantities of p30. Intracellular processing of viral precursor polyproteins to the mature virion structural proteins was not altered in the presence of interferon. However, an accumulation of the viral p30 and p12E proteins was observed in interferon-treated cells, consistent with an increase in cell-associated virions. Immunoprecipitation analysis of the tissue culture fluids from [35S]methionine-labeled control and interferon-treated cells revealed marked decrease in p30 and p15E/p12E released after interferon treatment. In contrast, gp70 did not accumulate in interferon-treated cells, but was released into the culture medium in a form that was neither pelletable nor associated with p15E/p12E.
The transforming proteins of PRCII virus and Rous sarcoma virus form a complex with the same two cellular phosphoproteins
Authors: Adkins B , Hunter T , Sefton B M
Published: January 2020

P105 and P110, the presumptive transforming proteins of PRCII avian sarcoma virus, have been found to be present in transformed chicken cells in two forms: as monomers and as part of a complex which contains both a 50,000-dalton and a 90,000-dalton cellular phosphoprotein. The 90,000-dalton cellular protein was found to be identical to one of the proteins in chicken cells whose synthesis is induced by stress. The 50,000-dalton protein was found to contain phosphotyrosine when isolated from the complex and therefore may be a substrate for the tyrosine protein kinase activity which is associated with P105 and P110. These same two cellular phosphoproteins have previously been shown to be present in a complex with pp60src, the tyrosine protein kinase which is the transforming protein of Rous sarcoma virus. However, not all avian sarcoma virus transforming proteins with associated tyrosine protein kinase activities form a complex efficiently with these cellular proteins. Little if any of P90, the putative transforming protein of Yamaguchi 73 virus, was found in a complex with the 50,000-dalton and 90,000-dalton cellular phosphoproteins.
Polypurine tract adjacent to the U3 region of the Rous sarcoma virus genome provides a cis-acting function
Authors: Sorge J , Hughes S H
Published: January 2020

The v-src coding region was deleted from cloned Rous sarcoma virus DNA, and the deleted clones were tested for infectivity by transfection. All of the coding region, as well as most of the sequences lying between v-src and the unique 3' region (U3), could be deleted without affecting viability. However, at least 9 and at most 29 of the nucleotides in the purine-rich tract adjacent to U3 were necessary for growth, even in the presence of a helper virus. It is concluded that these nucleotides (the polypurine tract) provide a cis-acting function necessary for retrovirus replication.
Endo-N-acetylneuraminidase associated with bacteriophage particles
Authors: Kwiatkowski B , Boschek B , Thiele H , Stirm S
Published: January 2020

A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-). phi 1.2 is specific for the host capsular polysaccharide (colominic acid). The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues. Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate. Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated. No NeuNAc monomers, however, are formed. The capsules of E. coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme. Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D. E. Bradley, Bacteriol. Rev. 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes. By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes.
Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro
Authors: Tuschall Deborah M. , Hiebert Ernest , Flanegan James B.
Published: January 2020

The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate· oligouridylate [oligo(U)], polycytidylate · oligoinosinate, and polyinosinate· oligocytidylate templates to about the same extent. The observed activity on polyuridylate· oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg
2+
concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested.
Two colinear and spliced viral transcripts are present in non-virus-producing benign and malignant neoplasms induced by the shope (rabbit) papilloma virus
Authors: Nasseri M , Wettstein F O , Stevens J G
Published: January 2020

The nature of Shope virus-specific RNA was investigated in non-virus-producing Shope (rabbit) papilloma virus-induced benign and malignant domestic rabbit tumors and in a cell line derived from the VX-7 transplantable carcinoma. RNA transfer (Northern) blot analysis of polyadenylated RNA isolated from whole cell extracts of all three sources was resolved into two major bands of 1.3 and 2.0 kilobases. Two additional minor bands of 3.5 and 4.8 kilobases could be seen in some analyses. RNA of the VX-7 cell line was further separated into nuclear, cytoplasmic, and polysomal fractions. The cytoplasmic fraction only contained the 1.3- and 2.0-kilobase species, and virus-specific RNA was found to be associated with polysomes. The two major transcripts present in VX-7 cells were mapped by hybridization of RNA transfer blots with subgenomic probes, and the results indicated that both transcripts are spliced and are most likely colinear. Our results are consistent with the suggestion that the two viral transcripts are necessary for induction and maintenance of the neoplasms.
Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA
Authors: Khan A S , Rowe W P , Martin M A
Published: January 2020

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.