bluuebunny/crossref_metadata_2025 huggingface dataset:
Helper-independent transformation by unintegrated Harvey sarcoma virus DNA
Authors: Lowy D R , Rands E , Scolnick E M
Published: January 2020

We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins.
Abrogation of Fv-1b restriction with murine leukemia viruses inactivated by heat or by gamma irradiation
Authors: Bassin R H , Duran-Troise G , Gerwin B I , Rein A
Published: January 2020

Fv-1b restriction in BALB/3T3 cells is temporarily abrogated following infection with N-tropic murine leukemia virus. The mechanism of this phenomenon was investigated by comparing the inactivation rates for viral infectivity and for the ability of the same virus to abrogate Fv-1 restriction. Inactivation of the abrogating ability of N-tropic murine leukemia virus following graduated doses of gamma radiation proceeded at half the rate of that for viral infectivity. This result indicates that viral RNA must function in abrogating Fv-1b restriction but that only a portion of the viral genome is required. The inactivation kinetics of N-tropic murine leukemia virus were also determined following incubation of virus at 43 degrees C. Abrogating ability of N-tropic murine leukemia virus was found to be about six times as stable under these conditions as was viral infectivity. Interestingly, virion-associated reverse transcriptase activity was inactivated at the same rate as was viral infectivity, indicating that this enzyme may not need to function during abrogation. Virus heated at 43 degrees C was used to study the kinetics of the abrogation phenomenon itself. Abrogation was shown to be transient, requiring 6 to 9 h after virus infection to become maximally effective and beginning to disappear after about 18 h. The data reported here confirm the idea that abrogation of Fv-1 restriction can be separated experimentally from virus replication, and they raise the possibility that a separate biochemical pathway exists for incoming viral RNA in Fv-1 restrictive cells.
Purification of the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components
Authors: Collins J K , Knight C A
Published: January 2020

Hemagglutinin from influenza A/PR8 virus was purified after treatment of the virus with sodium deoxycholate followed by extraction with tri-n-butyl phosphate. This fully disrupted the virus while preserving hemagglutinating activity. The hemagglutinin was obtained in the form of small aggregates that could be separated from other viral components. Purified hemagglutinin was hydrolyzed to determine carbohydrate composition and digested with Pronase to analyze oligosaccharide structures. Sugars present in the hemagglutinin were galactose, mannose, fucose, and glucosamine in molar rates of about 6:11:2:5, and these comprised 16% of the hemagglutinin glycoprotein. Oligosaccharides obtained from virus included a major component of a molecular weight of 2,800, composed of glucosamine, galactose, mannose, and fucose, and a minor heterogenous component of a molecular weight of 1,500 to 2,000, containing predominantly mannose. The 2,800-molecular-weight oligosaccharide was a constituent of the hemagglutinin, and treatment of this large oligosaccharide with specific exo-glycosidases demonstrated the presence of terminal galactose and fucose and allowed the deduction of a general structure for this component.
Control of protein synthesis in extracts from poliovirus-infected cells. I. mRNA discrimination by crude initiation factors
Authors: Helentjaris T , Ehrenfeld E
Published: January 2020

By using cell-free systems prepared from uninfected and poliovirus-infected cells, we have been able to demonstrate that crude preparations of initiation factors from infected cells do not stimulate the initiation of translation by polyribosomes containing endogenous host cell mRNA. When tested with polysomes containing endogenous viral mRNA, however, they were able to stimulate initiation of translation nearly as well as uninfected cell initiation factors. The uninfected cell initiation factor preparations were able to stimulate initiation of translation of both cell and viral mRNA. The results indicate an mRNA-specific activity present in crude initiation factor preparations from infected cells. Furthermore, the ability of eIF2 from infected cells to form a ternary complex with GTP and formyl [35S]methionine-tRNAfmet, an mRNA-independent step in initiation, was found not to be deficient. Implications of these data for proposed mechanisms of poliovirus-induced host cell shutoff are discussed.
Sequence complexity and relative abundance of vaccinia virus mRNA's synthesized in vivo and in vitro
Authors: Boone R F , Moss B
Published: January 2020

