bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of endogenous avian myeloblastosis virus information in different chicken cells
Authors: Chen J H
Published: January 2020

Uninfected chicken cells were found to contain endogenous avian myeloblastosis virus (AMV)-specific information. Different tissues from chicken embryos and chickens expressed different amounts of the AMV-specific information. The endogenous AMV-related RNA was most abundant in bone marrow cells, which contained about 20 copies per cell. About 5 to 10 copies of AMV endogenous RNA per cell were found in embryonic yolk sac cells and bursa cells. The spleen, muscle, liver, and kidney cells of chickens and the fibroblasts of chicken embryos contained about two copies per cell. The amounts of AMV endogenous RNA in bone marrow, yolk sac, and bursa varied with age. From 19-day-old embryos to 2-week-old chickens, the bone marrow contained 20 copies of AMV RNA per cell. Bone marrow cells from 2-year-old chickens contained five copies per cell. Yolk sac cells of 10-day-old embryos and 1-day-old chickens were found to contain two copies per cell, whereas in 15- to 17-day-old embryos, these cells contained 5 to 10 copies. These results indicate that the level of endogenous AMV expression correlates with the development of granulopoiesis of the chicken hemopoietic system. The results of experiments on the thermostability of RNA-DNA hybrids indicated that the endogenous AMV RNA is closely related to viral AMV RNA. The expression of endogenous AMV information is independent of the activity of the chick helper factor. This endogenous AMV information is expressed as 20 to 21S RNA in both bone marrow and yolk sac cells.
Lack of focus formation of S+L- mink cells by the ecotropic murine leukemia viral genome
Authors: Ishimoto A
Published: January 2020

Infection of mink S+L- cells with ecotropic murine leukemia virus, achieved by phenotypic mixing with xenotropic virus, did not result in the induction of transformed foci. Also, clonal line of S+L- mink cells, chronically infected with ecotropic murine leukemia virus, which produce both the ecotropic virus and its murine sarcoma virus pseudotype are morphologically indistinguishable from normal S+L- mink cells. Neutralizing antiserum added to S+L- mink cells inoculated with xenotropic virus 24 h earlier prevented the formation of foci of transformed cells. Together, these observations indicate that focus formation in S+L- cells requires a regional spread of infection, with the insertion of additional murine sarcoma virus genomes and resultant transformation occurring from a gene dosage effect.
Oncogenic transformation by by equine herpesviruses. II. Coestablishment of persistent infection and oncogenic transformation of hamster embryo cells by equine herpesvirus type 1 preparations enriched for defective interfering particles
Authors: Robinson R A , Vance R B , O'Callaghan D J
Published: January 2020

Infection of permissive hamster embryo cells with virus preparations enriched for defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) resulted in persistent infection and oncogenic transformation. Six cell lines, designated DI-5 to -10, exhibited biological properties (immortality, increased saturation density, growth in soft agar, etc.) inherent to transformed cells, but 2 to 18% of the total cells in these cell lines were shown to release virus as judged by electron microscope studies and infectious center assays. The released virus was shown to be standard EHV-1 and not to contain DI particles as determined by density measurements of the viral DNA in the analytical ultracentrifuge and by interference assays using the released virus. Tumorigenicity studies revealed that inoculation of these persistently infected cells into newborn LSH inbred hamsters resulted in a lethal, fulminating hepatitis, whereas inoculation into older immunocompetent hamsters (+4 weeks) led to the development of metastatic fibrous sarcomas. Tumor cell lines (DI-5T to -10T) established from these sarcomas were shown to be transplantable and virus nonproducers. Hybridization analyses of cellular DNAs from DI transformed and tumor cell lines using 32P-labeled genomic EHV-1 DNA as probes indicated that the whole virus genome was detectable in multiple copies (23 to 45) in the transformed cells and that DNA sequences representing only 43.5 to 56.6% of the virus genome were present in amounts of 2 to 4 copies per cell in the DI tumor cells. Expression of these viral DNA sequences as demonstrated by the detection of virus-neutralizing antibodies, 50% neutralizing dose titers ranging from 1:50 to 1:1,000, in the sera of animals inoculated with either the virus-producing transformed cells or the virus-nonproducing tumor cells. Further, EHV-1-specific proteins were detected in the membrane and the perinuclear region of bothDI transformed and tumor cells by indirect immunofluorescent assays using antisera against EHV-1 structural antigens, EHV-1 nonstructural antigens, or preparations of EHV-1 DI particles. The roles of DI particles in mediating persistent infection and cellular transformation are discussed.
Molecular cloning of unintegrated and a portion of integrated moloney murine leukemia viral DNA in bacteriophage lambda
Authors: Berns A J , Lai M H , Bosselman R A , McKennett M A , Bacheler L T , Fan H , Maandag E C , van der Putten H V , Verma I M
Published: January 2020

