bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphism of avian sarcoma virus src proteins
Authors: Beemon K , Hunter T , Sefton B M
Published: January 2020

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.
Gene 24-controlled osmotic shock resistance in bacteriophage T4: probable multiple gene functions
Authors: Leibo S P , Kellenberger E , Kellenberger-van der Kamp C , Frey T G , Steinberg C M
Published: January 2020

By use of mixed infections with conditional lethal mutations in the head genes and an osmotic shock-resistant mutant we have demonstrated that osmotic shock resistance is controlled by gene 24. Using acrylamide gel electrophoresis combined with the "immune replicate" technique, we confirmed the positions of gene products 24 and 24* (P24 and P24*). In this paper we have still used the notation "P24," etc., for designating the product of gene 23, etc., although we prefer and use in general the designation "gp23" as introduced by Casjens and King (Annu. Rev. Biochem. 44:585, 1975). The reason for using the old notation is because the illustrations were prepared several years ago.) P24 ts showed a significantly slower mobility. Both osmotic shock-resistant and -sensitive mature phages contain 24*. Giants constructed with the Osr phage showed the same surface lattice as normal phage. Through temperature-shift experiments with 24(tsL90) alone and in combinations, we studied the phages which are matured after the shift to permissive temperature in the absence of new protein synthesis. Our results strongly suggest that only a fraction of the total phage complement of gene 24-controlled proteins is involved in determining the phenotype of shock resistance, and the remainder is necessary to mature the head.
Differential translation in normal and adenovirus type 5-infected human cells and cell-free systems
Authors: Cherney C S , Wilhelm J M
Published: January 2020

When uninfected or adenovirus 5-infected KB cells are exposed to hypertonic medium, the incorporation of radioactive amino acids into protein decreases in both, but more severely in the uninfected cells. Although the effect of hypertonic medium on the synthesis of specific polypeptides varies, the translation of viral polypeptides as a class is less inhibited. The same patterns of proteins are synthesized regardless of the solute used in the hypertonic medium. The mechanism by which hypertonic conditions exert their effect on whole cells was investigated in K cell-free systems. It was possible to simulate the differential patterns of protein synthesis obtained in whole cells in hypertonic medium by increasing ion concentrations in cell-free extracts which are capable of initiating polypeptide chains on exogenous templates. However, in cell lysates which only elongate proteins, the same patterns were not obtained. Certain host and viral polypeptides displayed striking responses to increased ionic conditions in whole cells and cell-free systems. The synthesis of a host 44K protein, actin, appeared to be most sensitive; lower-molecular-weight proteins were fairly resistant. Among the viral proteins, the synthesis of 100K was inhibited, but most notable was the marked resistance of the synthesis of polypeptide IX. Possible mechanisms for differential synthesis and their significance are considered.
Glycoprotein encoded by the Friend spleen focus-forming virus
Authors: Dresler S , Ruta M , Murray M J , Kabat D
Published: January 2020

The Friend spleen focus-forming virus (F-SFFV) released from cultured erythroleukemia cells (cell line F4-6/K) was cloned free of its helper lymphatic leukemia virus (F-MuLV). After allowing adsorption to Sc-1 fibroblasts at a low multiplicity of infection, the cells were seeded individually into wells of a microtitier test plate and the resulting colonies were grown into large cultures. Among 14 of these cell cultures that have been analyzed thoroughly, 6 contained F-SFFV alone, 1 contained F-MuLV plus F-SFFV, and 7 were uninfected. Each of the Sc-1 cell lines which had been infected with cloned F-SFFV contained a glycoprotein with an apparent molecular weight of 55,000 (gp55) that was absent from the cell lines that lacked F-SFFV. gp55 was also present in Friend erythroleukemia cells and in fibroblasts infected with an F-SFFV that had been doubly cloned in another laboratory. These results indicate that gp55 is encoded by the F-SFFV genome. gp55 has the following additional properties. It can be immunoprecipitated with antiserum made to the F-MuLV virion envelope glycoprotein (gp75). Its unglycosylated polypeptide, formed in cells treated with 2-deoxy-D-glucose, has a molecular weight of approximately 45,000. Its tryptic peptide map contains peptides in common with F-MuLV gp75 but it also contains unique peptides. It appears to be absent or present in only low concentrations in erythroleukemia cell plasma membranes as determined by lactoperoxidase-catalyzed iodination, and it accumulates intracellularly in large amounts. In addition, it is absent from released virions. The majority of the cellular gp55 has an isoelectric point of 8.5 to 9.0. These results are consistent with the idea that an env gene recombination event was involved in the origin of F-SFFV.
Use of UV irradiation to identify the genetic information of vesicular stomatitis virus responsible for shutting off cellular RNA synthesis
Authors: Weck P K , Carroll A R , Shattuck D M , Wagner R R
Published: January 2020