The sequence complexity and relative abundance of vaccinia virus mRNA's, synthesized in vivo and in vitro, have been measured by DNA-RNA hybridization. Up to 42% of [3H]thymidine-labeled virus DNA can be protected from digestion with nuclease S1, a single-strand specific nuclease, after annealing to excess polyadenylylated mRNA obtained at 7 h after infection. In contrast, only 26% of vaccinia virus DNA is protected when hybridized to polyadenylylated RNA obtained at 2 h after infection in the presence of an inhibitor of DNA synthesis. That the 94 kilobases transcribed early are a subset of the 152 kilobases present late was suggested by hybridization of DNA with a mixture of early and late RNAs. Some control of transcription is lost when virus purified by procedures that include sonic treatment is used for infection since under these conditions similar proportions of DNA are protected by either excess early or late RNA. Excess RNA, synthesized in vitro by enzymes within purified vaccinia virus particles, hybridized to approximately the same fraction of the DNA as did RNA present at late times in vivo. A second type of transcriptional control was demonstrated by kinetic analysis of the hybridization of polyadenylylated RNA to labeled DNA. With virion DNA used as the probe, a single abundance class for early RNA, two classes differing 11-fold in abundance for late RNA, and two classes differing 43-fold in abundance for in vitro RNA were found. To be able to detect high-abundance RNAs of very low sequence complexity, labeled complementary DNA probes to early, late, and in vitro polyadenylylated RNA were used. Evidence that, at late times, RNAs totaling 9 kilobases of sequence complexity are present 40 to 500 times more frequently than the bulk of the virus-specific RNA was obtained. In contrast, the highest abundance class of RNA present at 2 h after infection corresponded to 7 kilobases present in only a 13-fold molar excess over the majority of virus-specific sequences. RNA synthesized in vitro was found to contain a small amount of sequence information, approximately 2 kilobases, which occurred 150 times more frequently than the majority of viral sequences. Studies using hybridization of viral DNA to labeled complementary DNA probes also suggested that 52 to 59% of the polyadenylylated RNA present at 2 h after infection and 82 to 92% of that at 7 h are virus specific.
Detection of proviral DNA in horse cells infected with equine infectious anemia virus
Authors: Rice N R , Simek S , Ryder O A , Coggins L
Published: January 2020

Equine infectious anemia virus (EIAV) recently has been shown to possess a high-molecular-weight RNA genome and a virion reverse transcriptase. We completed the demonstration that EIAV is a retrovirus by showing the presence of proviral DNA in equine cells infected in vitro, but not in normal horse DNA. These studies were performed by using a highly representative cDNA probe synthesized by the virion polymerase. It was found that this cDNA reassociated extensively, and with high thermal stability, with either viral RNA or DNA extracted from infected cells, but showed no detectable reassociation with DNA from uninfected horse cells. Similarly, sequences related to EIAV were neither found in the DNA of four other Equus species, nor in a variety of other mammals including sheep, cows, pigs- dogs, cats, and humans; nor did EIAV cDNA hybridize with a variety of other retrovirus RNAs. These experiments were performed under conditions of very low stringency to enable detection of distantly related sequences, with a sufficient ratio of DAN to cDNA to allow detection of less than one viral copy per haploid genome. We conclude that EIAV is not an endogenous virus of the horse or of the other species tested.
Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts
Authors: Verma I M
Published: January 2020

