bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro transcription catalyzed by heat-treated human rotavirus
Authors: Spencer E , Arias M L
Published: January 2020

The in vitro characteristics of human rotavirus transcription have been examined. The virus has an associated RNA polymerase activity which was activated after a heat shock treatment. The enzyme required the presence of the four ribonucleoside triphosphates and a divalent cation (Mg2+), and it required an optimum pH of 8.5. The polymerase was activated by monovalent salts and inhibited by Na PPi. The addition of actinomycin D, alpha-amanitin, or rifampin did not inhibit the polymerase activity. After thermal shock of the virus, at least eight different RNA species were synthesized which may correspond to independent transcripts. Transcription also requires a hydrolyzable form of ATP. Analogs such as beta,gamma-imido ATP or beta,gamma-methylene ATP were inhibitory, whereas others, such as the beta-gamma-imido or methylene analogs of CTP, UTP, or GTP, were not inhibitory. This suggests that ATP is related to reactions other than polymerization, probably to initiation or elongation of RNA molecules, as has been described for vesicular stomatitis virus or vaccinia virus.
In vitro translation of avian myeloblastosis virus RNA
Authors: Anderson S M , Chen J H
Published: January 2020

Avian myeloblastosis virus (AMV)-infected cells contain two viral mRNA's, a genome-sized 34S (7.5-kilobase) mRNA and a 21S (2.5-kilobase) subgenomic mRNA, which contains the AMV-specific sequences (myb sequences). We found that AMV virions packaged both the 7.5-kilobase full-length genomic RNA and the 2.5-kilobase subgenomic RNA. In vitro translation of AMV virion RNA sized by sucrose density gradient centrifugation yielded 76,000-, 56,000-, 48,500-, 47,000-, and 32,000-dalton products. The 76,000-dalton protein was coded for by RNA throughout the gradient, but the peak of activity was at 34S to 35S. The 56,000-, and 48,500-, and 32,000-dalton proteins were encoded in a 21S RNA, and 47,000-dalton protein was encoded in an RNA of approximately 24S. The 76,000-dalton protein was identified as Pr76gag, based upon immunoprecipitation with specific antiserum and the presence of the 19* dipeptide. 7-Methylguanosine triphosphate inhibited the syntheses of Pr76gag and the 56,000-, 48,500-, and 32,000-dalton proteins, but not the synthesis of the 47,000-dalton protein. The 56,000-, 48,500-, 47,000-, and 32,000-dalton proteins were not immunoprecipitated by anti-gag, anti-reverse transcriptase, or anti-gp85 antiserum. Two-dimensional peptide maps of the 56,000- and 48,500-dalton proteins indicated that they were unique. In vitro translational products of myeloblastosis-associated virus 1 were also analyzed to aid in the identification of the AMV myb gene product(s); the translational products analyzed included Pr76gag, p60env, and a 56,000-dalton polypeptide which apparently was not identical to the 56,000-dalton AMV translational product, as determined by two-dimensional peptide mapping. Our data indicated that one of these proteins (56,000, 48,500, or 32,000 daltons) may represent the product of the AMV myb gene and, therefore, the putative transforming protein(s) of AMV.
Evidence that the UV endonuclease activity induced by bacteriophage T4 contains both pyrimidine dimer-DNA glycosylase and apyrimidinic/apurinic endonuclease activities in the enzyme molecule
Authors: Warner H R , Christensen L M , Persson M L
Published: January 2020