A covalently closed circular form of unintegrated viral DNA obtained from NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV) and a port of the endogenous M-MLV from the BALB/Mo mouse strain have been cloned in bacteriophage lambda. The unintegrated viral DNA was cleaved with restriction endonuclease HindIII and inserted into the single HindIII site of lambda phage Charon 21A. Similarly high-molecular-weight DNA from BALB/Mo mice ws cleaved sequentially with restriction endonucleases EcoRI and HindIII and separated on the basis of size, and one of the two fractions which reacted with an M-MLV-specific complementary DNA was inserted into the HindIII site of Charon 21A. Recombinant clones containing M-MLV-reacting DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and infectivity assays. The restriction endonuclease map of the insert derived from unintegrated viral DNA, lambda x MLV-1, was comparable to published maps. Electron microscope analysis of the hybrid formed between lambda x MLV-1 DNA and 35S genomic M-MLV RNA showed a duplex structure. The molecularly cloned lambda x MLV-1 DNA contained only one copy of the long terminal repeat and was not infectious even after end-to-end ligation of the insert DNA. The insert DNA derived from endogenous M-MLV, lambda x MLVint-1, contained a DNA stretch measuring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in length. The viral part of the insert showed the typical restriction pattern of M-MLV DNA except that a single restriction site, PvuII, in the 5' long terminal repeat was missing. Reconstructed genomes containing the 5' half derived from the integrated viral DNA and the 3' half derived from the unintegrated viral DNA were able to induce XC plaques after transfection in uninfected mouse fibroblasts.
Synthesis and processing of viral glycoproteins in two nonconditional mutants of Rous sarcoma virus
Authors: Linial M , Fenno J , Burnette W N , Rohrschneider L
Published: January 2020

We have studied the pattern of glycoprotein synthesis in two nonconditional mutants of Rous sarcoma virus. One mutant, SE33, produces no viral particles but synthesizes Pr92env, which is cleaved intracellularly to mature glycoproteins. The second mutant, SE521, encodes a gPr92env which is not cleaved to gp85 or gp37 and therefore produces virions with the phenotype of Bryan RSV(-) or NY8. Neither of these mutants have detectable genomic deletions. The study of these mutants has led to the following conclusions. (i) In the absence of particle production or p15 synthesis, gPr92env can be cleaved to the mature glycoprotein which is found on the cell surface. (ii) Noncleaved gPr92env is not packaged into virions but is found on the cell surface. (iii) gPr92env alone can account for subgroup specific viral interference. (iv) gPr92env is probably transported to the cell surface before additional glycosylation or cleavage to mature virion glycoprotein. The nonprocessed precursor of SE521 appears to be glycosylated normally, and thus far we have been unable to determine the basis for the defect in this mutant.
Separation of cyanogen bromide-cleaved peptides of the vesicular stomatitis virus glycoprotein and analysis of their carbohydrate content
Authors: Kingsford L , Emerson S U , Kelley J M
Published: January 2020

The purified glycoprotein of vesicular stomatitis virus was cleaved at methionine residues with cyanogen bromide, and the resultant peptides were analyzed by two-dimensional electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Five peptide bands were resolved in cylindrical gels run under nonreducing conditions. After reduction and electrophoresis in the second dimension, 11 peptides were resolved, indicating that several were originally linked by disulfide bonds. Double-label experiments indicated that at least 8 of the 11 peptides were unique. The major oligosaccharide chains were attached to two different cyanogen bromide peptides. In addition, six other peptides contained small amounts of sialic acid, fucose, and mannose, indicating that the glycoprotein contains more carbohydrate chains than the two major ones which have been reported previously.
Unique species of mRNA from adenovirus type 7 early region 1 in cells transformed by adenovirus type 7 DNA fragment
Authors: Yoshida K , Fujinaga K
Published: January 2020

Adenovirus type 7 (Ad7) early region 1 mRNA species transcribed in rat cell lines transformed by the HindIII-I . J fragment (the left 7.8% of the viral genome) and in human KB cells infected with Ad7 were mapped on the viral genome, using S1 nuclease gel and diazobenzyloxymethyl paper hybridization techniques. At the early stage of productive infection, two mRNA's (950 and 840 nucleotides long) with the common 5' and 3' ends but different internal splicings were mapped from region 1A (map units 1.4 to 4.3), and one mRNA (2,310 nucleotides long, with the internal splicing between map units 9.9 to 10.1) was mapped from region 1B (map units 4.6 to 11.4). At the late stage, these early spliced mRNA's were also found and at least three additional Ad7 mRNA's were identified: 700-nucleotide-long mRNA in region 1A; and 1,100- and nucleotide-long mRNA's in region 1B. In transformed rat cell lines, two early region 1A mRNA's (950 and 840 nucleotides long) were also transcribed. Surprisingly, in addition, several unique Ad7 mRNA's, not found in productivity infected cells, were identified in all of the transformed cell lines. Their molecular sizes and coding sequences varied in individual cell lines. However, these mRNA's had the 5' end-proximal portion in region 1B and the 3' end-proximal portion in region 1A, these portions being transcribed by extending from region 1B to 1A on viral DNA fragments joined in a tandem array in transformed cells.
Heterogeneity of the 3' end of minus-strand RNA in the poliovirus replicative form
Authors: Richards O C , Ehrenfeld E
Published: January 2020

