bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of spleen focus-forming virus-specific RNA sequences coding for spleen focus-forming virus-specific glycoprotein with a molecular weight of 55,000 (gp55)
Authors: Yoshida M , Yoshikura H
Published: January 2020

The 32S RNA of the Friend strain of spleen focus-forming virus (SFFV) contains two sets of sequences: about half is specific to SFFV, and the other half is in common with the sequence of the helper lymphatic leukemia virus. Fingerprinting analysis of RNase T1 oligonucleotides showed that the SFFV-specific sequences were located in two distinct regions: in the 3' half and near the 5' terminus of the genome. Translation of SFFV RNA in a cell-free system yielded three SFFV-specific polypeptides: two main products with molecular weights of about 47,000 (P47) and 16,000 (P16) and a variable amount of a product with a molecular weight of 40,000 (P40). P47 was translated from polyadenylic acid-containing fragments of 1,500 to 3,000 nucleotides with SFFV-specific sequences from the 3' half of the genome, whereas P16, which contained peptides in common with those of P47, was synthesized by smaller RNA. P47 formed in vitro was found to be structurally related to the protein portion of a glycoprotein, gp55, specifically found in SFFV-infected cells in vitro. It is concluded from the results that a defective env gene containing SFFV-specific sequences in the 3' half of the genome codes for SFFV-specific gp55.
Defective interfering particle generated by internal deletion of the vesicular stomatitis virus genome
Authors: Epstein D A , Herman R C , Chien I , Lazzarini R A
Published: January 2020

The genome structure of the long, truncated defective interfering particle derived from the heat-resistant strain of vesicular stomatitis virus has been examined. Stocks of this defective interfering particle are shown to contain several different species having information primarily from the 3' half of the vesicular stomatitis virus genome; the proportions of these components vary depending on the passage history of the stock. The two most abundant types have been identified and characterized. One has complementary 5' and 3' termini and consequently appears as a circular molecule when examined by electron microscopy. The other cannot circularize and remains linear. The circular forms are consistently 8 to 10% longer than the linear molecules. Rapid sequencing analyses reveal that both forms retain the 5' parental viral terminal sequence, but only the linear form retains the parental 3'-terminal sequence which is the complement of the 5' end. Hybridization experiments and electron microscopic analyses indicate that the linear form has retained 320 to 350 nucleotides of the 5' parental sequence and was probably generated by an internal deletion of the vesicular stomatitis virus genome.
Localization of Three Major Capped 5′ Ends of Polyoma Virus Late mRNA's Within a Single Tetranucleotide Sequence in the Viral Genome
Authors: Flavell Andrew J. , Cowie Alison , Arrand John R. , Kamen Robert
Published: January 2020

The nucleotide sequences at the 5′ ends of polyoma virus late mRNA's were determined by direct RNA sequencing of decapped and 5′-
32
P-labeled RNase T1 oligoribonucleotides. Virus-specific polyadenylated cytoplasmic RNA, which was isolated from mouse cells late during productive infection, was enzymatically or chemically treated to specifically remove the cap structure (m
7
Gppp). The unblocked 5′ ends of the viral mRNA's were then labeled enzymatically with
32
P, and the RNAs were digested with RNase T1 and fingerprinted. Three oligonucleotides derived from capped termini were isolated, and their sequences were determined to be pGmACAUUUUCUAUUUUAAGp, p(m)AmCAUUUUCUAUUUUAAGp, and p(m)AmUUUUCUAUUUUAAGp. These oligonucleotides comprise a staggered set with members 15, 17, and 18 nucleotides long, which share a common 3′ sequence for 15 nucleotides. The sequences correspond exactly to the polyoma virus DNA sequence (Arrand et al., J. Virol.
33
:606-618, 1980) from 66.79 to 66.46 map units (between 75 and 92 nucleotides preceding the ATG initiation codon for the capsid protein VP2). Previous results showed that the sequence between 13 and 64 nucleotides preceding the VP2 initiation codon corresponds to oligonucleotides reiterated in the leader sequence which is spliced onto the bodies of the three functionally distinct viral late mRNA's (Flavell et al., Cell
16
:357-372, 1979; Legon et al., Cell
16
:373-388, 1979). The three capped oligonucleotides we sequenced are derived from the first large predicted T1 oligonucleotide 5′ to those detected in the leader sequence. The occurrence of a cap at each purine of a single tetranucleotide sequence reflects micro-heterogeneity either in transcriptional initiation or in processing cleavage involved in cap syntheses.
Purification of Theiler's Murine Encephalomyelitis Virus and Analysis of the Structural Virion Polypeptides: Correlation of the Polypeptide Profile with Virulence
Authors: Lipton Howard L. , Friedmann Adam
Published: January 2020

Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs
2
SO
4
gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates.
Coding assignments of double-stranded RNA segments of SA 11 rotavirus established by in vitro translation
Authors: Smith M L , Lazdins I , Holmes I H
Published: January 2020

The segmented double-stranded (ds) RNA genome of the simian rotavirus SA 11, after denaturation, can be translated in a cell-free protein synthesizing system. Of the 11 genome segments, 9 can be resolved on polyacrylamide gels and thus could be individually isolated and translated, providing a means of identifying the polypeptide encoded by each segment. On the basis of electrophoretic mobility of products in sodium dodecyl sulfate-polyacrylamide gels, the probable gene-coding assignments of dsRNA segments 1 to 6 were determined. RNA segments 1 to 4 code for polypeptides I1, I2, I3, and I4, respectively; segment 5 codes for a polypeptide very similar in mobility to a minor polypeptide present in SA 11-infected cells, O1A; and segment 6 codes for the major inner-capsid polypeptide I5.
env Gene of Rous sarcoma virus: identification of the gene product by cell-free translation
Authors: Pawson T , Mellon P , Duesberg P H , Martin G S
Published: January 2020

Cell-free translation of polyadenylic acid-selected, denatured virion 70S RNA of the Schmidt-Ruppin strain of Rous sarcoma virus (subgroup A) yields a 64,000-Mr polypeptide which is specifically immunoprecipitated by a group-specific serum raised against envelope glycoprotein gp85. This polypeptide is not synthesized from the virion RNA of the replication-defective mutant rdNY8SR-A, which contains an extensive deletion within the envelope (env) gene. From this genetic evidence we conclude that the 64,000-Mr polypeptide represents the nonglycosylated product of the env gene and propose the designation of P64env. The 64,000-Mr polypeptide is translated from a 26S to 28S polyadenylated RNA species, whereas the p60src product is synthesized from a 20S to 22S RNA, and both Pr76gag and P180gag-pol are synthesized predominately from 34S RNA. The product of the env gene of Rous-associated virus-2 was also identified by cell-free translation.
Role of two particle types in bovine rotavirus morphogenesis
Authors: Clark S M , Spendlove R S , Barnett B B
Published: January 2020

The involvement of light (L) and dense (D) bovine rotavirus particle types during virus replication has been studied. It was found that infectious parental L virions are uncoated in vivo to a particle similar to native D particles. Differences in the rate of synthesis and relative yields of L and D particles in MDBK and MA-104 cells have been detected. Results from pulse-chase labeling experiments indicate that D particles serve as morphogenic precursors to the complete L virion.
Temperature-sensitive mutants of foot-and-mouth disease virus with altered structural polypeptides. I. Identification by electrofocusing
Authors: King A M , Newman J W
Published: January 2020

The structural polypeptides of foot-and-mouth disease virus were analyzed by electrofocusing in a polyacrylamide gel containing 9 M urea. Three versions of the technique were used to accomodate the widely differing isoelectric points of the four polypeptides. VP2 was identified by comparing mature virus with procapsids. The selective actions of proteases on virions of two serotypes and on their 12S particles were examined. From this emerged a simple test for distinguishing the similarly sized polypeptides: VP1, VP2, and VP3. The effects of carbamylation and succinylation on the charge of the polypeptides were investigated. From the properties of polypeptides modified either chemically or by mutation, it was concluded that all amino acid substitutions that might be expected to cause a charge change would be detected except for neutral-to-histidine substitutions in the most basic polypeptide, VP1. In a sample of 73 temperature-sensitive mutants, 11 classes of variant polypeptides were distinguished on the basis of charge. Their molecular weights were unchanged. Alterations were found in all structural polypeptides except VP4. Mutations affecting VP2 caused similar shifts in the precursor, VP0.
Temperature-sensitive mutants of foot-and-mouth disease virus with altered structural polypeptides. II. Comparison of recombination and biochemical maps
Authors: King A M , Slade W R , Newman J W , McCahon D
Published: January 2020