UV irradiation of infectious vesicular stomatitis virus was employed to study the relationship between the expression of certain viral gene functions and viral inhibition of RNA synthesis in mouse myeloma (MPC-11) cells. Viral infectivity, protein synthesis, and viral mRNA synthesis were all highly susceptible to inactivation by UV radiation; however, low levels of viral transcriptase activity were detected in vitro in virus preparations subjected to large doses of UV radiation. In sharp contrast, the capacity of vesicular stomatitis virus to shut off cellular transcription was quite resistant to UV radiation. The data presented here indicate that viral transcription is essential to inhibit host RNA metabolism, even though synthesis of viral polypeptides in the inhibited cells could not be detected. At those levels of UV radiation that inactivated all viral gene functions, except viral inhibition of cellular RNA synthesis, the only viral product detected was non-adenylated, low-molecular-weight RNA species.
Biosynthesis of an unglycosylated envelope glycoprotein of Rous sarcoma virus in the presence of tunicamycin
Authors: Diggelmann H
Published: January 2020

Cells stably infected with Rous sarcoma virus were treated with tunicamycin to prevent the glycosylation of the precursor (pr92gp) to the two viral envelope glycoproteins gp85 and gp35. Pretreatment of the cells for 4 h with the antibiotic resulted in a 90% reduction in [3H]mannose incorporation into total cellular glycoproteins, intracellular viral glycoproteins, and released virus particles. Protein synthesis and virus particle formation were not significantly affected by the treatment. A new polypeptide made in the presence of the drug was identified by immunoprecipitation of pulse-labeled cell lysates with monospecific anti-gp85 and anti-gp35 sera. This polypeptide, migrating on sodium dodecyl sulfate-polyacrylamide gels as a molecule of 62,000 daltons (pr62), contained no [3H]mannose, was labeled with [S35]methionine and [3H]arginine, could not be chased into the higher-molecular-weight glycosylated form, and contained the same [3H]arginine tryptic peptides as pr92gp. The unglycosylated pr62 was still detectable 2 h after the pulse labeling of the cells. The lack of glycosylation of pr62 did not seem to reduce its stability. No clear evidence for the incorporation of this molecule or its cleavage products into viral particles could be obtained. To code for an envelope polypeptide of 62,000 daltons, only about 1,500 nucleotides or 15% of the total coding capacity of the virus are needed.
DNA of Epstein-Barr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA
Authors: Given D , Yee D , Griem K , Kieff E
Published: January 2020

Previous data indicated that Epstein-Barr virus DNA is terminated at both ends by direct or inverted repeats of from 1 to 12 copies of a 3 X 10(5)-dalton sequence. Thus, restriction endonuclease fragments which include either terminus vary in size by 3 X 10(5)-dalton increments (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; S. D. Hayward and E. Kieff, J. Virol. 23:421--429, 1977). Furthermore, defined fragments containing either terminus hybridize to each other (Given and Kieff, J. Virol. 28:524--542, 1978). The 5' ends of the DNA are susceptible to lambda exonuclease digestion (Hayward and Kieff, J. Virol. 23:421--429, 1977). To determine whether the terminal DNA is a direct or inverted repeat, the structures formed after denaturation and reannealing of the DNA from one terminus and after annealing of lambda exonuclease-treated DNA were examined in the electron microscope. The data were as follows. (i) No inverted repeats were detected within the SalI D or EcoRI D terminal fragments of Epstein-Barr virus DNA. The absence of "hairpin- or pan-handle-like" structures in denatured and partially reannealed preparations of the SalI D or EcoRI D fragment and the absence of repetitive hairpin- or pan-handle-like structures in the free 5' tails of DNA treated with lambda exonuclease indicate that there is no inverted repeat within the 3 X 10(5)-dalton terminal reiteration. (ii) Denatured SalI D or EcoRI D fragments reanneal to form circles ranging in size from 3 X 10(5) to 2.5 X 1O(6) daltons, indicating the presence of multiple direct repeats within this terminus. (iii) Lambda exonuclease treatment of the DNA extracted from virus that had accumulated in the extracellular fluid resulted in asynchronous digestion of ends and extensive internal digestion, probably a consequence of nicks and gaps in the DNA. Most full-length molecules, after 5 min of lambda exonuclease digestion, annealed to form circles, indicating that there exists a direct repeat at both ends of the DNA. (iv) The finding of several circularized molecules with small, largely double-strand circles at the juncture of the ends indicates that the direct repeat at both ends is directly repeated within each end. Hybridization between the direct repeats at the termini is likely to be the mechanism by which Epstein-Barr virus DNA circularizes within infected cells (T. Lindahl, A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn, J. Mol. Biol. 102:511-530, 1976).
Comparison of late mRNA splicing among class B and class C adenoviruses
Authors: Kilpatrick B A , Gelinas R E , Broker T R , Chow L T
Published: January 2020