We performed experiments to determine whether the phage T4-induced UV endonuclease activity is a single protein containing both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities. The UV endonuclease activity is induced by the denV gene and codes for the glycosylase activity. We obtained several kinds of evidence that the protein containing the glycosylase activity also contains the apyrimidinic endonuclease activity. After chromatography on DEAE-cellulose, the two activities copurified during phosphocellulose chromatography and Sephadex G-100 chromatography, with a constant ratio of activities across the activity peaks. On Sephadex G-100 columns the molecular weights of the two activities agreed within 2,500 or less. When an extract of cells infected with the T4 V1 mutant was purified in exactly the same way as an extract of cells infected with T4 V1+, neither glycosylase nor apyrimidinic endonuclease activity was detected in the normal elution position of the T4 UV endonuclease activity. The glycosylase and apyrimidinic endonuclease activities were induced with similar kinetics, which were characteristic of immediate early rather than delayed early enzymes. This correlated well with the presumed major role of these activities in repairing thymine dimers in parental DNA before DNA replication begins. Finally, glycosylase and apyrimidinic endonuclease activities were lost in parallel during incubation of the enzyme at 46 degree C. Our results indicated that both of these enzyme activities are contained in the same enzyme molecule and, probably, in the same polypeptide.
Release of a Group 1 Mycoplasma Virus from
Acholeplasma laidlawii
After Treatment with Mitomycin C
Authors: Liss Alan
Published: January 2020

A rod-shaped group 1 mycoplasma virus was released from
Acholeplasma laidlawii
strain JA2 after treatment with 2.5 μg of mitomycin C per ml. Similar treatment of
A. laidlawii
strain Bju failed to stimulate release of any PFU.
Baculovirus induction of a DNA polymerase
Authors: Miller L K , Jewell J E , Browne D
Published: January 2020

The baculovirus, Autographa california nuclear polyhedrosis virus, induced a new aphidicolin-sensitive, alpha-like, DNA polymerase upon infection of the lepidopteran noctuid, Trichoplusia ni. The new virus-induced DNA polymerase could be separated from the host alpha-like polymerase by phosphocellulose chromatography. The two polymerases differed in their sensitivities to heat inactivation, high salt concentrations, and 0.1 M phosphate buffer.
Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 das mutation
Authors: Mickelson C , Wiberg J S
Published: January 2020

Lethal, amber mutations in T4 genes 46 and 47 cause incomplete degradation of host DNA, premature arrest of phage DNA synthesis, accumulation of abnormal DNA replication intermediates, and defective recombination. These phenotypes can be explained by the hypothesis that genes 46 and 47 control a DNA exonuclease, but in vitro demonstration of such a nuclease has not yet been reported. Membrane and supernatant fractions from 46- and 47- mutant-infected and 46+ 47+ control-infected cells were assayed for the presence of the protein products of these genes (i.e., gp46 and gp47) and for the ability to degrade various DNA substrates to acid-soluble products in vitro. The two proteins were found only on membranes. The membrane fraction from 46- 47- mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower that the membrane fraction from control-infected cells. No such effect was found in the cytoplasmic fractions. The effect on nuclease activity in membranes was the same whether 46- and 47- mutations were present singly or together. NaClO4, a chaotropic agent, released both gp46 and gp47 from 46+ 47+ membranes, as well as the DNase activity controlled by genes 46 and 47. DNA cellulose chromatography of proteins released from membranes by NaClO4 showed that gp46 and gp47 bound to the native DNAs of both E. coli and T4. Thus, the overall enrichment of gp46 and gp47 relative to total T4 protein was 600-fold (10-fold in membranes, 2-fold more upon release from membranes by NaClO4, and 30-fold more upon elution from DNA cellulose). T4 das mutations, which partially suppress the defective phenotype of 46- and 47- mutants, caused a considerable increase in vitro DNase activity in both membrane and cytoplasmic fractions, We obtained evidence that the das+ gene does not function to inhibit E. coli exonuclease I or V, endonuclease I, or the UV endonuclease of gene uvrA or to decrease the activity of T4 exonuclease A or the T4 gene 43 exonuclease.
Isolation and Characterization of an RNA-Dependent RNA Polymerase from Black Beetle Virus-Infected
Drosophila melanogaster
Cells
Authors: Guarino Linda A. , Kaesberg Paul
Published: January 2020