The structural polypeptides of foot-and-mouth disease virus were digested with Staphylococcus aureus V8 protease in the presence of sodium dodecyl sulfate. The protease-resistant peptides derived from temperature-sensitive mutants were compared with those of the wild type by electrofocusing in a polyacrylamide gel. Covariation between the charge shifts of different peptides indicated that they shared common sequences: only five independent peptides in all were derived from VP1, VP2, and VP3, accounting for approximately 50% of the polypeptide sequences. In two instances, amino acid substitutions that caused similar shifts in the isoelectric point were found to be located in different peptides. However, 15 mutants that possessed identical shifts in VP2 could not be distinguished by peptide analysis. The polypeptides of revertants able to grow at the nonpermissive temperature were compared with those of the parental mutants. By this test, 6 of the 12 distinguishable classes of coat protein mutations were found to covary with temperature sensitivity. In addition to true revertants, several phenotypic revertants which possessed a second charge change, either in a different structural polypeptide or in a different region of the same polypeptide, were isolated. The orientation of the recombination map was deduced from the loci of the coat protein mutations.
Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA
Authors: Lusby E , Fife K H , Berns K I
Published: January 2020

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).
Genome of porcine transmissible gastroenteritis virus
Authors: Brian D A , Dennis D E , Guy J S
Published: January 2020

The Purdue strain of transmissible gastroenteritis virus, a porcine coronavirus, was grown to titers of greater than 10(8) PFU/ml in a swine testicle cell line, and the RNA was isotopically labeled with [3H]uridine. The RNA was extracted from purified virus and was found to have the following properties. (i) It consisted primarily of a homogeneous large-molecular-weight species which electrophoretically migrated with an apparent molecular weight of 6.8 X 10(6) under denaturing conditions. (ii) It migrated electrophoretically at the same rate on nondenaturing gels before and after heat denaturation, suggesting that it does not consist of subunits. (iii) It was susceptible to pancreatic RNase A digestion in high (0.3 M) NaCl. (iv) It was polyadenylated to the extent that greater than 60% of the native RNA bound to oligodeoxythymidilic acid-cellulose under conditions of high (0.5 M) NaCl. RNA extracted from virions was infectious. This coronavirus can therefore be characterized as a positive-strand RNA virus.
Novel p19-related protein in Rous-associated virus type 61: implications for avian gag gene order
Authors: Shealy D J , Mosser A G , Rueckert R R
Published: January 2020

Virions of Rous-associated virus type 61 contain a previously unrecognized p19-related protein, called p19f, which comigrates with gag protein p12 during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels but can be separated by gel filtration chromatography in 6 M guanidine hydrochloride. It is shown that the existence of p19f accounts for the earlier inability to order p27 and p12 by the pactamycin mapping procedure. Remapping with pactamycin by using methods which take this new protein into account yielded a gag gene order of NH2-p219-p27-p12-p15-COOH. It also confirmed earlier positions for the env and pol genes and placed unclassified protein p10 near a translational initiation site. The pactamycin-derived mapping position of p12 differs from reports based on tryptic analysis. An analysis of procedural shortcomings emphasizes the need for more definitive determinations of the avian gag gene order.
Assembly of avian reticuloendotheliosis virus: association of the core precursor polypeptide with the intracellular ribonucleoprotein complex
Authors: Wong T C , Lewis R B , Bose H R , Kang C Y
Published: January 2020

A virus-specific ribonucleoprotein complex is present in the cytoplasm of reticuloendotheliosis virus-transformed chicken bone marrow cells. This ribonucleoprotein complex contains viral reverse transcriptase activity and may represent a precursor to the budding virion. The major viral polypeptide associated with the ribonucleoprotein complex was a polypeptide with a molecular weight of 63,000. This protein exhibited a precursor-product relationship with the major reticuloendotheliosis virus structural core protein p29. Core polypeptides were not associated with the intracellular ribonucleoprotein complex. Thus, p29 was incorporated into the virion in the form of its precursor Pr63. The cleavage of Pr63 in the ribonucleoprotein complex was accomplished either during the budding process of shortly after the release of particles from the cell.
Reovirus progeny subviral particles synthesize uncapped mRNA
Authors: Zarbl H , Skup D , Millward S
Published: January 2020

Reovirus progeny subviral particles were isolated from L-cells at late times postinfection. It has been shown (D. Skup and S. Millward, J. Virol. 34: 490--496, 1980) that these progeny subviral particles have masked capping enzymes, indicating that mRNA synthesized by these particles should be uncapped. When progeny subviral particles were used for mRNA synthesis in vitro, they failed to incorporate the beta-phosphate of [beta-32P]GTP into the 5' terminal. Direct analysis of reovirus mRNA synthesized by progeny subviral particles in the presence of either [alpha-32P]GTP or [alpha-32P]CTP indicated that the 5' terminal was uncapped, having the structure pGpC... The implications of this finding to the reovirus replicative cycle are discussed.
Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2
Authors: Cohen G H , Ponce de Leon M , Diggelmann H , Lawrence W C , Vernon S K , Eisenberg R J
Published: January 2020