Adenovirus class B (Ad3 and Ad7) and class C (Ad1, Ad5, and Ad6) late r-strand mRNA's were found to have segmented 5' leaders. These leaders were very similar among serotype within a class but differed in sequence from the leaders on late mRNA's of a different class. However, the leader components of class B viruses mapped at essentially the same map coordinates as those of class C viruses. The 5' coordinates of the main bodies of class B messages to which the tripartite leaders are attached as well as the map positions of several of their early mRNA's were very similar to those of Ad2 transcripts. Infrequent examples of late r-strand polysomal RNAs of Ad3 and Ad7 had, in addition to the three common leader segments, a fourth leader segment derived from RNA encoded at various sites between the second and third leaders. The extra components formed several distinct groups. These molecules are presumably intermediates in the splicing processes that generate mature messages.
Polarized Injection of the Bacteriophage T5 Chromosome
Authors: Shaw Alan R. , Davison John
Published: January 2020

Examination of the first-step-transfer DNA of T5
ris
mutants which carry new
Eco
RI sites showed that the left end of the chromosome is injected first.
Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the region coding for the carboxyl terminus of the T antigen
Authors: Van Heuverswyn H , Cole C , Berg P , Fiers W
Published: January 2020

Nucleotide sequence analysis of two simian virus 40 early mutants dl1263 and dl1265, which lack a DNA segment around map positions 0.21 and 0.18, respectively (C. Cole, T. Landers, S. Goff, S. MAnteuil-Brutlag, and P. Berg, J. Virol. 24:277--294, 1977), revealed in-phase deletions of 33 nucleotide pairs for dl1263 and 39 nucleotide paris for dl1265. The 33-base-pair deletion in dl1263 does not correspond to an apparent shortening by 6,000 daltons observed for the mutant T antigen. In dl1265 the normal termination signal as well as most of the proline-rich terminal tryptic peptide has been removed, and the carboxyl terminus of the mutant T antigen is a series of three cysteine residues.
Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase
Authors: Cheng Y C , Tsou T Y , Hackstadt T , Mallavia L P
Published: January 2020

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.
Separation and characterization of herpes simplex virus type 1 immediate-early mRNA's
Authors: Watson R J , Preston C M , Clements J B
Published: January 2020

Polyadenylated immediate-early transcripts of herpes simplex virus type 1, made in BHK cells infected and maintained in the presence of cycloheximide, have been separated on denaturing agarose gels containing methyl mercuric hydroxide. Three virus-specific mRNA bands of estimated sizes 4.7, 3.0, and 2.0 kilobases (kb) were detected, and these mRNA's were mapped on the virus genome and also used to direct protein synthesis in vitro. The 4.7- and 3.0-kb mRNA's hybridized predominantly to certain DNA fragments which are located in the short and long repetitive regions of the genome, respectively, whereas the 2.0-kb mRNA's mapped to three discrete regions of the virus DNA. In vitro translation of these separated mRNA size classes indicated that the 3.0-kb mRNA specified the synthesis of virus polypeptide Vmw 110, whereas the 2.0-kb mRNA's specified Vmw 68, 63, and 12. The synthesis of small amounts of Vmw 175 was specified by the 4.7-kb mRNA. In contrast with the mRNA's which specify these other immediate-early polypeptides, that specifying Vmw 12 is much larger than required for its coding sequences.
Physical Map of the Channel Catfish Virus Genome: Location of Sites for Restriction Endonucleases
Eco
RI,
Hin
dIII,
Hpa
I, and
Xba
I
Authors: Chousterman Suzanne , Lacasa Michel , Sheldrick Peter
Published: January 2020

The overall arrangement of nucleotide sequences in the DNA of channel catfish virus has been studied by cleavage with four restriction endonucleases. Physical maps have been developed for the location of sites for
Eco
RI,
Hin
dIII,
Hpa
I, and
Xba
I. The sum of the molecular weights of fragments generated by each restriction enzyme indicates a molecular weight of approximately 86 × 10
6
for the channel catfish virus genome. Fragments corresponding to the molecular ends of channel catfish virus DNA have been identified by their sensitivity to exonuclease treatment. The distribution of restriction sites in the genome shows that sequences included in a 12 × 10
6
-molecular weight region at one end are repeated with direct polarity at the other end, and that the overall genomic sequence order is nonpermuted.
Production of plasminogen activator by human and hamster cells infected with human cytomegalovirus
Authors: Yamanishi K , Rapp F
Published: January 2020