Crude lysates of black beetle virus (BBV)-infected cells of
Drosophila melanogaster
contain an RNA-dependent RNA polymerase activity not detectable in uninfected cells. The activity (designated BBV polymerase) sedimented at 20,000 ×
g
, indicating an association with particulate material. It was solubilized from the pellet by sonication in a magnesium-deficient buffer. Differential centrifugation resulted in a 43-fold purification with 84% recovery of polymerase activity. The effects of divalent and monovalent cations, time, temperature, and pH on the activity of the partially purified polymerase were examined. RNA synthesis was not stimulated by the addition of exogenous BBV RNA, suggesting that an enzyme-template complex existed. Analysis of the RNA products of the RNA polymerase reaction indicated that full-length “negative” strand BBV RNAs were synthesized.
Localization of the Abelson murine leukemia virus protein in a detergent-insoluble subcellular matrix: architecture of the protein
Authors: Boss M A , Dreyfuss G , Baltimore D
Published: January 2020

We examined the interaction of Abelson murine leukemia virus protein P120 with other cellular components after extraction with the nonionic detergent Triton X-100. Most of the Abelson murine leukemia virus P120-associated kinase activity was found in the detergent-insoluble matrix in both lymphoid and fibroblast cell lines. The P120 labeled during a short exposure of cells to [35S]-methionine was mainly in the detergent-insoluble matrix (lymphoid cells) or equally distributed in the detergent-insoluble matrix and the soluble fraction (fibroblasts). Steady-state-labeled P120 was distributed equally in the two fractions (lymphoid cells) or mostly in the soluble portion (fibroblasts). Thus, there was an apparent movement of P120 from the detergent-insoluble matrix to the detergent-soluble fraction and a concomitant loss of enzymatic activity. When the detergent-insoluble matrix was incubated with [32P]ATP in situ, phosphorylation of tyrosine residues of P120 was observed. We found an 80,000-molecular-weight fragment of P120 (designated F80) after extraction of fibroblast cells with detergent. F80 was not found in extracted lymphoid cells, but mixing labeled lymphoid cells and unlabeled fibroblasts before extraction produced the fragment. F80 contained the gag determinants of P120 but did not react with Abelson-specific serum. These data allowed us to assign various features of the protein to regions of the P120 molecule and to localize the Abelson-specific antigenic determinants to the C-terminal region of the molecule.
Characterization of two temperature-sensitive mutants of type 5 adenovirus with mutations in the 100,000-dalton protein gene
Authors: Oosterom-Dragon E A , Ginsberg H S
Published: January 2020

Complementation analysis assigned the mutations of strains H5ts115 and H5ts116, two hexon-minus mutants, to the 100,000-dalton (100K) protein gene. Heterotypic marker rescue (i.e., type 5 adenovirus [Ad5] temperature-sensitive mutants DNA X EcoRI restriction fragments of Ad2 DNA) confirmed the results of previous marker rescue mapping studies, and the heterotypic recombinants yielded unique hybrid (Ad5-Ad2) 100K proteins which were intermediate in size between Ad5 and Ad2 proteins and appeared to be as functionally active as the wild-type 100K protein. Phenotypic characterization of these mutants showed that both the hexon polypeptides and the 100K polypeptides were unstable at the nonpermissive temperature, whereas fiber and penton were not degraded, and that the 100K protein made at 39.5 degrees C could not be utilized after a shift to the permissive temperature (32 degrees C). The role of the 100K protein in the assembly of the hexon trimer was also examined by in vitro protein synthesis. Normally, hexon polypeptides synthesized during an in vitro reaction are assembled into immunoreactive hexons. However, this assembly was inhibited by preincubation of the cell extract with anti-100K immunoglobulin G; neither anti-fiber immunoglobulin G nor normal rabbit immunoglobulin G inhibited hexon assembly. It is postulated that an interaction between the 100K protein and hexon polypeptides is required for effective assembly of hexon trimers.
Epstein-Barr virus DNA. X. Direct repeat within the internal direct repeat of Epstein-Barr virus DNA
Authors: Cheung A , Kieff E
Published: January 2020