The 3' terminus of the strand (minus strand) complementary to poliovirion RNA (plus strand) has been examined to see whether this sequence extends to the 5'-nucleotide terminus of the plus strand, or whether minus-strand synthesis terminates prematurely, perhaps due to the presence of a nonreplicated nucleotide primer for initiation of plus-strand synthesis. The 3' terminus was labeled with 32P using [5'-32P]pCp and RNA ligase, and complete RNase digests were performed with RNases A, T1, and U2. 32P-oligonucleotides were analyzed for size by polyacrylamide-urea gel electrophoresis. The major oligonucleotide products formed were consistent with the minus strand containing 3' ends complementary and flush with the 5' end of the plus strand. However, a variable proportion of the isolated minus strands from different preparations were heterogeneous in length and appeared to differ from each other by the presence of one, two, or three 3'-terminal A residues.
Synthesis and processing of glycoproteins gD and gC of herpes simplex virus type 1
Authors: Cohen G H , Long D , Eisenberg R J
Published: January 2020

Herpes simplex virus type 1 (HSV-1) contains five glycoproteins, designated gA, gB, gC, gD, and gE. The present studies focused on the synthesis and processing of two of these, gC and gD. By using monoprecipitin antibody to gC, we demonstrated an antigenic and structural relationship between the precursor, pgC(110), and the product, gC(130). Tryptic peptide analysis showed that pgC and gC shared methionine peptides and that these molecules had the same fingerprint pattern as that of gC(130) extracted from the purified virion. These results suggested that post-translational processing of gC involved no major changes in methionine-containing tryptic peptides or in the cleavage sites required to generate those peptides. The syntheses of gC and gD were compared. We found that the glycoproteins were synthesized starting at different times in the infectious cycle; pgD was detected by 2 h postinfection, whereas pgC was first detected at 4 to 6 h postinfection. Both precursor molecules, pgC(110) and pgD(52), are basic glycopolypeptides, and in both cases processing involved changes in molecular weight and charge. These changes were detected by two-dimensional gel electrophoresis. Both glycoproteins exhibited heterogeneity, displayed as a series of spots (6 for gD and 15 to 20 for gC) of increasing negative charge and molecular weight. Neuraminidase treatment decreased the size, number, and acidic charge of the spots, suggesting that processing was due in part, but not entirely, to addition of sialic acid to pgD and pgC.
Epstein-Barr virus RNA. V. Viral RNA in a restringently infected, growth-transformed cell line
Authors: King W , Thomas-Powell A L , Raab-Traub N , Hawke M , Kieff E
Published: January 2020

A continuous lymphoblastoid cell line, IB-4, was established by infection and growth transformation of normal neonatal B lymphocytes with the B95-8 isolate of Epstein-Barr virus (EBV). The IB-4 cells contained the intranuclear antigen, EBNA, but not early antigen, EA. The fragments produced by the digestion of intracellular episomal viral DNA (density, 1.700 to 1.720 g/cm3) with EcoRI restriction endonuclease were identical in size to the A, B, C, E, F, G, and H fragments of virion DNA. As expected from the previous observation that episomal intracellular DNA is circular, the fragment containing the rightward terminal sequences of EBV DNA in IB-4 cells was larger than the corresponding fragment of linear viral DNA, probably as a consequence of covalent linkage to the leftward terminal fragment. Also, two fragments, EcoRI-I and -J, which were adjacent to each other in the virion DNA, were absent from the intracellular DNA. The labeled EcoRI-J of viral DNA hybridized instead to a new fragment equal in size to EcoRI-I and -J combined. Analysis of viral RNA in IB-4 cells showed that RNAs encoded by more than 30% of the viral DNA comprised approximately 0.06% of the nuclear RNA, whereas RNAs encoded by 20% and 10% of the viral DNA comprised approximately 0.06% and 0.003% of the polyadenylated and polyribosomal RNAs, respectively. Viral mRNA (polyribosomal RNA) was encoded by DNA which mapped at 0.05 x 10(8) to 0.36 x 10(8) daltons and to a lesser extent by DNAs which mapped at 0.62 x 10(8) to 0.67 x 10(8), 0.70 x 10(8) to 0.73 x 10(8), and 1.13 x 10(8) to 1.15 x 10(8) daltons in the B95-8 genome. The most agundant nuclear viral RNAs were encoded primarily by DNA which mapped at the same loci; but RNAs encoded by many other fragments of viral DNA could also be detected among nuclear RNAs. Viral mRNA(s) (polyribosomal) was encoded by about 40% of the internal reiteration and by 25% of the BamHI-H fragments which mapped from 0.32 x 10(8) to 0.36 x 10(8) daltons, nuclear RNAs were encoded by at least 57% of the internal reiteration and 40% of BamHI-H. These data indicate that there is selective accumulation of some viral RNAs within the nucleus of IB-4 cells and that there is selective post-transcriptional processing of these RNAs. Finer mapping of the DNA which encodes mRNA (polyribosomal) in IB-4 cells indicated that some of this DNA is deleted in the DNA of the P3 HR-1 virus, the only isolate of EBV which cannot initiate growth transformation. These data, therefore, support the hypothesis that expression of this region of EBV genome is important for growth transformation or for the maintenance of restrigent infection.
A cloned polyoma DNA fragment representing the 5' half of the early gene region is oncogenic
Authors: Chowdhury K , Light S E , Garon C F , Ito Y , Israel M A
Published: January 2020