Plasminogen activator was produced by both human embryo fibroblasts (a permissive system) and hamster embryo fibroblasts (a nonpermissive system) after exposure to human cytomegalovirus. The level of this activator was measured by using plates coated with [125I]fibrin. The production of plasminogen activator was enhanced when the human cells were exposed to human cytomegalovirus previously irradiated with UV light (5,520 to 55,200 ergs/mm2).
Protein induced by bacteriophage T4 which is absent in Escherichia coli infected with nuclear disruption-deficient phage mutants
Authors: Koerner J F , Thies S K , Snustad D P
Published: January 2020

A protein induced by wild-type T4 phage which is absent in Escherichia coli infected with nuclear disruption-deficient phage (with mutations in gene ndd) was identified by polacrylamide gel electrophoresis. This protein was synthesized at maximum rate at 3 to 6 min after infection. It had a molecular weight of 15,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was associated with sedimentable fractions of the cell from which it can be dissociated with 1 M guanidine-hydrochloride. The dissociated protein can be partly recovered in a form soluble in dilute buffer after partial purification and dialysis. The occurrence of this protein in a particulate cell fraction is of interest because of the postulated role of the bacterial cell membrane in nuclear disruption.
Epstein-Barr virus DNA is amplified in transformed lymphocytes
Authors: Sugden B , Phelps M , Domoradzki J
Published: January 2020

Leukocytes isolated from two adult donors who lacked detectable antibodies to antigens associated with Epstein-Barr virus were exposed to an average of 0.02 to 0.1 DNA-containing particles of Epstein-Barr virus per cell and immediately clones in agarose. Within about 30 generations all transformed cell clones contained between 5 and 800 copies of viral DNA per cell. Only 1 in 10(4) to less than 1 in 10(5) of the cells of each clone release virus, and the frequency of release did not correlate with the average number of copies of viral DNA in the cells of each clone. One clone that had an average of five copies of viral DNA per cell was recloned, and the average number of copies in four of six subclones increased 15-to 50-fold while the subclones were being propagated sufficiently to study them. These results indicate that Epstein-Barr virus DNA can undergo amplification relative to cell DNA at different times after it transforms cells.
Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1
Authors: Eisenberg R J , Hydrean-Stern C , Cohen G H
Published: January 2020

The type-common CP-1 antigen of herpes simplex virus type 1 (HSV-1) is associated in the infected cell with two components, a 52,000-molecular-weight glycoprotein (gp52 or pD) and a 59,000-molecular-weight glycoprotein (gp59 or D). The larger form (D) is also found in the virion envelope. It was postulated that pD is a precursor of D. We found that pD shared methionine and arginine tryptic peptides with D isolated from infected cell extracts. D isolated from infected extracts had the same trypric methionine peptide profile as D isolated from the virion envelope. Thus, processing of pD to D does not involve any major alterations in polypeptide structure. Furthermore, D did not share tryptic methionine peptides with the other major glycoproteins of HSV-1. Using [2-3H]mannose as a specific glycoprotein label, we found that pD, which is a basic protein (isoelectric point = 8.0) contained a 1,800-molecular-weight oligomannosyl core moiety and was processed by further glycosylation and sialyation to a more acidic and heterogeneous molecule D, which as a molecular weight of at least 59,000.
Characterization of an immune complex kinase in immunoprecipitates of avian sarcoma virus-transformed fibroblasts
Authors: Richert N D , Davies P J , Jay G , Pastan I H
Published: January 2020

Kinase activity detected in immune complexes containing the src gene product of the avian sarcoma virus has been reported. To further characterize this immune complex kinase, we developed a routine quantitative assay involving trichloroacetic acid precipitation on filters. The enzyme reaction required either Mg2+ or Mn2+, but was inactive with Ca2+. The kinetics of the phosphorylation reaction indicated a transient enzyme activity limited by rapid substrate-dependent inactivation of the enzyme. A variety of nucleoside and deoxyribonucleoside triphosphates (dATP, ATP, GTP, CTP, dGTP, TTP, dCTP) served as phosphoryl donors. The phosphorylation of immunoglobulin G was inhibited by the presence of nucleoside diphosphates. Deoxyribonucleoside diphosphates can either stimulate or inhibit the kinase reaction depending upon the concentration used. The unusual enzymatic properties of the immune complex kinase raise the possibility that the enzyme does not function as a protein kinase in vivo, but rather belongs to a different class of kinases (nucleotide kinases) which adventitiously phosphorylates immunoglobulin G when immunoprecipitated with immune serum.
Persistence of the cytomegalovirus genome in human cells
Authors: Mocarski E S , Stinski M F
Published: January 2020