Capsids of herpes simplex virus (HSV) types 1 and 2 contain seven polypeptides ranging in molecular weight from 154,000 to 12,000 (termed NC-1 through NC-7 in order of descending molecular weight). Antibodies prepared to HSV-1 capsid polypeptides isolated from sodium dodecyl sulfate-polyacrylamide gels reacted in an immunofluorescence assay against HSV-1-infected KB cells. Three of the antibodies (anti-NC-1, anti-NC-2, and anti-NC-3,4) also reacted with HSV-2-infected cells. Tryptic peptide analysis showed that each of the HSV-1 capsid polypeptides had a unique methionine peptide profile, and none appeared to be derived from the major capsid polypeptide. Comparative peptide analysis of HSV-1 and HSV-2 showed that one polypeptide (NC-7, 12,000 molecular weight) had an identical methionine peptide profile and a very similar arginine peptide profile in both virus types. The arginine peptide profile of NC-7 of HSV-1 was very different from the arginine profile of KB histone H4. Although there were certain intertypic similarities in the methionine peptide profiles of the other capsid components especially in NC-1 (the major capsid protein), there was no case where the tryptic peptides were identical in the two virus types.
Relationship between integrated and nonintegrated viral DNA in rat cells transformed by polyoma virus
Authors: Gattoni S , Colantuoni V , Basilico C
Published: January 2020

Fischer rat fibroblasts transformed by polyoma virus contain, in addition to viral sequences integrated into the host genome, nonintegrated viral DNA molecules, whose presence is under the control of the viral A gene. To understand the mechanism of production of the "free" viral DNA, we have characterized the DNA species produced by several rat lines transformed by wild-type virus or by ts-a polyoma virus and compared them with the integrated viral sequences. Every cell line tested yielded a characteristic number of discrete species of viral DNA. The presence of defectives was a very common occurrence, and these molecules generally carried deletions mapping in the viral "late" region. The production of multiple species of free viral DNA was not due to heterogeneity of the transformed rat cell population, and its pattern did not change upon fusion with permissive mouse cells. Analysis of the integrated viral DNA sequences in the same cell lines showed, in most cases, a full head-to-tail tandem arrangement of normal-size and defective molecules. The free DNA produced by these lines faithfully reflected the integrated species. This was true also in the case of a cell line which contained a viral insertion corresponding to approximately 1.3 polyoma genomes, with each of the repeated portions of the viral DNA molecule carrying a different-size deletion. These results support the hypothesis that the free DNA derives from the integrated form through a mechanism of homologous recombination leading to excision and limited replication.
Use of [125I]deoxycytidine to detect herpes simplex virus-specific thymidine kinase in tissues of latently infected guinea pigs
Authors: Fong B S , Scriba M
Published: January 2020

The footpad skin and the lumbosacral dorsal root ganglia were removed from inbred guinea pigs at different times after subcutaneous infection with herpes simplex virus type 2 (HSV-2) in both hind footpads. These tissues, shown by our previous study to harbor latent HSV, were dispersed into single cells. The presence of virus-specific thymidine kinase (TK) in these cells was assayed by the uptake and phosphorylation of [125I]deoxycytidine in culture. [125I]deoxycytidine was shown to be a specific substrate for the HSV-coded TK. The method could detect herpes TK activity in a culture of 10(6) cells with less than 0.1% of the cells being virally infected. The enzyme was readily detected in footpad cells of acutely (24 h) but not of latently (14 days to 1 year) infected guinea pigs. No herpes TK was found either in the sensory ganglionic cells of guinea pigs during the early and late phases of latent infection. It is concluded that HSV-2, while residing in the footpads and the lumbosacral ganglia of the guinea pig during latent infection, does not express any viral TK function.
tRNA's and priming of RNA-directed DNA synthesis in mouse mammary tumor virus
Authors: Peters G , Glover C
Published: January 2020