The 3,360-base-pair internal direct repeat (IR) in Epstein-Barr virus DNA separates the short and long unique DNA domains. IR has a single BamHI site. The juncture between the short unique domain and IR has been mapped by restriction endonucleases and is less than 2,600 nucleotides before the BamHI site in IR. The junction between IR and the long unique domain has been sequenced and is approximately 650 nucleotides after the BamHI site in IR. Thus, relative to the start of IR at the juncture with the short unique domain, the last repeat is at least 90 base pairs short of being complete. There is homology between the 250-nucleotide fragments to the left and the right of the unique BamHI site in IR. A 35-base-pair sequence of the left fragment is directly repeated within the right fragment, once fully and once partially. The implications of these findings are discussed.
Structure of varicella-zoster virus DNA
Authors: Straus S E , Aulakh H S , Ruyechan W T , Hay J , Casey T A , Vande Woude G F , Owens J , Smith H A
Published: January 2020

Varicella-zoster virus (VZV) DNA was prepared from nucleocapsids and from enveloped virions of a laboratory strain (Ellen) and directly from the vesicle fluids of patients with zoster infections. VZV Ellen nucleocapsid DNA was cleaved with 11 different restriction endonucleases and electrophoresed in agarose gels. The restriction profiles of the nucleocapsid DNA were identical to those of the DNA recovered from purified virions, but differed from those of another VZV strain (KM). In vitro-labeled VZV K.M. DNA purified directly from vesicle fluid yielded a distinct restriction pattern which appeared to be unchanged after several tissue culture passages of the isolate from that fluid. Restriction endonuclease analysis (EcoRI or BglII) of VZV DNA revealed the presence of four cleavage fragments with a molar ratio of approximately 0.5. No individual fragments with molar ratios of 0.25 were noted. This observation suggests that the VZV genome may contain one invertible segment. Comparison of the electrophoretic migrations of VZV DNA fragments relative to those of DNAs of known size permitted calculation of the VZV genome size to be 72 X 10(6) to 80 X 10(6) daltons. These results were confirmed by electron microscopy which demonstrated a genome size of about 76 X 10(6) daltons for passaged and unpassaged VZV DNA. Electron microscopy also revealed that some of the DNA molecules recovered from nucleocapsids or directly from vesicle fluids were superhelical circles.
Intracellular vesicular stomatitis virus leader RNAs are found in nucleocapsid structures
Authors: Blumberg B M , Kolakofsky D
Published: January 2020

Previous studies demonstrated that cytoplasmic extracts of cells infected with vesicular stomatitis virus contain plus-strand leader RNAs which sediment at 18S on sucrose gradients as a complex with viral N protein. The work presented in this paper demonstrated that these 18S complexes were stable on CsCl density gradients, banding at a buoyant density near that of genome nucleocapsids, and exhibited a morphology in an electron microscope similar to the disk structures found in virus genome nucleocapsids. Minus-strand leader RNAs were also found in 18S complexes on sucrose gradients. Quantitation of intracellular leader RNA suggested that, late in infection, approximately three-quarters of total intracellular leader RNA was encapsidated.
Experiments with cloned complete tumor-derived bovine leukemia virus information prove that the virus is totally exogenous to its target animal species
Authors: Deschamps J , Kettmann R , Burny A
Published: January 2020

Taking advantage of the existence of a unique SacI restriction site in the long terminal repeats of the integrated bovine leukemia virus proviral DNA isolated from a bovine tumor, the total viral information (about 9.2 kilobases) was cloned in the lambdoid vector lambda WES. lambda B. Use of this cloned bovine leukemia virus DNA allowed us, for the first time, to definitely rule out the existence of any endogenous bovine leukemia virus sequence in the bovine, ovine, caprine, murine, feline, chicken, or human genomes. These data prove the absence of acquired cellular information in the provirus that has given rise to a tumor.
Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species
Authors: Carlock L R , Jones N C
Published: January 2020

A mutant of adenovirus type 5 containing an octanucleotide insert in region E1a of the viral genome was constructed. The insert was present in only one (13s) of the three overlapping mRNA's synthesized from this region. The insert was within the sequences removed by RNA splicing during the production of the other two nRNA's. The insertion resulted in a shift in the translational reading frame of the 13s mRNA and the probable premature termination of translation. The mutant was defective for viral DNA replication in HeLa cells and the transformation of rat embryo and baby rat kidney cells, indicating that a product encoded by the 13s nRNA is required for these two processes. Other early regions of the genome were expressed in HeLa cells infected by this mutant although in some cases the expression was decreased as compared with wild-type-infected cells.
In vitro transcription of adenovirus
Authors: Fire A , Baker C C , Manley J L , Ziff E B , Sharp P A
Published: January 2020