A small percentage of human fibroblast cells survived high-multiplicity infection by cytomegalovirus and were isolated as persistently infected cultures. Approximately 30% of the cells were in the productive phase of infection, since virus-specific structural antigens and virions were associated with these cells. The remaining cells contained neither viral structural antigens nor particles. Nuclear DNA from these nonproductive cells contained approximately 120 genome equivalents of viral DNA per cell as determined by reassociation kinetics. In situ hybridization confirmed that nuclei from nonproductive cells contained a significant amount of viral DNA that was distributed in most of these cells. Early virus-induced proteins and antigens were also detected. Nonproductive cells continued to grow, and there was a slow, spontaneous transition of some of these cells to productive viral replication. The majority of the viral DNA in nonproductive cells persisted with restricted gene expression. When infectious virus production was eliminated by growing the persistently infected cultures in the presence of anticytomegalovirus serum, approximately 45 genome equivalents of the viral DNA persisted per cell. The reassociation reaction approached completion. After removal of the antiserum and subculturing, infectious virus production resumed. Therefore, it was assumed that all sequences of the viral genome remained associated with these cells. Restriction of cytomegalovirus gene expression in persistently infected cell cultures is discussed.
Preparation of syngeneic tumor regressor serum reactive with the unique determinants of the Abelson murine leukemia virus-encoded P120 protein at the cell surface
Authors: Witte O N , Rosenberg N , Baltimore D
Published: January 2020

Antisera reactive with the Abelson murine leukemia virus (A-MuLV)-specified P120 (anti-AbT sera) were produced in C57L/J mice. Of many strains tested, only C57L/J reproducibly rejected syngenic A-MuLV-induced tumor cells; after multiple immunizations their sera would immunoprecipitate both P120 and Moloney-MuLV (M-MuLV) proteins. Using labeled A-MuLV-induced nonproducer cells, only P120 could be detected by anti-AbT sera, suggesting that it may be the only A-MuLV-specified protein. Reactivity of anti-AbT sera with P120 was not blocked by M-MuLV virion proteins, implying that the sera recognize a portion of P120 that is not homologous to any M-MuLV product. Anti-AbT sera stained the surface of live, A-MuLV-transformed nonproducer cells in a two-stage immunofluorescence assay, and such staining was not blocked by M-MuLV protein. Also, intact A-MuLV-transformed cells absorbed much of the reactivity of certain anti-AbT sera for P120. Thus a portion of P120 appears to be exposed on the surface of transformed cells. P120 lacks detectable carbohydrate, is not affected by endoglycosidase H, and cannot be labeled by lactoperoxidase-catalyzed iodination. Thus P120 is an unusual surface protein.
Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization
Authors: Hager G L , Chang E H , Chan H W , Garon C F , Israel M A , Martin M A , Scolnick E M , Lowy D R
Published: January 2020

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.
Efficient propagation of measles virus in suspension cultures
Authors: Baczko K , Lazzarini R A
Published: January 2020

Suspension cultures of a human prostate cell line (MA160) supported abundant growth of the Edmonston strain of measles virus. The virus yields obtained with these suspension cultures (150 to 800 PFU/cell) were at least 20- to 100-fold higher than those frequently reported in the literature. Monolayer cultures of MA160 cells did not support a virus replication nearly as efficiently (progeny yield, 25 PFU/cell).
Tryptic peptide analysis of avian oncovirus gag and pol gene products
Authors: Rettenmier C W , Karess R E , Anderson S M , Hanafusa H
Published: January 2020

Radiolabeled tryptic peptides of the gag and pol gene products of avian oncoviruses were examined. This analysis included Rous-associated virus 2 structural proteins and the Pr76gag and P180gag-pol proteins in Rous-associated virus 2-infected chicken embryo cells. The methionine- and cysteine-containing tryptic peptides of virion internal structural proteins were present in both Pr76gag and P180gag-pol, suggesting that there was no loss of gag gene-coding sequences during the generation of P180gag-pol. No overlap of gag and pol gene structural information was detected. Analysis of intermediates in the processing of Pr76gag and translation inhibition mapping with pactamycin yielded the following order of structural proteins within the Rous-associated virus 2 Pr76gag precursor: NH2-p19-p12-p27-p15-COOH. The gag and pol sequences missing in the endogenous gsmp120 protein of uninfected gs+ chicken cells were identified by comparison with those of Rous-associated virus 2 P180gag-pol.
Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro
Authors: Flanegan J B , Van Dyke T A
Published: January 2020