Genome-length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of avian myeloblastosis virus. Moloney murine leukemia virus, and clone 124 mouse sarcoma virus. The size of the genomelenth cDNA transcripts was measured on either alkaline sucrose gradients or alkaline agarose gels. The longest cDNA transcripts synthesized by using avian myeloblastosis virus, Moloney murine leukemia virus, and clone 124 mouse sarcoma virus were 7, 9 and 6 kilobases (kb), respectively. The in vitro system used was capable of synthesizing double-stranded DNA, but the plus strands (same polarity as the viral RNA) were only 0.5 to 1.5 kb long. Lone Moloney murine leukemia virus cDNA transcripts were used as templates to synthesize the second plus strand. Essentially two strategies were employed as follows. (i) The 3' ends of the cDNA transcripts were extended by addition of 50 to 100 dAMP residues by terminal deoxynucleotidyl transferase. The (dA)n-tailed cDNA transcripts were used as templates along with an oligomer of dT as primer and Escherichia coli DNA polymerase to synthesize the plus strands. (ii) DNase-digested calf thymus DNA was used to prime the synthesis of plus strands on long cDNA with E. coli DNA polymerase I. In both cases, the synthesis of the plus strands was monitored by increased resistance of the cDNA templates to single-strand-specific S1 nuclease. The double-stranded DNA was fractionated on neutral sucrose gradients. Analysis of the double-stranded DNA synthesized by using oligo(dT) primer showed the plus strands to be about 5 to 6 kb long, whereas the plus strands synthesized by using DNase-digested calf thymus DNA primers were only 0.3 to 0.5 kb long. Double-stranded DNA synthesized by either method has an average size of 6 x 10(6) daltons. Double-stranded DNA was also synthesized by using cDNA transcripts as templates without the addition of any primers. In this case, the plus strands were covalently linked to the template strand and were not representative of the whole parent strand.
Simian virus 40 DNA extracted from infected cells with sodium deoxycholate no longer reflects its in vivo superhelix density
Authors: Germond J E
Published: January 2020

When simian virus 40 DNA is extracted from infected cells with low concentrations of sodium deoxycholate, which selectively extract non-encapsidated simian virus 40 DNA, the DNA has a lower average number of superhelical turns than the DNA extracted from purified viral particles. During extraction, a partial deproteinization of the DNA by a concentration of detergent that did not inactivate a nicking-closing activity led to the removal of some superhelical turns. The DNA extracted in this way no longer reflected its in vivo number of superhelical turns.
Peptide analysis of the transformation-specific antigen from avian sarcoma virus-transformed cells
Authors: Brugge J , Erikson E , Collett M S , Erikson R I
Published: January 2020

Sera from rabbits bearing tumors induced by avian sarcoma virus (ASV) were ussed to immunopecipitate virus-specific proteins from extracts of chicken, hamster, and field vole cells transformed by ASV. Two virus-specific proteins having molecular weights of 76,000 and 60,000 were found in all cell lines examined. The 76,000-molecular-weight protein, Pr76, is the precursor to the internal core proteins of ASV. The 60,000-molecular-weight (60K) transformation-specific antigen from each cell line was subjected to peptide analysis, using chymotrypsin and Staphylococcus aureus V8 protease. The resulting peptide maps of the 60K protein from the different ASV-infected cell types were similar for each enzyme, strongly suggesting that the 60K protein is virus coded. Two-dimensional analysis of chymotryptic peptides from Pr76 and 60K reveals that 60K is not related to the gs antigen precursor. Radiolabeling of ASV-transformed cells with inorganic phosphate revealed that 60K is phosphorylated in vivo. The 60K proteins isolated from both ASV-transformed chicken and field vole cells were found to contain one tryptic phosphopeptide. The tryptic phosphopeptides of 60K from both cell lines migrated identically upon two-dimensional peptide analyses, and their migration differed from that of the principal phosphopeptide of Pr76.
Human cytomegalovirus DNA. I. Molecular weight and infectivity
Authors: Geelen J L , Walig C , Wertheim P , van der Noordaa J
Published: January 2020

Human cytomegalovirus DNA (strain AD 169) was isolated from purified virions and further purified by sucrose density gradient centrifugation. The viral DNA molecules were studied by electron microscopy and found to be linear and to have a length of 76.22 +/- 5.22 micron, corresponding to a molecular weight of 147 +/- 6.2 x 10(6). The DNA was infectious when tested in human embryonic lung cells.
Anomalous behavior of the major avian myeloblastosis virus glycoprotein in the presence of sodium dodecyl sulfate
Authors: Marciani D J , Papamatheakis J D
Published: January 2020