A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters.
Rous sarcoma virus mutant LA3382 is defective in virion glycoprotein assembly
Authors: Hardwick J M , Hunter E
Published: January 2020

LA3382 is a temperature-sensitive replication-defective mutant of Rous sarcoma virus that contains four active mutations. In this study we performed experiments to determine the biochemical defect that blocks the synthesis of infections virus late in the replication cycle. At the nonpermissive temperature (41 degrees C) cells infected with LA3382 synthesized virus particles which were noninfectious and exhibited significant reductions in the amounts of gp85 and gp37 present in the virions. An analysis of the intracellular viral polypeptides indicated that the precursor of the viral glycoproteins (Pr95) were synthesized normally but underwent cleavage at a reduced rate at the restrictive temperature. Pr95 did not accumulate in infected cells ans was inserted into mutant virions at 41 degrees C; however, Pr95 was cleaved in such a way that gp85 was released from the viruses and could be detected in the supernatant medium by immunoprecipitation. The virus-free glycoprotein was indistinguishable from wild-type gp85 and may have been released due to an anomalous cleavage. Pulse-chase experiments also indicated that the Pr180 polyprotein precursor of the reverse transcriptase was cleaved to the active form of the enzyme more slowly at 41 degrees C in LA3382-infected cells. This accounted for the twofold lower level of polymerase activity found in mutant virions at 41 degrees C, defect which probably did not account for the observed 20- to 50-fold reduction in infectivity. Furthermore, the replication defect was not complemented by an env deletion mutant Rous sarcoma virus [RSV(-)[, which should complement a pol defect. Therefore, we conclude that the major lesion that impairs replication in LA3382 is within the env gene.
Monocistronic and polycistronic bacteriophage T4 gene 23 messages
Authors: Young E T , Menard R C , Harada J
Published: January 2020

We studied transcription of T4 late genes by in vitro translation of size-fractionated late RNA and by hybridization of T4 late RNA to plasmids containing identified T4 late genes. We identified mRNA species that coded for the late proteins gp10, gp18, gp21, gp22, gp23, and gp24. Functional mRNA's that coded for the early proteins gp32 and IPIII were also detected after fractionation of late RNA. As in preparations of early RNA, gene 32 message activity was present in two species of RNA, which had molecular weights of approximately 0.5 x 10(6) and 0.8 x 10(6), and IPIII message activity was present in multiple species of RNA. Gene 24 and gene 10 message activities migrated as single species that had approximate molecular weights of 0.5 x 10(6) and 1.2 x 10(6), respectively. mRNA activity for gp18 migrated heterogeneously. We detected multiple transcripts from gene 23 by in vitro translation and by hybridization of late RNA to plasmids containing genes 21 through 23. Both types of analysis indicated that the major gene 23 transcript had a molecular weight of 0.75 x 10(6). In addition, two gene 23 transcripts having molecular weights of about 1.0 x 10(6) and 1.3 x 10(6) were present; these RNA species also coded for gp21 and gp22. Physical linkage of transcripts from genes 21, 22, and 23 was demonstrated by hybridization.
Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning
Authors: Chen I S , Mak T W , O'Rear J J , Temin H M
Published: January 2020

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.
Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells
Authors: Roth M G , Compans R W
Published: January 2020

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.
5'-terminal deletions are a common feature of endogenous retrovirus loci located on chromosome 1 of White Leghorn chickens
Authors: Tereba A
Published: January 2020