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.
Construction of a specific amber codon in the simian virus 40 T-antigen gene by site-directed mutagenesis
Authors: Rawlins D R , Muzyczka N
Published: January 2020

The site-directed bisulfite mutagenesis technique has been used to construct a specific mutation, am404, at nucleotide position 3124 in the simian virus 40 genome. The mutation was contained within a PstI restriction site (map position 0.27) and prevented cleavage by PstI at that position. Nucleotide sequence analysis of the mutagenized region indicated that only a single base pair change had occurred: a guanosine x cytosine leads to adenine x thymine transition. Comparison of the nucleotide sequence of am404 with the known DNA sequence of simian virus 40 indicted that the mutation in am404 resulted in the conversion of a glutamine codon to an amber codon. am404 could not replicate autonomously when transfected into monkey cells (BSC-40) but did replicate when it was cotransfected with the late deletion helper virus dl1007. On the basis of its position in the T-antigen, gene am404 should produce a T-antigen 24% shorter than the wild-type protein.
Phosphorylation of the nonstructural proteins encoded by three avian acute leukemia viruses and by avian fujinami sarcoma virus
Authors: Bister K , Lee W H , Duesberg P H
Published: January 2020

The gag gene-related, nonstructural proteins of three avian acute leukemia viruses (namely, myelocytomatosis viruses MC29 and CMII and avian erythroblastosis virus) and of avian Fujinami sarcoma virus (FSV) isolated by immunoprecipitation from cellular lysates with anti-gag serum were shown to be phosphoproteins in vivo. The specific 32P radioactivity of the nonstructural proteins of MC29, CMII, and FSV was significantly higher than that of helper viral, intracellular gag proteins. Two of these proteins, i.e., the 140,000-dalton FSV and the 110,000-dalton MC29 proteins, were also phosphorylated in vitro by a kinase activity associated with immunocomplexes. This kinase activity is either separated from these proteins or inactivated by incubation of cellular lysates with normal serum followed by adsorption to staphylococcal protein A or sedimentation at 100,000 x g or both. It remains to be resolved whether the 110,000-dalton MC29 and 140,000-dalton FV proteins, in addition to being substrates for phosphorylation, also have intrinsic kinase activity.
gA and gB glycoproteins of herpes simplex virus type 1: two forms of a single polypeptide
Authors: Eberle R , Courtney R J
Published: January 2020

Utilizing a combination of preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-hydroxylapatite column chromatography, we have separated and purified the gA and gB glycoproteins of the major virus-specific glycoprotein region from herpes simplex virus type 1-infected cells. By using purified antigen preparations, antisera specific to each of these glycoproteins were produced. Immunoprecipitation from detergent extracts of infected cells and radioimmune precipitation of the purified antigens have shown that the anti-gA and anti-gB sera each recognize both the gA and the gB glycoproteins. The anti-gA serum was also shown to neutralize virus despite the presence of only minute quantities of the gA glycoprotein in virions. Pulse-chase studies have indicated that the gA and gB glycoproteins are synthesized from a common precursor polypeptide. Together, these data demonstrate that the gA and gB glycoproteins of herpes simplex virus type 1 are antigenically similar but not identical and probably represent two different forms of the same polypeptide which differ in their degree of glycosylation.
Avian erythroblastosis virus produces two mRNA's
Authors: Anderson S M , Hayward W S , Neel B G , Hanafusa H
Published: January 2020

We analyzed the viral mRNA's present in fibroblast nonproducer clones transformed by avian erythroblastosis virus. Two size classes of mRNA (28 to 30S and 22 to 24S) were identified by solution hybridization with both complementary DNA strong stop and complementary DNA made against the unique sequences of avian erythroblastosis virus. Based upon the kinetics of hybridization with complementary DNA made against the unique sequences of avian erythroblastosis virus, we estimated that there were 400 to 500 copies of the 28 to 30S RNA per cell and 200 to 250 copies of the 22 to 24S RNA per cell. Both RNA species were packaged in the virion. In vitro translation of the 28 to 30S virion RNA yielded a 75,000-dalton protein which was the 75,000-dalton gag-related polyprotein found in avian erythroblastosis virus-transformed cells. In vitro translation of the 22 to 24S virion RNA yielded two proteins (46,000 and 48,000 daltons). This indicates that there may be two genes in avian erythroblastosis virus, one coding for the 75,000-dalton gag-related polyprotein and the second coding for the 46,000- or 48,000-dalton protein or both.
Simian virus 40-associated small RNA: mapping on the simian virus 40 genome and characterization of its synthesis
Authors: Alwine J C , Khoury G
Published: January 2020