The low-molecular-weight RNAs of mouse mammary tumor virus (MuMTV) were examined by two-dimensional acrylamide gel electrophoresis. Unlike other retroviruses, MuMTV was found to contain only two major fractions of tRNA. These have been fully characterized and shown to correspond to the published sequences for tRNA1+2Lys and tRNA3Lys. By determining which of these tRNA's was most tightly associated with the MuMTV genome and which of them acquired label from [alpha-32P]deoxynucleoside triphosphates in limited DNA synthesis reactions, we identified tRNA3Lys as the primer for MuMTV reverse transcription in vitro. tRNA3Lys does not share any unusual sequence feature with the other previously characterized retrovirus primers, tRNATrp and tRNAPro.
Exclusion of bacteriophage T1 by bacteriophage lambda. II. Synthesis of T1-specific macromolecules under N-mediated excluding conditions
Authors: Gawron M C , Christensen J R , Shoemaker T M
Published: January 2020

The results of experiments investigating T1 macromolecular synthesis under N-mediated excluding conditions failed to demonstrate a substantial alteration in the T1 mRNA production in excluding cultures at any stage in the T1 infectious cycle. The number of T1 DNA sequences in the excluding culture was found to be one-third to one-half that found in T1-infected cultures. The most severe reduction in T1-specific macromolecules was seen in protein synthesis. Total incorporation of labeled amino acids was reduced sixfold, and gel experiments confirmed that the T1-specific proteins capable of detection are reduced in excluding cells.
Analysis of secondary modifications of mouse mammary tumor virus proteins by two-dimensional gel electrophoresis
Authors: Nusse R , Janssen H , de Vries L , Michalides R
Published: January 2020

The structural proteins of mouse mammary tumor virus (MMTV) were analyzed by two-dimensional electrophoresis on isoelectric focusing and sodium dodecyl sulfate gels. Many of the viral proteins displayed heterogeneity in charge due to variable contents of carbohydrates (in particular, sialic acid) and phosphate residues. Neuraminidase treatment of the virions influenced the isoelectric pattern of the envelope glycoproteins. The glycoproteins of an MMTV variant which was attenuated by replication in feline kidney cells had different isoelectric points. This suggested that the acquisition of an altered carbohydrate configuration had changed the host range of the virus. The major MMTV structural core protein, p27, consisted of two species, which had identical iodinated tryptic peptide compositions but differed in phosphate contents. Another MMTV phosphoprotein, p21, was separated into four different phosphorylated species. Phosphorylation of p21 could be performed in vitro by the MMTV virion-associated protein kinase. This enzyme also has a high affinity for MMTV p30 as a substrate. Possible functions of this enzyme are discussed.
Characterization of JC papovavirus adapted to growth in human embryonic kidney cells
Authors: Miyamura T , Yoshiike K , Takemoto K K
Published: January 2020

Human papovavirus JC virus was adapted to growth in human embryonic kidney (HEK) cells. After eight passages, the HEK-adapted JC virus produced high virus yields and was capable of forming plaques in HEK monolayer cultures. Eleven plaque-purified stocks were prepared and characterized. Biologically, the plaque-purified virus induced tumor and viral antigens in HEK cells earlier and in a higher percentage of cells than uncloned virus. Cytopathic changes were also evident sooner and were more extensive. The DNA from uncloned as well as plaque-purified isolates was analyzed by restriction endonuclease cleavage followed by gel electrophoresis. The DNA from uncloned HEK-adapted virus was heterogeneous. Plaque-purified virus isolates yielded DNA which, although much less heterogeneous than the uncloned stock, still consisted of two or more species of viral DNA.
Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit
Authors: Luka J , Jörnvall H , Klein G
Published: January 2020

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified 700-fold to apparent homogeneity from Raji and Namalwa cell extracts by a three-step procedure involving heat treatment, DNA-cellulose chromatography, and hydroxyapatite chromatography. Acid-fixed nuclear binding and complement fixation were used to monitor antigenic specificity. Purified EBNA was also capable of specifically inhibiting the regular anticomplement immunofluorescence reaction for EBNA against Raji target cells. The purified antigen had a molecular weight of 170,000 to 200,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it yielded a single 48,000-dalton (48K) monomer. An EBNA-associated protein was also purified from the same cell extract. It had a molecular weight of about 200,000 and yielded a single 53K protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same protein was also found in Epstein-Barr virus negative B-cell lymphoma lines. The two types of protein were characterized by amino acid composition and peptide mapping. The results showed that the 53K and 48K protein components have no long regions in common; this excludes that the smaller product arises by breakdown of the larger product. Residue distributions were different, but an excess of hydrophilic residues was found in both proteins, suggesting a certain overall similarity in properties. 53K components from different cell lines appeared to differ somewhat. Epstein-Barr virus-positive lines carry two 53K components, one of which may be a slightly modified 53K product. Immunocomplexing assay showed that the 48K, but not the 53K, protein carries EBNA specificity. In mixtures, the 53K protein is co-precipitated with the 48K protein. The data suggest that EBNA may form a complex with the 53K proten within the cell.
Use of lambda pMu bacteriophages to isolate lambda specialized transducing bacteriophages carrying genes for bacterial chemotaxis
Authors: Kondoh H , Paul B R , Howe M M
Published: January 2020