My previous studies demonstrated that chromosome 1 has all five of the endogenous retrovirus loci associated with nonexpression of viral proteins (gs- chf-) in White Leghorn chickens. The current study was done to localize the two defective endogenous retrovirus loci, ev3 and ev6, to determine whether nonexpression of the viral loci on chromosome 1 is a transcriptional prerequisite or a result of an underlying structural defect. The structure of ev6 is every similar to that of two other gs- chf- -associated loci, ev4 and ev5; all three contain a 5'-terminal deletion that eliminates the viral transcriptional promoter. Unlike the gs- chf- -associated loci, ev6 apparently uses an adjacent cell promoter to produce high levels of viral glycoprotein. The ev6 locus was located on the long arm of chromosome 1 by in situ hybridization, thus indicating that transcription of an endogenous retrovirus locus on chromosome 1 can occur. However, the 5'-terminal deletion in several viral loci on chromosome 1, but not on other chromosomes, suggests that the deletion may result from the events that generate or maintain these loci in the White Leghorn population. The additional finding of ev3, which has an internal deletion, on a microchromosome suggests that the mechanism that produced deletions in many chromosome 1 loci is specific for the 5' terminus.
Cloning overlapping DNA fragments from the B95-8 strain of Epstein-Barr virus reveals a site of homology to the internal repetition
Authors: Buell G N , Reisman D , Kintner C , Crouse G , Sugden B
Published: January 2020

Overlapping, sheared DNA fragments from the B95-8 strain of Epstein-Barr virus were cloned in Charon 4A. Eleven recombinant phages plus one recombinant plasmid contained all of the sequences found in B95-8 virion DNA. Analysis of recombinant DNA molecules revealed a previously undetected site of homology to the internal repetition found in Epstein-Barr virus DNA. This site was adjacent to or at a site which was unstable when the recombinant DNA was propagated as phage DNA in procaryotic hosts.
Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation
Authors: Purchio A F
Published: January 2020

pp60src, the product of the rous sarcoma virus src gene, was purified greater than 100,000-fold by a combination of ion-exchange and immunoaffinity chromatography. Incubation of pp60src purified in this fashion with [32P-gamma]ATP resulted in a single 32P-labeled protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of these gels with silver nitrate showed a predominant 60,000-dalton polypeptide which comigrated with the protein labeled with 32P in vitro. Partial digestion of this protein with V8 protease after in vitro iodination indicated that it was pp60src. These results suggest that pp60src is able to autophosphorylate.
Comparative restriction endonuclease maps of proviral DNA of the primate type C simian sarcoma-associated virus and gibbon ape leukemia virus group
Authors: Trainor C D , Wong-Staal F , Reitz M S
Published: January 2020

Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or point mutations or both exist throughout the entire unique portion of the genome among these viruses.
Identification of adenovirus 2 early region 4 polypeptides by in vitro translation and tryptic peptide map analysis
Authors: Matsuo T , Hashimoto S , Wold W S , Symington J , Rankin A , Green M
Published: January 2020

The mRNA species encoded by early region 4 (E4) (map position [mp] 91.5 to 99.3) of adenovirus 2 were isolated from the polysomes of infected KB cells and were purified by hybridization to the cloned HindIII-F fragment (mp 89.5 to 97.3) or to EcoRI-C fragment (mp 89.7 to 100). The mRNA's were translated in vitro using [35S]methionine as a labeled precursor in rabbit reticulocyte lysates treated with micrococcal nuclease as well as in wheat germ lysates. Five major (35,000-molecular-weight [35K], 23K, 22K, 21K, 18K) polypeptides were observed when the reticulocyte lysate was used. The 23K, 22K, 21K, and 18K polypeptides were also observed with the wheat germ lysate, as well as a very prominent 11K polypeptide; the 35K polypeptide was not observed. Assignment of these polypeptides to E4 was further established by hybrid arrested translation. Two-dimensional gel electrophoresis of a wheat germ translate resolved five polypeptides ranging from 18K to 23K, the major 11K polypeptide, and polypeptides of 10K and 9K. The in vitro 23K to 18K and 11K polypeptides migrated to approximately the same positions on two-dimensional gels as did seven 26K to 21K polypeptides and an 11K polypeptide synthesized in vivo (Brackmann et al., J. Biol. Chem, 255:6772--6779, 1980). Two-dimensional tryptic peptide maps demonstrated that the 35K, 23K, 22K, 21K, and 18K polypeptides are related. The peptide map of 11K is different from those of the above polypeptides, although 11K may share one tryptic methionine polypeptide with them. These results indicate that E4 encodes a major 11K polypeptide, as well as major 35K, 23K, 22K, 21K, and 18K polypeptides.
Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence
Authors: Galibert F , Chen T N , Mandart E
Published: January 2020