The simian virus 40 (SV40)-associated small RNA (SAS-RNA) has previously been shown to arise late in SV40 lytic infection and to bear homology with the SV40 early mRNA's, or the SV40 genome, at map position 0.21. By using hybridization analysis, we determined that the SAS-RNA is between 62 and 65 nucleotides in length and its homology region lies between nucleotides 2760 and 2825 of the SV40 late(+) DNA strand. Viable deletion mutants which lacked part or all of this region made no SAS-RNA, strongly indicating that this is the coding region of the SAS-RNA. The expected sequence for the SAS-RNA, determined from the DNA sequence between nucleotides 2760 and 2825, appeared to be very pyrimidine rich (76% uridine and cytidine). Deletion or alteration of sequences immediately preceding the SAS-RNA coding region (approximately nucleotides 2716 to 2748) resulted in the loss of SAS-RNA production. These sequences may be part of a promotor for SAS-RNA synthesis or a processing site for its excision from long nuclear late transcripts. Under growth conditions where late transcription was not fully initiated (tsA58 at 41 degrees C; wild-type SV40 in the presence of 1-beta-D-arabinofuranosylcytosine), no SAS-RNA was produced, indicating that the expression of the SAS-RNA is regulated by a mechanism related to the control of late transcription.
Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones
Authors: Groner B , Buetti E , Diggelmann H , Hynes N E
Published: January 2020

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.
Properties of Cells Carrying the Herpes Simplex Virus Type 2 Thymidine Kinase Gene: Mechanisms of Reversion to a Thymidine Kinase-Negative Phenotype
Authors: Bastow K. F. , Darby G. , Wildy P. , Minson A. C.
Published: January 2020

We have isolated cells with a thymidine kinase-negative (tk
−
) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10
−3
to 10
−4
, and resistance to either compound conferred simultaneous resistance to the other. tk
−
revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences.
Nucleic acids of respiratory syncytial virus
Authors: Lambert D M , Pons M W , Mbuy G N , Dorsch-Hasler K
Published: January 2020

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.
Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. II. Evidence for a strand displacement mechanism in plus-strand synthesis
Authors: Boone L R , Skalka A M
Published: January 2020

Analyses of the native DNA product of mellitin-activated avian retrovirus reverse transcription have revealed a unique structure. The vast majority of the molecules were linear, either 7.7 (genome) or 8.0 (extended genome) kilobases in length, and contained single-stranded DNA branches distributed throughout. These conclusions are based on electrophoretic properties of intact and restriction endonuclease-treated molecules before and after treatment with single-strand-specific nuclease S1. Preliminary data from linear viral DNA extracted from infected cells suggest that these molecules have a similar structure. The findings summarized in this report and those in the preceding paper indicated that the single-stranded branches are of positive polarity and are generated by a strand displacement mechanism. The existence of these branches suggests a role for strand displacement in replication and recombination.
Transformation-dependent alterations in the oligosaccharides of Prague C Rous sarcoma virus glycoproteins
Authors: Hunt L A , Lamph W , Wright S E
Published: January 2020

The influence of cell transformation on the glycosylation of viral envelope glycoproteins was examined by high-resolution gel filtration and specific glycosidase digestions of 3H-sugar-labeled glycopeptides from nondefective and transformation-defective Prague C strains of Rous sarcoma virus replicated in fibroblasts from the same chicken embryo. The major difference in glycosylation attributable to the viral transformation of the host cells was an increase in this relative amount of larger acidic-type oligosaccharides containing additional "branch" sugars (NeuNAc-Gal-GlcNAc-) compared with the smaller acidic-type and neutral-type oligosaccharides. There was also a shift in size distribution of neutral-type oligosaccharides toward smaller oligomannosyl cores in the transforming versus nontransforming virus glycopeptides. These alterations were consistent with a transformation-dependent increase in the extent of intracellular processing of a common precursor structure for the asparagine-linked oligosaccharides of Rous sarcoma virus.
Endodeoxyribonuclease activity associated with Rauscher murine leukemia virus
Authors: Kopchick J J , Harless J , Geisser B S , Killam R , Hewitt R R , Arlinghaus R B
Published: January 2020

Preparations of purified Rauscher murine leukemia virus were found to contain an endodeoxyribonuclease after disruption of the virus with nonionic detergents. The enzyme makes single-strand breaks in linear or covalently closed circular phage double-stranded DNA molecules. The enzyme was partially purified by ion-exchange chromatography on DEAE- and carboxymethyl-Sepharose columns followed by electrophoresis in DNA-containing polyacrylamide gels. The enzyme was separated from reverse transcriptase (p80pol), and the final endonuclease preparation contained no detectable reverse transcriptase activity. The DEAE-Sepharose column-purified endonuclease activity contained a polypeptide of about 40,000 Mr that we term p40. Peptide mapping experiments demonstrated that p40 shares methionine-labeled tryptic peptides with Pr200gag-pol and Pr135pol. Six major methionine-labeled tryptic peptides derived from p40 were found in Pr200gag-pol, but only five of these were detected in Pr135pol. The four core proteins (p30, p15, pp12, and p10) and p80pol plus p40 account for most, but not all, of the peptide sequences of Pr200gag-pol. The endonuclease-associated p40 is similar in size and precursor origin to the avian retrovirus-coded endonuclease (p32). In view of these similarities to the avian p32 endonuclease and its association with partially purified Rauscher murine leukemia virus-associated endonuclease preparations, we propose that p40 is the Rauscher murine leukemia virus-coded endonuclease.
Stable hairpin structure within the 5'-terminal 85 nucleotides of poliovirus RNA
Authors: Larsen G R , Semler B L , Wimmer E
Published: January 2020