The sodium dodecyl sulfate (SDS) complex of the major glycoprotein of avian myeloblastosis virus exhibited an anomalously low free electrophoretic mobility compared with those of non-glycosylated protein standards. The apparent molecular weight of the glycoprotein calculated from the relation between log molecular weight and electrophoretic mobility depended on the acrylamide concentration and reached a lower limit of 80,000. The molecular weight was also estimated from the retardation coefficients of protein standards and the viral glycoprotein. This method yielded a molecular weight of 64,000 for the avian myeloblastosis virus glycoprotein. When gel chromatography in SDS was used to determine the apparent molecular weight of the glycoprotein from its hydrodynamic properties alone, the estimated value was 50,000. The generally assigned value of 80,000 daltons for the avian myeloblastosis virus major glycoprotein, as determined by SDS electrophoresis, may be an overestimate due to its relatively low free electrophoretic mobility and peculiar conformation in SDS.
Oncogenicity of AKR endogenous leukemia viruses
Authors: Nowinski R C , Hays E F
Published: January 2020

Four biologically distinct groups of endogenous murine leukemia virus (MuLV) have been isolated from AKR mice. These viruses included (i) ecotopic XC+ MuLV that occur in high titer in normal tissues and serum of AKR mice throughout their life span, (ii) ecotropic XC- MuLV that are produced in high titers by leukemia cells, (iii) xenotropic MuLV that are readily demonstrable only in aged mice, and (iv) polytropic MuLV thatarise in the thymuses of aged mice as a consequence of genetic recombination between ecotropic and xenotropic MuLV. Virus of each of these biological classes were assayed in AKR mice for their ability to accelerate the occurrence of spontaneous leukemia. Certain isolates of ecotropic XC- MuLV and polytropic MuLV were found to have high oncogenic activity. These viruses induced 100% leukemias within 90 days of inoculation. In contrast, ecotropic XC+ MuLV that were obtained from AKR embryo fibroblasts and xenotropic MuLV that were obtained from the lymphoid tissues of aged AKR mice did not demonstrate oncogenic activity. These findings demonstrate fundamental differences between XC- and XC+ ecotropic MuLV that are found in leukemic and normal tissues, respectively. Furthermore, these findings point to the role of ecotropic XC- and polytropic MuLV in the spontaneous leukemogenesis of AKR mice.
Anatomy of bovine mammillitis DNA II. Size and arrangements of the deoxynucleotide sequences
Authors: Buchman T G , Roizman B
Published: January 2020

We previously reported that bovine mammillitis virus (BMV) DNA consists of two covalently linked components designated L and S and estimated to be 71.5 x 10(6) and 15.7 x 10(6) in molecular weight, respectively; the components invert relative to each other, giving rise to four equimolar populations differing soley in the relative orientation of the two components. We now report that (i) BMV DNA has a contour length corresponding to a molecular weight of 89 x 10(6). (ii) Component L consists of a unique sequence (Ul) bracketed by sequences ab and its inverted repeat b'a', estimated to be of molecular weights 66.1 x 10(6), 2.7 x 10(6), and 2.7 x 10(6), respectively. (iii) Component S consists of a unique sequence (Us) bracketed be sequence ca and its inverted repeat a'c', estimated to be of molecular weights 8.3 x 10(6), 3.7 x 10(6), and 3.7 x 10(6), respectively. (iv) The a sequences present at the termini of a complete linear molecule (abUlb'a'a'c'Usca) are arranged in tandem so that the DNA can circularize after limited digestion with arranged in tandem so that the DNA can circularize after limited digestion with lambda 5'-exonuclease. The size of the a sequences was estimated to be 0.7 x 10(6) in molecular weight. (v) At least portions of the a sequences are repeated in an inverted orientation immediately adjacent to or near the a sequence. Thus, BMV DNA mimics herpes simplex virus type 1 DNA with respect to the arrangement but not size of deoxynucleotide sequences. The evolutionary relationship of BMV DNA relative to other herpesvirus DNAs is discussed.
Transfer of Fv-1 locus-specific resistance to murine N-tropic and B-tropic retroviruses by cytoplasmic RNA
Authors: Yang W K , Tennant R W , Rascati R J , Otten J A , Schluter B , Kiggans J O , Myer F E , Brown A
Published: January 2020