A general method for constructing lambda specialized transducing phages is described. The method, which is potentially applicable to any gene of Escherichia coli, is based on using Mu DNA homology to direct the integration of a lambda pMu phage near the genes whose transduction is desired. With this method we isolated a lambda transducing phage carrying all 10 genes in the che gene cluster (map location, 41.5 to 42.5 min). The products of the cheA and tar genes were identified by using transducing phages with amber mutations in these genes. It was established that tar codes for methyl-accepting chemotaxis protein II (molecular weight, 62,000) and that cheA codes for two polypeptides (molecular weights, 76,000 and 66,000). Possible origins of the two cheA polypeptides are discussed.
Acetylation of histone-like proteins of adenovirus type 5
Authors: Fedor M J , Daniell E
Published: January 2020

We examined acetylation of the histone-like adenovirus core proteins VII and V and the precursor of the major core protein, pVII, by measuring the incorporation of [14C]acetate. Adenovirus proteins pVII and V appeared to be acetylated, whereas protein VII was not. Label incorporated into these viral proteins in the form of acetate was metabolically stable, and labeling was not enhanced by treatment with sodium butyrate, an inhibitor of histone deacetylases. Viral protein acetylation therefore differs from the reversible acetylation of histones that has been implicated in transient alterations of chromatin structure. Inhibition of protein synthesis in infected cells resulted in a proportional reduction in [14C]acetate uptake into pVII and V, suggesting that these proteins undergo acetylation during protein synthesis and not as a post-translational modification. Therefore, these viral proteins are probably acetylated amino-terminally, a characteristic shared by three of the five major histone classes.
Role of the host cell in bacteriophage T4 development. II. Characterization of host mutants that have pleiotropic effects on T4 growth
Authors: Stitt B L , Revel H R , Lielausis I , Wood W B
Published: January 2020

Mutant host-defective Escherichi coli that fail to propagate bacteriophage T4 and have a pleiotropic effect on T4 development have been isolated and characterized. In phage-infected mutant cells, specific early phage proteins are absent or reduced in amount, phage DNA synthesis is depressed by about 50%, specific structural phage proteins, including some tail and collar components, are deficient or missing, and host-cell lysis is delayed and slow. Almost all phage that can overcome the host block carry mutantions that map in functionally undefined 'nonessential' regions of the T4 genome, most near gene 39. The mutant host strains are temperature sensitive for growth and show simultaneous reversion of the ts phenotype and the inability to propagate T4+. The host mutations are cotransduced with ilv (83 min) and may lie in the gene for transcription termination factor rho.
Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes
Authors: Milavetz B I , Spotila L D , Thomas R , Huberman J A
Published: January 2020

The nonhistone proteins sedimenting in low-salt glycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradients as the first dimension and sodium dodecyl sulfate-gel electrophoresis as the second dimension. By densitometric quantitation of the radiolabeled proteins present in each fraction of the gradients, it was possible to identify sedimenting with all or a fraction of the simian virus 40 chromosomes. VP-1 sedimented with simian virus 40 chromosomes; additional evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI 6.0; Mr 32,000, pI 7.2; Mr 35,000, pI 8.5; and Mr 80,000, pI 7.2) sedimented with specific subsets of chromosomes.
Detection and characterization of mouse mammary tumor virus cell surface antigens: estimation of antigen abundance by protein A assay
Authors: Callis A H , Ritzi E M
Published: January 2020