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.
Terminal sequences of vesicular stomatitis virus RNA are both complementary and conserved
Authors: Keene J D , Schubert M , Lazzarini R A
Published: January 2020

The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).
Oligoribonucleotide map and protein of CMII: detection of conserved and nonconserved genetic elements in avian acute leukemia viruses CMII, MC29, and MH2
Authors: Bister K , Löliger H C , Duesberg P H
Published: January 2020

RNA and protein of the defective avian acute leukemia virus CMII, which causes myelocytomas in chickens, and of CMII-associated helper virus (CMIIAV) were investigated. The RNA of CMII measured 6 kilobases (kb) and that of CMIIAV measured 8.5 kb. By comparing more than 20 mapped oligonucleotides of CMII RNA with mapped and nonmapped oligonucleotides of acute leukemia viruses MC29 and MH2 and with mapped oligonucleotides of CMIIAV and other nondefective avian tumor viruses, three segments were distinguished in the oligonucleotide map of CMII RNA: (i) a 5' group-specific segment of 1.5 kb which was conserved among CMII, MC29, and MH2 and also homologous with gag-related oligonucleotides of CMIIAV and other helper viruses (hence, group specific); (ii) an internal segment of 2 kb which was conserved specifically among CMII, MC29, and MH2 and whose presence in CMII lends new support to the view that this class of genetic elements is essential for oncogenicity, because it was absent from an otherwise isogenic, nontransforming helper, CMIIAV; and (iii) a 3' group-specific segment of 2.5 kb which shared 13 of 14 oligonucleotides with CMIIAV and included env oligonucleotides of other nondefective viruses of the avian tumor virus group (hence, group specific). This segment and analogous map segments of MC29 and MH2 were not conserved at the level of shared oligonucleotides. CMII-transformed cells contained a nonstructural, gag gene-related protein of 90,000 daltons, distinguished by its size from 110,000-daltom MC29 and 100,000-dalton MH2 counterparts. The gag relatedness and similarity to the 110,000-dalton MC29 counterpart indicated that the 90,000-dalton CMII protein is translated from the 5' and internal segments of CMII RNA. The existence of conserved 5' and internal RNA segments and conserved nonstructural protein products in CMII, MC29, and MH2 indicates that these viruses belong to a related group, termed here the MC29 group. Viruses of the MC29 group differ from one another mainly in their 3' RNA segments and in minor variations of their conserved RNA segments as well as by strain-specific size markers of their gag-related proteins. Because (i) the conserved 5' gag-related and internal RNA segments and their gag-related, nonvirion protein products correlate with the conserved oncogenic spectra of the MC29 group of viruses and because (ii) the internal RNA sequences and nonvirion proteins are not found in nondefective viruses, we propose that the conserved RNA and protein elements are necessary for oncogenicity and probably are the onc gene products of the MC29 group of viruses.
Comparative analysis of GS and BK virus genomes
Authors: Pater M M , Pater A , di Mayorca G
Published: January 2020

Analysis of heteroduplexing between the genomes of GS virus, a BK-group virus, and the prototype BK virus revealed one region of nonhomology. Further analysis by cleavage of viral DNA with the restriction endonucleases EcoRI, HindIII, and HaeIII revealed that base changes in the GS virus genome spanned 0.6 to 0.7 map unit from the EcoRI site. Large T and small t antigens of GS virus appear to be similar in size to the BK virus antigens.
Mappings of adenovirus type 7 cytoplasmic RNA species synthesized early in lytically infected cells and synthesized in transformed cells
Authors: Yoshida K , Sekikawa K , Fujinaga K
Published: January 2020

Early virus-specific RNA synthesized in KB cells infected with adenovirus type 7 and virus-specific RNA synthesized in rat embryo cells (71JY1-2) transformed by the adenovirus type 7 HindIII-I.J fragment (left-hand 8.1% of the viral genome) have been mapped on the viral genome. About 25% of the viral genome, four discrete regions, two on each strand of the viral genome, are expressed as "early" mRNA. Almost similar regions in the left-hand 8.1% of the viral genome are transcribed both in KB cells at early times after infection and in 71JY1-2 cells.
Expression of simian virus 40 gene A affects tubulin stability
Authors: Wiche G , Furtner R , Steinhaus N , Cole R D
Published: January 2020