The complete nucleotide sequence of a woodchuck hepatitis virus genome cloned in Escherichia coli was determined by the method of Maxam and Gilbert. This sequence was found to be 3,308 nucleotides long. Potential ATG initiator triplets and nonsense codons were identified and used to locate regions with a substantial coding capacity. A striking similarity was observed between the organization of human hepatitis B virus and woodchuck hepatitis virus. Nucleotide sequences of these open regions in the woodchuck virus were compared with corresponding regions present in hepatitis B virus. This allowed the location of four viral genes on the L strand and indicated the absence of protein coded by the S strand. Evolution rates of the various parts of the genome as well as of the four different proteins coded by hepatitis B virus and woodchuck hepatitis virus were compared. These results indicated that: (i) the core protein has evolved slightly less rapidly than the other proteins; and (ii) when a region of DNA codes for two different proteins, there is less freedom for the DNA to evolve and, moreover, one of the proteins can evolve more rapidly than the other. A hairpin structure, very well conserved in the two genomes, was located in the only region devoid of coding function, suggesting the location of the origin of replication of the viral DNA.
In vitro studies of the mechanism of leukemogenesis. II. Characterization of endogenous murine leukemia viruses isolated from AKR thymic epithelial reticulum cell lines
Authors: Yanagihara K , Hamada K , Seyama T , Imamura N , Yokoro K
Published: January 2020

Thymic epithelial reticulum (TER) cell lines were established from thymuses of a young healthy AKR mouse (A2T), a preleukemic AKR mouse (A6T), and two lymphoma-bearing AKR/Ms mice (ASLT-1 and ASLT-2). Numerous type-C virus particles with occasional budding forms were observed in all cell lines. Expression of XC-detectable, N-tropic, ecotropic virus was observed in every cell line, whereas the presence of xenotropic and mink cell focus-inducing (MCF) viruses could be detected only in TER cells derived from preleukemic and leukemic mice. Expression of xenotropic virus in various cells of newborn and young AKR mice could readily be induced by IUdR treatment, whereas MCF virus was never detected in these cells, with the exception of the A2T cell line after more than 20 passages, in which MCF virus with dual-tropic infectivity emerged in addition to ecotropic and xenotropic viruses. These spontaneous and induced MCF viruses were purified, and their virological properties were characterized. The cloned MCF viruses (MCFs AT1, AT2, AT3, and AT4-IU) showed dual tropism and produced cytopathic effect-like foci in mink lung cells. Preinfection with either ecotropic or xenotropic virus interfered with the infectivity of MCF viruses. Spontaneous leukemogenesis in AKR mice was accelerated by the inoculation of MCF viruses. These findings indicate that TER cells could serve as the host cells for the genetic recombination of the endogenous MuLV; the recombinant MuLV, MCF virus, appears to be most closely associated with leukemogenesis in AKR mice.
Deletions of specific regions of the simian sarcoma-associated virus genome are found in defective viruses and in the simian sarcoma virus
Authors: Gelmann E P , Petri E , Cetta A , Wong-Staal F
Published: January 2020