Antisera against the following mouse mammary tumor virus (MMTV) structural proteins were used to detect MMTV cell surface antigens: (i) the 27,000-dalton nucleoid protein, p27; (ii) the 36,000-dalton envelope glycoprotein, gp36; and (iii) the 52,000-dalton exterior envelope glycoprotein, gp52. We report here the development of an adherent-cell isotopic staphylococcal protein A (SPA) test (ISPAT) for MMTV structural proteins which allows for the detection of an MMTV membrane-associated antigen as well as an estimate of its relative abundance on the cell surface. This test demonstrated that the gp52 was the predominant MMTV cell surface antigen detected on both C3H and GR mouse mammary tumor cells. In a comparative study with anti-gp52 and anti-gp36 sera, SPA-specific binding with anti-gp36 serum was found to be only 5 to 6% of that obtained for the external virion glycoprotein, gp52. Both direct and indirect ISPAT indicated the presence of a low but detectable number of gp36 determinants on GR-MMTV cells; however, these gp36 determinants, unlike gp52 determinants, appeared to be exposed by the fixation procedure used. Only 0.9 to 1.1% of the gp52-specific binding was detected when anti-gp36 serum was allowed to react with viable cells. The binding of [125I]SPA achieved with anti-p27 serum was even less than that detected with gp36-directed reagents, indicating that p27 is not a cell surface antigen. The use of fluoresceinated SPA further demonstrated that p27 and gp36 reactivity was only associated with a small number of cells in each of the mammary cultures tested. When N-[4-(5-nitro-2-furyl)-2-thiazoly]-formamide-induced C3H bladder tumor cells were subjected to a gp52-directed ISPAT, the failure to detect gp52-specific binding demonstrated the specificity of this assay for MMTV gp52-expressing cells. In addition to detecting and characterizing MMTV cell surface antigens, the newly developed adherent cell assay could measure changes in the abundance of cell surface gp52. When dexamethasone-treated and untreated GR cells were compared, measurements of gp52-specific SPA binding indicated that dexamethasone stimulation leads to a 12.2-fold increase in the amount of cell surface gp52 detected.
DNA of avian myeloblastosis-associated virus type 2 integrates at multiple sites in the chicken genome
Authors: Bergmann D G , Baluda M A
Published: January 2020

The cellular sites of integration of the avian myeloblastosis-associated virus type 2 (MAV-2) DNA have been examined by Southern blot analysis of cellular DNA from infected cloned and uncloned chicken embryonic fibroblasts. Provirus-cell juncture fragments were not detected in restriction enzyme digests of DNA from MAV-2-infected uncloned cells. However, each MAV-2-infected cell clone examined produced a unique set of junctive bands. Thse findings indicate that multiple sites of integration exists for MAV-2 proviruses in cellular DNA.
Selective amplification of mouse mammary tumor virus in mammary tumors of GR mice
Authors: Fanning T G , Puma J P , Cardiff R D
Published: January 2020

DNAs extracted from the mammary tumors of GR mice were analyzed for mouse mammary tumor virus proviral sequences by the restriction enzyme-Southern blot procedure. The tumor DNAs contain more proviral copies of mouse mammary tumor virus than DNA from a nonmalignant tissue. The degree of proviral amplification is small (ca. one to five additional copies) and appears to be variable from tumor to tumor. The restriction patterns of the amplified proviral sequences suggest a clonal origin for the tumor mass. In addition, the restriction patterns observed after digestion with the enzymes BglII and SacI indicate that only one of the proviruses endogenous to GR mice is amplified. The amplified provirus found in GR mammary tumors is identical to the provirus that is missing in GR-Mtv-2- mice, a congenic line exhibiting a low mammary tumor incidence.
Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells
Authors: Stabel S. , Doerfler W. , Friis R. R.
Published: January 2020

The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.
Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones
Authors: Highfield P E , Rafield L F , Gilmer T M , Parsons J T
Published: January 2020

Unintegrated, circular viral DNA, isolated from Prague A avian sarcoma virus (PrA-ASV)-infected quail cells (QT6), was cloned in the lambda vector lambda gtWES x lambda B. Three independent lambda-ASV recombinants were identified, and each contained a complete copy of the PrA-ASV genome. The arrangement of the ASV sequences within the recombinants was determined by restriction enzyme analysis and hybridization with labeled ASV-specific complementary DNA. One of the recombinants (lambda RPA101) resulted from cloning at the EcoRI site located within the terminally repeated sequence and therefore was virtually co-linear with PrA-ASV virion RNA. The other two recombinants (lambda RPA102 and 103) resulted from cloning at the EcoRI site located within the viral env gene. By restriction enzyme analysis and by measurement of R-loops formed between lambda RPA101 and PrA-ASV virion 35S RNA, the viral genome was estimated to be 9,100 bases in length. Genome length viral DNA purified from clones lambda RPA102 and 103 was biologically active. Transfection of chicken embryo cells with viral DNA, in the form of either circles or linear dimers, produced foci of transformed cells within 8 to 10 days. Linear DNA was much less efficient at inducing transformation. Viral DNA from the clone lambda RPA101 was unable to cause transformation; the basis for this defect is unknown.
Identification and initial characterization of a new low-molecular-weight virus-encoded T antigen in a line of simian virus 40-transformed cells
Authors: Spangler G J , Griffin J D , Rubin H , Livingston D M
Published: January 2020