The stability of tubulins present in crude extracts of untransformed BALB/c-3T3 mouse fibroblasts, Chinese hamster lung cells, and various of their simian virus 40 transformants was assessed by measurement of their individual colchicine-binding decay rates. In all cases studied the decays followed the kinetics of first-order reactions, and rates were reduced at low temperatures and by vinblastine sulfate. Under all assay conditions, including different temperatures and protein concentrations, tubulins of normal cells decayed considerably faster than those of simian virus 40-transformed cells. Experiments performed with a number of Chinese hamster lung cell clones transformed with temperature-sensitive simian virus 40 gene A mutants showed a clear correlation between increased tubulin stability and the expression of gene A function. These results suggest that it is T-antigen, the viral gene A product, that affects tubulin.
Persistent vesicular stomatitis virus infection mediates base substitutions in viral RNA termini
Authors: Semler B L , Holland J J
Published: January 2020

We have sequenced (via a product RNA) the 3' RNA terminus of a defective interfering particle that was generated from the standard virus isolated from a culture of BHK-21 cells persistently infected with vesicular stomatitis virus for over 5 years. By hybridization and RNA sequencing, seven mutations were identified in the 46 nucleotides at the terminus of this defective-interfering-particle RNA. It is likely that these mutations are a reflection of altered protein-nucleic acid interactions that the virus has evolved to maintain its persistently infected carrier state in vitro.
Polypeptides of the Epstein-Barr virus membrane antigen complex
Authors: Thorley-Lawson D A , Edson C M
Published: January 2020

Epstein-Barr virus (EBV)-associated membrane antigens have been purified from the plasma membranes of the producer cell line P3HR-1 NONO. The antigens were assayed with a specific rabbit anti-ebv antiserum using an 125I-labeled staphylococcal protein A binding assay. The antigens have been shown to be present on purified plasma membranes. Treatment of the plasma membranes with Triton X-100 allows the separation of two antigenically distinct classes of antigens, one soluble and one insoluble in the detergent. Immunoprecipitates of [125I5- and [35S]methionine-labeled, detergent-soluble antigens contained three major polypeptides of molecular weights of 350,000, 140,000, and 75,003 (on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and several minor components. These polypeptides were all specifically precipitated from four EBV-producer cell lines, P3HR-1, P3HR-1 NONO, B95-8, and 7744. They could not be precipitated from producer cell lines treated with phosphonoacetic acid, which inhibits late viral functions, nor could they be precipitated from nonproducer cell lines. The 350,000 and 75,000 molecular weight polypeptides bound to Ricin and lentil lectin columns; however, most of the 140,000 molecular weight material did not. A component of molecular weight 220,000 (prominent only in P3HR-1 NONO) was probably a degradation product of the 350,000 molecular weight polypeptide.
Deletion mutants of polyoma virus defining a nonessential region between the origin of replication and the initiation codon for early proteins
Authors: Bendig M M , Folk W R
Published: January 2020

Mutants of polyoma virus with deletions as large as 90 base pairs were isolated by selecting spontaneously arising genomes resistant to endonuclease HaeII or by treating HaeII- or BglI- cleaved linear DNAs with S1 nuclease and exonuclease III. All of the mutants were viable and, therefore, defined a nonessential region in the polyoma genome between the origin of DNA replication and the initiation codon for translation of early proteins. Several mutants with large deletions had altered growth properties, giving smaller plaques and lower virus yields than the parental wild-type virus. These viruses may lack sites that are important for DNA replication or for transcription and translation of early mRNA's. All of the mutants tested could transform BHK-21 cells to anchorage independence.
Regulation of the Primary Expression of the Early Adenovirus Transcription Units
Authors: Nevins Joseph R. , Ginsberg Harold S. , Blanchard J.-M. , Wilson Michael C. , Darnell James E.
Published: January 2020

The time course of appearance of transcriptional activity from five early adenovirus type 2 transcription units has been determined. RNA complementary to region 1A (1-4.4 map units), the first region to be transcribed, was detectable at 45 min after infection; a maximal rate of RNA synthesis was reached at 3 h after infection and was maintained thereafter for at least 6 h. RNA from region 2 (75-56 map units), which encodes the mRNA for the 72,000-dalton DNA-binding protein, was the last to be synthesized; transcription commenced at about 2 h postinfection, reached a maximum at 7 h, and then declined. Transcription of regions 3 (76-86 map units) and 4 (99-91 map units) reached a maximal value at 3 h postinfection. The rates of RNA synthesis from these regions then declined over the next 6 h. The decline of transcription from regions 2 and 4 appeared to be a specific repression of these transcription units. The repression did not occur in the absence of protein synthesis, suggesting that a viral protein might be involved. Transcription of all early regions was initiated and continued for at least 2 to 3 h in cells that were treated with cycloheximide or emetine before and during infection, suggesting that at least the initiation of RNA synthesis from the five early adenovirus type 2 transcription units does not depend on the formation of a viral protein. Moreover, mRNA was formed in the absence of protein synthesis that hybridized to DNA fragments representing each of the five early transcription units. The increase in mRNA accumulation in the presence of cycloheximide (or emetine) does not appear to be due to increased RNA synthesis; thus, either increased mRNA stability or increased efficiency of nuclear RNA processing must occur.
Analysis of cellular integration sites in avian sarcoma virus infected duck embryo cells
Authors: Gilmer T M , Parsons J T
Published: January 2020