The primary sequence of a 5'-terminal fragment of poliovirus type 1 RNA, generated by digestion with RNase III, has been determined. This sequence reveals the presence of a stable hairpin structure beginning nine nucleotides from the terminally linked protein VPg. The sequence does not contain (i) the initiation codons AUG or GUG or (ii) the putative ribosome-binding sequence complementary to the 3' end of eucaryotic ribosomal 18S RNA. The stem-and-loop structure identified can be drawn in either plus or minus RNA strands. It is unclear to which strand functional significance (if any) can be assigned. It is possible that the hairpin structure is involved in ribosomal recognition and translation or in RNA synthesis by interacting with replicase molecules.
Protein-coding potential of mouse mammary tumor virus genome RNA as examined by in vitro translation
Authors: Dickson C , Peters G
Published: January 2020

The protein-coding capacity of the mouse mammary tumor virus genome has been examined by in vitro translation of genome length and polyadenylated subgenomic fragments of viral RNA. Intact genome RNA of about 35S programmed synthesis of the Pr77gag, Pr110gag and Pr160gag/pol precursors seen in infected cells in vivo. Polyadenylated RNA fragments of 18 to 28S encoded products whose tryptic peptide maps resembled those of the nonglycosylated precursor to the envelope glycoproteins, confirming the gene order 5'-gag-pol-env-3'. Translation of polyadenylated RNA fragments smaller than 18S yielded a series of related proteins whose peptide maps bore no resemblance to any of the virion structural proteins. Thus, a region of the mouse mammary tumor virus genome distal to the env gene appears to have an open reading frame sufficient to encode at least 36,000 daltons of protein as of yet unknown function.
Analysis of bacteriophage SP82 major "early" in vitro transcripts
Authors: Panganiban A T , Whiteley H R
Published: January 2020

A restriction map was constructed for the 17.5-kilobase SalI C fragment of SP82 DNA. Unfractionated SP82 DNA, the SalI C fragment, and restriction fragments derived from SalI-C were used as templates for in vitro synthesis by the Bacillus subtilis RNA polymerase, and the resulting transcripts were analyzed by gel electrophoresis. Comparison of the RNA species obtained from SalI-C with those produced from unfractionated DNA indicated that most of the RNAs and all of the major transcripts originate from the SalI C fragment; this fragment contains one copy of the terminally redundant portion of the genome. Seven major transcripts, a bidirectional terminator site, and 5 of the 13 minor transcripts were located within the 13-kilobase redundant region. The binding of polymerase to fragments of DNA produced by digestion of SalI-C with HpaII and HhaI was used to identify promoter-bearing regions within SalI-C.
Frog virus 3 requires RNA polymerase II for its replication
Authors: Goorha R
Published: January 2020

The involvement of host cell RNA polymerase II in the replication of frog virus 3 (FV 3) was examined in alpha-amanitin-sensitive or -resistant Chinese hamster ovary (CHO) cells in the presence and absence of alpha-amanitin. In the presence of alpha-amanitin, FV 3 replicated normally in resistant CHO cells but failed to do so in sensitive CHO cells. Synthesis of virus-specific RNAs and proteins was inhibited in sensitive cells infected in the presence of alpha-amanitin, but in alpha-amanitin-resistant cells, as expected, virus-specific protein synthesis and, by implication, virus-specific RNA synthesis were not affected by the presence of the drug. Inhibition of FV 3 replication was maximum when alpha-amanitin was added to sensitive CHO cells before virus adsorption, but the drug had no effect on virus replication if added after the adsorption. These data indicate that host RNA polymerase II was required for early transcription of the FV 3 genome and confirm a nuclear requirement for FV 3 RNA synthesis (R. Goorha et al., Virology 82:34-52, 1978).
Stability of bacteriophage phi X174-specific mRNA in vivo
Authors: Hayashi M N , Hayashi M
Published: January 2020

Different-size species of phi X174-specific mRNA's decayed exponentially, with half-lives ranging from 4.5 to 11 min.
Spleen focus-forming virus: specific neutralization by antisera to certain gag gene-encoded proteins
Authors: Anand R , Ruscetti S , Steeves R A , Lilly F
Published: January 2020

Immunization of rats with syngeneic cells infected with spleen focus-forming virus (SFFV) but not with its helper, Friend murine leukemia virus (FMuLV), produces antisera which specifically neutralize SFFV, and not FMuLV, in the Friend virus complex. To determine which SFFV-encoded protein molecule bears the antigen recognized by these neutralizing antibodies, we studied different lots of rat anti-SFFV antiserum by immunoprecipitation and virus neutralization assays. The ability of these sera to neutralize SFFV correlated with the titer of antibodies to p45gag and not with the titer of those to gp52, suggesting that the neutralizing antibodies recognize the p45gag molecule. To verify this specificity for p45gag, we tested antisera to various MuLV gag gene-encoded proteins for neutralization of SFFV. Goat anti-Rauscher murine leukemia virus (RMuLV) p30 and goat anti-RMuLV p10 sera neither precipitated p45gag from SFFV-infected nonproducer cells nor neutralized SFFV. In contrast, goat anti-RMuLV Pr65gag and goat anti-RMuLV p12 sera precipitated p45gag from SFFV-infected cells and also specifically neutralized SFFV in the Friend virus complex. These findings suggest that, unlike the gag proteins coded for by FMuLV, the proteins coded for by defective SFFV are incorporated into the envelope of virions carrying the SFFV genome, but not into the envelope of those carrying the helper FMuLV genome.
Inheritance of susceptibility to the myeloproliferative sarcoma virus: effect of the Fv-2 locus and evidence for a myeloproliferative sarcoma virus resistance locus
Authors: Ostertag W , Odaka T , Smadja-Joffe F , Jasmin C
Published: January 2020