SV80 cells, a simian virus 40 (SV40)-transformed derivative of a strain of human fibroblasts, synthesize an 8-kilodalton anti-T reactive polypeptide in addition to large T and small t antigens. Although not observed during lytic infection carried out under a variety of conditions, an anti-T reactive molecule which comigrated with the SV80 8-kilodalton protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis was synthesized by one of five other SV40-transformed cell lines studied. The SV40 8-kilodalton protein was present in lysates of cells exposed to a brief pulse of radioactive methionine and did not accumulate during an extended chase period. This polypeptide could not by generated by mixing an unlabeled extract of SV80 cells with a labeled extract of infected monkey cells. The 8-kilodalton molecule reacts with antibody raised against homogeneous large T antigen, is present only in the cytoplasm, is not complexed with T, lacks DNA-binding properties, and is not phosphorylated. This protein could be translated in a cell-free system programmed by SV40-specific mRNA. At least two messenger species (approximately 19S and approximately 22S) directed its synthesis. Tryptic peptide analysis of [35S]methionine-labeled proteins demonstrated that the 8-kilodalton protein contains all eight of the common T/t peptides and one additional peptide not present in the maps of t or T. It lacks both of the t-unique peptides. The organization of the integrated viral sequences which encode this molecule was determined by restriction endonuclease analysis. In particular, SV80 cells contain at least two integrated SV40 genomes which are oriented in tandem, with an intervening cellular sequence..
Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity
Authors: Gomatos P J , Stamatos N M , Sarkar N H
Published: January 2020

We previously reported that virus-specific particles with polycytidylate [poly(C)]-dependent RNA polymerase activity accumulated at 30 degrees C in reovirus-infected cells. These particles sedimented heterogeneously from 300 to 550S and traversed through a 40% glycerol cushion to the pellet in 3 h at 190,000 x g. In the present report, we found that smaller particles with poly(C)-dependent RNA polymerase activity remained in the glycerol cushion. These smaller, enzymatically active particles, when purified, sedimented at 15 to 1S. They were spherical or triangular with a diameter of 11 to 12 nm. They were comprised mostly, and likely solely, of one reovirus protein, sigma NS. No particles with poly(C)-dependent RNA polymerase activity were found in mock-infected cells. Chromatography on the cation exchanger, CM-Sephadex, ascertained that sigma NS was the poly(C)-dependent RNA polymerase and showed its existence in two forms. In one form, it was enzymatically active and eluted from the column at 0.5 M KCl. In the enzymatically inactive state, it did not bind to the column. Our results suggest that the enzymatically active form of sigma NS carries a greater net positive charge than the inactive form. They also suggest that both forms of sigma NS are associated with a particle which has poly(C)-dependent RNA polymerase activity.
RNA synthesis of vesicular stomatitis virus. X. Transcription and replication by defective interfering particles
Authors: Rao D D , Huang A S
Published: January 2020

In cells coinfected by standard vesicular stomatitis virus (VSV) and defective interfering (DI) T particles, small RNA consisting of 46 nucleotides was synthesized in molar excess over other VSV-specific RNAs. Although its rate of synthesis increased over time, small RNA accumulated linearly, suggesting that the molecule is unstable. In contrast, replication of the genome RNA of DI T particles was relatively constant after 3 h of infection, resulting in the intracellular accumulation of stable genomic and antigenomic RNA of DI T particles. Coinfection of cells with DI T particles and selected temperature-sensitive mutants from all five complementation groups of VSV indicated that the replication of DI genomes was controlled separately from the synthesis of small RNA. Also, when viral RNA replication was inhibited by cycloheximide, small RNA continued to be synthesized as long as there were enough templates present. These results indicate that small RNA is synthesized by the enzyme(s) involved in VSV transcription and that its dependence on RNA replication is due to the requirement for template amplification.
Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants
Authors: Erwin C , Brown D T
Published: January 2020

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.
Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells
Authors: Condra J H , Lazzarini R A
Published: January 2020

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.