The cellular sites of integration of avian sarcoma virus (ASV) have been examined in clones of duck embryo cells infected with the Bratislava 77 strain of ASV using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled ASV complementary DNA probes. DNA prepared from 11 clones of duck embryo cells infected with the Bratislava 77 strain of ASV was digested with the restriction enzymes HpaI, which cleaves once within the viral genome, and Hind III, which cleaves twice within the viral genome, and the virus-cell DNA juncture fragments were resolved by agarose gel electrophoresis. Analysis of the virus-cell junctures present in individual ASV-infected duck embryo clones revealed that all clones contain at least one copy of nondefective proviral DNA with some clones containing as many as 5 to 6 copies of proviral DNA. A comparison of the virus-cell juncture fragments present in different ASV-infected clones showed that each clone contains a unique set of virus-cell junctures. These data suggest that ASV DNA can integrate at multiple sites within the duck embryo cell genome and that these sites appear to be different as defined by digestion with the restriction enzymes HpaI and HindIII.
Stabilization of "A" particles of coxsackievirus B3 by a HeLa cell plasma membrane extract
Authors: McGeady M L , Crowell R L
Published: January 2020

Previous studies in our laboratory showed that HeLa cell plasma membranes were recovered from sucrose gradients in two major bands and that the heavier band possessed a putative inhibitor of uncoating of coxsackievirus B3. It has now been found that the mechanism of inhibition is the stabilization of "A" particles against inactivation at 37 degrees C. [3H]uridine-labeled virions converted to A particles by band 4, the heavier band, were four times more stable at 37 degrees C than those produced by band 3. Partially purified A particles from both bands were equally unstable. It was found that the stabilizing factor was extractable by saline from band 4 and remained soluble after centrifugation (109,000 X g for 2 h). Addition to A particles of this soluble factor isolated from either band 4 or band 3 stabilized the A particles. The stabilizing factor could not be replaced by an extract from band 3 or by bovine serum albumin. Thus, the finding that the membrane factor inhibits virus uncoating by stabilizing A particles against spontaneous disruption at 37 degrees C focuses attention on an inherent problem associated with defining receptor-mediated virus uncoating.
Persistence of infectious Friend virus in spleens of mice after spontaneous recovery from virus-induced erythroleukemia
Authors: Chesebro B , Bloom M , Wehrly K , Nishio J
Published: January 2020

Persistent infectious virus was detected in the majority of spleens of (C57BL/10 X A.BY)F1 mice after spontaneous recovery from Friend virus-induced erythroleukeima. The Friend murine leukemia helper virus (F-MuLV) was detected in titers up to 3 X 10(5) PFU/g of spleen. The defective spleen focus-forming virus (SFFV) was present in much lower titers and could be detected in cell-free spleen homogenates only after amplification of virus titer by growth of virus in vitro on SC1 cells. The incidence of cells producing F-MuLV alone in spleens after recovery from leukemia was 0.003 to 0.3%, and the incidence of cells producing both F-MuLV and SFFV was less than 0.0001 to 0.01%. In most recovered mouse spleens there appeared to be a selective reduction of SFFV relative to F-MuLV.
Viable deletion mutant of human papovavirus BK that induces insulinomas in hamsters
Authors: Watanabe S , Yoshiike K , Nozawa A , Yuasa Y , Uchida S
Published: January 2020

A plaque morphology mutant (pm-522) of human papovavirus BK, which was rescued from a human papovavirus BK-induced hamster pineocytoma, was characterized and compared with a cloned wild-type virus (wt-501). Mutant pm-522 formed turbid plaques and grew more slowly than wt-501 in human embryonic kidney (HEK) cells. The immunofluorescence assay revealed that more HEK cells underwent abortive infection with pm-522 than with wt-501. Whereas wt-501 induced brain tumors and osteosarcomas, but no insulinomas, in hamsters, pm-522 induced brain tumors and insulinomas. The DNA of pm-522 was found by electrophoresis and electron microscopy to have a deletion (85 +/- 15 base pairs) and an insertion (40 +/- 10 base pairs) between map coordinates 0.708 and 0.725 from the endonuclease EcoRI cleavage site. These results demonstrate the presence of a viable deletion human papovarivus BK mutant capable of inducing insulinomas in hamsters.