Myeloproliferative sarcoma virus (MPSV) causes a generalized stem cell leukemia with erythroid and myeloid hyperplasia in adult mice. MPSV also transforms fibroblasts. Mice congenic for the Fv-2 locus showed marked differences in susceptibility to MPSV according to the Fv-2 genotype. MPSV was injected into C57BL/6 Fvs and C57BL/6 Fv-2r mice congenic except for the Fv-2 locus. C57BL/6 mice with the Fvs genotype were much more susceptible to MPSV than were those with the Fvr genotype. Both DDD Fv-2r mice congenic with DDD Fv-2s mice except for the Fv-2 locus and DDD Fv-2s mice, however, were sensitive to spleen focus formation by MPSV. These data indicate that at least one additional resistance locus to MPSV is present in C57BL/6 mice but not in DDD mice. Both the Fv-2 locus and the putative MSPV resistance locus (loci) Mpsvr appear to be epistatic to either of the sensitivity loci. Fibroblast focus formation by MPSV was obtained well in C57BL/6 Fv-2r and C57BL/6 Fvs fibroblasts, indicating that the genes for MPSV resistance (Fv-2r and Mpsvr) were not operating in fibroblast cells. A model is proposed which may account for the differences in response of genetically different mice to MPSV and Friend spleen focus-forming virus.
Glycosylation sites of influenza viral glycoproteins: characterization of tryptic glycopeptides from the A/USSR(H1N1) hemagglutinin glycoprotein
Authors: Basak S , Pritchard D G , Bhown A S , Compans R W
Published: January 2020

Glycosylated tryptic peptides of the hemagglutinin (HA) glycoprotein of influenza A/USSR/90/77(H1N1) virus were separated by a combination of ion-exchange chromatography and gel filtration. Seven different glycosylated tryptic peptide classes were obtained from the HA1 polypeptide, and only one glycosylated peptide was obtained from the HA2 polypeptide. Several of the tryptic fragments of HA1 and the HA2 glycopeptides were sulfated. The nature of the carbohydrate chain in each of the glycosylated tryptic peptides was determined from observations of the incorporation of different sugar precursors and susceptibility to cleavage by the enzyme endoglycosidase H and by compositional analysis by gas chromatography. Such analyses showed that three types of carbohydrate chains were present in HA1 (type I [complex], type II [high mannose], and hybrid type), whereas HA2 contained only type I oligosaccharide chains. The amino acid composition of each of the glycosylated tryptic peptides was also determined.
Semliki Forest virus multiplication in clones of Aedes albopictus cells
Authors: Tooker P , Kennedy S I
Published: January 2020

A total of 115 clones of Aedes albopictus cells were examined for their response to infection with Semliki Forest virus. Virus yield and cytopathology showed a bimodal distribution. More than 68% of the clones gave low yields of virus (between 8 x 10(6) and 2 x 10(8) PFU/ml) with no discernable cytopathology, and 30% gave high yields of virus (between 1 x 10(9) and 8 x 10(9) PFU/ml) and showed moderate to severe cytopathology. To determine the level at which restriction in virus growth occurs in the low-virus-producing clones, we compared the nature and extent of several virus-directed events in selected low-virus-producing clones with the same events in high-virus-producing clones. Specifically, we compared virus-specified polypeptide synthesis, positive- and negative-strand RNA synthesis, adsorption, uncoating, and transfection with virion 42S RNA. These studies showed that whereas events before negative-strand RNA synthesis and all subsequent virus-specified events were markedly reduced in the low-virus-producing lines, compared with the high-virus-producing lines. Thus, the restriction in virus growth in the low-virus-producing lines occurs at the level of synthesis of negative-strand RNA. The consequence of this restriction in an early step in the virus multiplication cycle is discussed in terms of the survival of invertebrate cells after alphavirus infection.
Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus
Authors: Acheson N H
Published: January 2020