A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.
Induction of type C RNA virus from cultured rabbit lymphosarcoma cells
Authors: Bedigian H G , Fox R R , Meier H
Published: January 2020

Type C RNA virus(es) was readily released from primary lymphosarcoma cell cultures of WH/J rabbits after induction with halogenated pyrimidines. The virus contained an RNA-directed DNA polymerase and the p30 structural protein. The rabbit virus RNA-directed DNA polymerase and p30 protein shared antigenic homologies with other mammalian type C oncornaviruses but appear to also possess unique antigenic determinants. Normal rabbit liver contained DNA homologous to a 3H-labeled complementary DNA transcript of the rabbit viral genome, indicating that type C viral genetic information is present in at least the WH/J strain of rabbits.
Interaction of vesicular stomatitis virus with lipid vesicles: depletion of cholesterol and effect on virion membrane fluidity and infectivity
Authors: Moore N F , Patzer E J , Shaw J M , Thompson T E , Wagner R R
Published: January 2020

Interaction with excess unilamellar phosphatidylcholine (PC) vesicles resulted in depletion of as much as 90% of the cholesterol from the membrane of intact vesicular stomatitis (VS) virus. The cholesterol depletion was not significantly influenced by the proteolytic removal of virion glycoprotein spikes, but it was temperature dependent. Cholesterol depletion caused substantial reduction in anisotropy of the VS virion membrane as measured by fluorescence depolarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene; residual adsorbed vesicles represent a significant factor in this apparent increase in virion membrane fluidity. Interaction with PC vesicles resulted in a substantial loss of VS viral infectivity as measured by plating efficiency on L-cell monolayers. Reduction in infectivity appeared to be related to temperature-dependent depletion of virion cholesterol by PC vesicles. Interaction of VS virions with cholesterol-containing PC vesicles resulted in significantly less decline in infectivity, but attempts to restore cholesterol and infectivity to depleted VS virions were unsuccessful. Depletion of virion cholesterol apparently results through collision with PC vesicles rather than movement of cholesterol monomers or micelles through the aqueous phase, because PC vesicle-virion interaction in the presence of cholesterol oxidase did not result in substantial oxidation of translocated cholesterol.
Shedding of the glycoprotein from vesicular stomatitis virus-infected cells
Authors: Little S P , Huang A S
Published: January 2020

In a culture of Chinese hamster ovary cells infected with vesicular stomatitis virus, there is specific shedding of viral antigens into the medium. This shedding appears to be unrelated to progeny formation or to cell lysis. Although all five of the virus-specific proteins are detected in the extracellular soluble fraction, the major antigen is the Gs protein. This protein has a molecular weight of 54,000. Indirect analysis of the content of sialic acid as well as peptide analysis of the Gs and G proteins of vesicular stomatitis virus suggest that the Gs protein is derived from the G protein by proteolysis. Both proteins are hydrophobic when analyzed by charge-shift electrophoresis. The presence of phenylmethylsulfonyl fluoride in the culture medium or the removal of serum from the culture medium partially reduces the shedding of Gs protein. Increased shedding of the Gs protein is seen when there is an unstable M or matrix protein synthesized by a temperature-sensitive mutant, tsG31. These results indicate that the G protein is cleaved at the cell surface, thus releasing Gs protein into the medium. Furthermore, the stability of G protein at the cell surface appears to be dependent on its association with the M protein.
DNA of Epstein-Barr virus. III. Identification of restriction enzyme fragments that contain DNA sequences which differ among strains of Epstein-Barr virus
Authors: Raab-Traub N , Pritchett R , Kieff E
Published: January 2020