Extrachromosomal DNA was isolated from tissue culture cells that were acutely infected with simian sarcoma virus (SSV) and its associated helper (simian sarcoma-associated virus [SSAV]). Two sizes of closed circular viral genomic DNA intermediates were isolated, cleaved at the single EcoRI site, and ligated to the Charon 21A phage lambda vector. Cloned molecules of the larger size all represented the full-length (9.0-kilobase [kb]) SSAV molecule. A heterogeneous group of clones was derived from the smaller DNA circles. These included the SSV genome and SSAV deletion mutants. When two SSV clones were compared with the helper, they contained the following three characteristic deletions: (i) a 250-base pair deletion in the gag gene about 1.0 kb from the 5' end of the genome; (ii) a 1.85-kb deletion in the pol gene; and (iii) a 1.9-kb deletion at the 3' end, which included part of the env gene. This latter deletion was the site of the onc gene substitution. Six other clones of the smaller molecules represented the following variants of the SSAV genome: (i) two clones of the entire genome containing only one long terminal repeat unit; (ii) one clone with the 1.85-kb deletion of the pol gene observed in SSSV; and (iii) three clones having a deletion of the 3' end of the SSAV genome. In each of the latter clones, the 5' border of the deletion was indistinguishable from the 5' border of the onc substitution in SSV. The fidelity of genetic deletions observed suggested that certain regions of the SSAV genome were deleted at a high frequency. In certain cases, these deletions may have been accompanied by a substitution of cellular sequences to generate SSV.
Induction of Epstein-Barr virus nuclear antigen and DNA synthesis in a human epithelial cell line after Epstein-Barr virus infection
Authors: Ben-Bassat H , Mitrani-Rosenbaum S , Goldblum N
Published: January 2020

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma is supported by the presence of EBV genomes in the epithelial elements of the tumor and by elevated antibody titers to EBV-specific antigens in the patients; the levels of these titers are related to the clinical course of the disease. However, since most laboratory data suggest that EBV is a B-lymphotropic virus, it is unclear how the virus becomes associated with the epithelial elements of the nasopharynx. The purpose of the present work was to find a human model system to study this association. A human epithelial line (U) was found that could be directly infected by EBV, and viral functions, the induction of EBV nuclear antigen and cellular DNA synthesis, were demonstrated. The U line was established in 1957 by the late H. J. Van Kooten (Kok-Doorschodt at the University of Utrecht), and although it is no longer diploid, it exhibits density inhibition. When U cells were infected with EBV, EBV nuclear antigen was expressed in 6 to 16% of the cells, 1 and 2 days after infection with B95-8 virus, but not with the P3HR-1 strain. No evidence for virus replication was obtained; immunofluorescence staining for early antigens and virus capsid antigens gave negative results. Quantitative adsorption experiments for EBV indicated that the adsorption capacity of U cells is significant (60% of Raji cells). The present results also demonstrated that infection with the virus overcomes block(s) in cellular DNA synthesis caused by 5-fluorodeoxyuridine. The induction of DNA synthesis was determined by increased incorporation of [3H]thymidine into the cells. The highest level of isotope incorporation was observed at about 15 h after infection and thereafter decreased. Analysis of the induced DNA indicated that it was of cellular origin.
Simian virus 40 mutant with transposed T-antigen and VP1 genes
Authors: Lazarowitz S G
Published: January 2020

A simian virus 40 mutant with the T-antigen gene transposed to the late region of the viral genome has been constructed. This transposed molecule directed the synthesis of a full-sized, 92,000-dalton large T antigen in both permissive and nonpermissive cells. This large T antigen functioned in the initiation of viral DNA replication and in the transformation of nonpermissive cells. T-antigen synthesis by this transposed genome had the characteristics of late transcription, thus indicating that functional large T antigen of simian virus 40 is not required for the initiation of late transcription.
MCF-specific murine monoclonal antibodies made against AKR-247 MCF virus recognize a unique determinant associated with the gp70-p-15(E) complex
Authors: Cloyd M W , Chesebro B , Portis J L , Weir M
Published: January 2020

Hybridomas obtained from (NFS X AKR)F mice immunized with syngeneic cells infected with AKR-247 MCF virus produced antibodies specific for only AKR-247 or closely related MCF viruses which hare a previously defined MCF antigen (MCFA-3). These monoclonal antibodies recognized a new type of viral antigenic determinant which appeared to be a conformational determinant associated with the env precursor polyprotein (pr80env) or its disulfide-linked gp70-p15(E) complex (gp80) but not with free gp70 or p15(E) or any other virion or virus-induced protein.
Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity
Authors: Centrella M , Lucas-Lenard J
Published: January 2020

Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.
Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids
Authors: Putnak J R , Phillips B A
Published: January 2020

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.