The efficiency of processing of polyoma viral RNA and of its export from nucleus to cytoplasm was measured in primary mouse kidney cells by comparing the initial rates of incorporation of [3H]uridine into cytoplasmic and nuclear viral RNA. Appropriate methods of cell fractionation were chosen to maximize yields of cytoplasmic RNA and to minimize leakage of nuclear RNA. Incorporation of [3H]uridine into cellular 4S RNA in the cytoplasm was followed to monitor pool equilibration and maintenance of an excess of radioactive precursor throughout the experimental period. During the early phase of infection (9 to 11 h, in the presence of 5-fluorodeoxyuridine), viral RNA was rapidly and efficiently exported from nucleus to cytoplasm. Viral RNA appeared in the cytoplasm within 6 min of its synthesis, greater than half of the viral RNA synthesized in the nucleus was exported to the cytoplasm. In contrast, during the late phase of infection (28 to 30 h), viral RNA was exported more slowly, appearing in the cytoplasm 12 to 20 min after its synthesis, and much less efficiently-only 5% of late nuclear transcripts was exported. The poor efficiency of processing of late viral RNA may be, in part, a result of (i) the presence in nuclear transcripts of non-mRNA sequences which are removed during processing; (ii) the presence in nuclear transcripts of multiple copies of mRNA sequences, only one of which is incorporated into mature mRNA; and (iii) inefficient polyadenylation of viral nuclear RNA.
Involvement of a high-molecular-weight polyprotein translational product of Snyder-Theilen Feline sarcoma virus in malignant transformation
Authors: Reynolds F H , Van de Ven W J , Blomberg J , Stephenson J R
Published: January 2020

The previously described high-molecular-weight polyprotein major translational product of the Snyder-Theilen strain of feline sarcoma virus (FeSV) was shown to possess protein kinase activity with specificity for tyrosine acceptor sites. Cells transformed by Snyder-Theilen FeSV exhibited constitutively elevated levels of phosphotyrosine and a concomitant reduction in epidermal growth factor (EGF) binding sites. By endpoint cloning in microtiter plates, a number of transformation-defective (tf) mutants of the Snyder-Theilen strain of FeSV were isolated. Mink cells nonproductively infected by such mutants were morphologically nontransformed, failed to grow in soft agar, bound EGF as efficiently as control mink cells, and lacked rescuable transforming virus. Although the level of expression of the major viral polyprotein translational product in td mutant-infected clones was comparable to that of wild-type (wt) transformants, the polyprotein in mutant clones lacked detectable protein kinase activity and total cellular phosphotyrosine levels were not elevated significantly above control values. Of a large number of wt Snyder-Theilen FeSV-transformed mink cell clones isolated, the majority were found to revert to a nontransformed morphology upon continuous passage in cell culture. Such nontransformed variants, as well as a Gardner FeSV-transformed mink cell revertant, lacked detectable polyprotein expression and exhibited levels of phosphotyrosine and EGF binding similar to those of control mink cells. These findings provide strong evidence favoring the involvement of the Snyder-Theilen FeSV-encoded high-molecular-weight polyprotein and its associated tyrosine-specific protein kinase activity in transformation.
Structure and origin of a snapback defective interfering particle RNA of vesicular stomatitis virus
Authors: Schubert M , Lazzarini R A
Published: January 2020

The nucleotide sequence of the region which covalently links the complementary strands of the "snapback" RNA of vesicular stomatitis virus, DI011, is (Formula: see text). Both strands of the defective interfering (DI) particle RNA were complementary for their full length and were covalently linked by a single phosphate group. Because the strands were exactly the same length and complementary, template strand and daughter strand nucleocapsids generated during replication of DI 011 were undistinguishable on the basis of sequence, a property not shared by other types of DI particle RNAs. Treatment of the RNA with RNase T1 in high-ionic-strength solutions cleaved the RNA only between positions 1 and 1'. These results and the availability of the guanosine residue in position 1' to kethoxal, a reagent that specifically derivatizes guanosines of single-stranded RNA, suggest that steric constraints keep a small portion of the "turnaround" region in an open configuration. The sequence of the turnaround region was not related in any obvious way to the sequences at the 3' and 5' termini and limited the number of possible models for the origin of this type of DI particle RNA. Two models for the genesis of DI 011 RNA are discussed. We favor one in which the progenitor DI 011 RNA was generated by replication across a nascent replication fork.
In vitro transcription of the inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites
Authors: Venkatesan S , Moss B
Published: January 2020

Specific RNAs synthesized in vitro by vaccinia virus cores were analyzed with the aid of DNA from the terminal 9,000 base pairs of the genome that was cloned in phage lambda, pBR322, and the single-stranded phage fl. Three mRNA's coding for polypeptides with molecular weights of 7,500 (7.5K), 19K, and 42K were shown to have sizes and map positions similar to those described for mRNA's made early in infection. A previously undescribed transcript made in vivo and in vitro, with a 5' end at about 8.7 kilobase pairs from the end of the genome, was also detected. After chemical removal of the terminal 7-methylguanosine residue, the 5' ends of the RNAs were specifically labeled by enzymatic capping and the mapped by gel electrophoresis of nuclease-resistant RNA.DNA hybrids, as well as by hybridization of the end-labeled RNA to immobilized DNA restriction fragments. Analysis of the purified cap structures demonstrated that three of the mRNA's have both m7G(5')pppAm and, m7G(5')pppGm ends, indicating some degree of terminal heterogeneity. The fourth transcript has exclusively m7G(5')pppAm ends. By synthesizing RNA in the presence of [beta-32P]GTP, it could be shown that cap sites correspond to sites of initiation of RNA synthesis.