Previous kinetic and absorption hybridization experiments had demonstrated that the DNA of the B95-8 strain of Epstein-Barr virus was missing approximately 10% of the DNA sequences present in the DNA of the HR-1 strain (R.F. Pritchett, S.D. Hayward, and E. Kieff, J. Virol. 15:556-569, 1975; B. Sugder, W.C. Summers, and G. Klein, J. Virol. 18:765-775, 1976). The HR-1 strain differs from other laboratory strains, including the B95-8 and W91 strains, and from virus present in throat washings from patients with infectious mononucleosis in its inability to transform lymphocytes into lymphoblasts capable of long-term growth in culture (P. Gerber, Lancet i:1001, 1973; J. Menezes, W. Leibold, and G. Klein, Exp. Cell. Res. 92:478-484, 1975; G. Miller, D. Coope, J. Niederman, and J. Pagano, J. Virol. 18:1071-1080, 1976; G. Miller, J. Robinson, L. Heston, and M. Lipman, Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974). In the experiments reported here, the restriction enzyme fragments of Epstein-Barr virus DNA which contain sequences which differ among the HR-1, B95-8, and W91 strains have been identified. The DNA of the HR-1, B95-8, and W91 strains each differed in complexity. The sequences previously shown to be missing in the B95-8 strain were contained in the EcoRI-C and -D and Hsu I-E and -N fragments of the HR-1 strain and in the EcoRI-C and Hsu I-D and -E fragments of the W91 strain. The HR-1 strain was missing DNA contained in EcoRI fragments A and J through K and Hsu I fragment B of the B95-8 strain and in the EcoRI-A and Hsu I-B fragments of the W91 strain. The relationship of these data to the linkage map of restriction enzyme fragments of the DNA of the B95-8 and W91 strains (E. Kieff, N. Raab-Traub, D. Given, W. King, A.T. Powell, R. Pritchett, and T. Dambaugh, In F. Rapp and G. de-The, ed., Oncogenesis and Herpesviruses III, in press; D. Given and E. Kieff, submitted for publication) and the possible significance of the data are discussed.
Polyoma virus has three late mRNA's: one for each virion protein
Authors: Siddell S G , Smith A E
Published: January 2020

Polyoma virus mRNA, isolated from the cytoplasm of 3T6 cells late after infection and purified by hybridization to HpaII fragment 3 of polyoma virus DNA, was separated on 50% formamide-containing sucrose density gradients, and the fractionated RNA was recovered and translated in vitro. Analysis of the cell-free products showed that the minor virion protein VP3 was synthesized from an mRNA sedimenting at approximately 18S betweeen the 19S VP2 mRN and the 16S VP1 mRNA. Other experiments showed that the VP2 and VP3 can be labeled with formyl methionine from initiator tRNA. We conclude that there are three late polyoma virus mRNA's, each directing the synthesis of only one viral capsid protein.
Proteolysis of noncapsid protein 2 of type 3 poliovirus at the restrictive temperature: breakdown of noncapsid protein 2 correlates with loss of RNA synthesis
Authors: Bowles S A , Tershak D R
Published: January 2020

Inhibition of RNA synthesis of the LH strain of type 3 poliovirus at the restrictive temperature occurs concurrently with the breakdown of noncapsid virus-specific peptide 2. This finding, along with other reported data (B.D. Korant, In Proteases and Biological Control, p. 621-644, 1975), suggests that noncapsid virus-specific peptide 2 is a component of the viral replicase.
env
Gene of Chicken RNA Tumor Viruses: Extent of Conservation in Cellular and Viral Genomes
Authors: Fujita Donald J. , Tal Jacov , Varmus Harold E. , Bishop J. Michael
Published: January 2020

The
env
gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode
env
; our work exploited the availability of radioactive DNA (cDNA
gp
) complementary to most or all of
env. env
sequences were detectable in the DNAs of chickens which synthesized an
env
gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no
env
gene product (chick helper factor negative).
env
sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral
env
only in chicken DNA contrasts sharply with the demonstration that the transforming gene (
src
) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London)
260
: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA
gp
and chicken DNA and also between cDNA
gp
and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA
gp
did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the
env
genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.
Poliovirus single-stranded RNA and double-stranded RNA: differential infectivity in enucleate cells
Authors: Detjen B M , Lucas J , Wimmer E
Published: January 2020

The ability of poliovirus virion RNA and double-stranded RNA (replicative form) to replicate in enucleate mouse L cells was investigated. Virion RNA replicated successfully in the absence of the cell nucleus, whereas replicative form infection did not produce any detectable progeny in enucleate cells. The results provide direct evidence of a nuclear requirement early in the infection initiated by replicative form RNA.
Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines
Authors: Luka J , Lindahl T , Klein G
Published: January 2020

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.
Specificity of interferon action in protein synthesis
Authors: Yau P M , Godefroy-Colburn T , Birge C H , Ramabhadran T V , Thach R E
Published: January 2020

Inhibitors of elongation steps in protein synthesis such as cycloheximide and anisomycin mimic interferon treatment in that they specifically inhibit the synthesis of certain viral proteins. These specific effects are seen only at very low concentrations of the antibiotics, under conditions where host cellular protein synthesis, as well as cell viability, are not severely reduced. A qualitatively as well as quantitatively close correlation between the effects of the two types of agents has been established for encephalomyocarditis virus, vesicular stomatitis virus and murine leukemia virus protein synthesis. It is concluded that one of the primary mechanisms of interferon action may be a nonspecific retardation of one or more elongation steps, and that this may be sufficient to account for its effects on the replication of certain viruses such as encephalomyocarditis and vesicular stomatitis viruses.
Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and phiX174 DNA: alignment with restriction enzyme maps
Authors: Rassart E , Spencer J H
Published: January 2020

Escherichia coli RNA polymerase has been shown to bind to a limited number of Hind and Hae III restriction enzyme fragments. On S13 replicative form DNA there are three major binding sites, and the locations correlate with promoter sites at the beginning of genes a and B and a site overlapping gene D and the beginning of gene E. Two less definite binding sites have been localized, one in gene F and one at the gene G-H junction. In phiX174 replicative form DNA, five sites, each with apparently similar binding properties, have been located, four of which correspond exactly to binding sites in S13. One site, at the beginning of the B gene, could not be assigned to exactly the same location found in S13. This was due in part to differences in the restriction cleavage maps in the area of the DNA and possibly to the higher background of nonspecific binding in the phiX174 experiments. The location of two of the phiX174 sites at the beginning of genes A and D-E corresponds very well with transcription data, but the site at the start of the B gene indicates the promoter site is closer to the initiation sequence of the B protein than was previously suggested on the basis of transcription data.
Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus
Authors: Lee H H , Miller L K
Published: January 2020

A nuclear polyhedrosis virus (MNPV) isolated from a lepidopteran (Noctuidae) insect, Autographa californica, was cloned by successive plaque purification using virions containing only one nucleocapsid per envelope as inoculum. The ability to clone the virus by this method was demonstrated by the isolation of nondefective, genotypic variants of the virus with similar but not identical restriction endonuclease fragment patterns. Five distinct variants were identified by genotypic analysis with HindIII, EcoRI, SalI, and Bam HI restriction endonucleases. The characteristic genotype of each variant was maintained upon passage in insect larvae. The isolation of these virus variants demonstrates (i) the heterogeneity of the uncloned virus preparation and (ii) the ability to clone MNPVs by plaque purification of media-derived nonoccluded virions. The A. californica MNPV is being considered for commercial use as a pesticide in the United States, and the cloning of the virus, in view of the heterogeneity detected, may be advisable. The cloning and genotype analyses are also significant with regard to understanding the genetic nature of multiply embedded NPVs (those NPVs containing more than one nucleocapsid per envelope in the occluded form of the virus) and indicate that further genetic analysis of these viruses is possible.
Phosphorylation of chromatin- and ribosome-associated proteins in cells transformed by adenovirus, murine sarcoma virus, and methylcholanthrene
Authors: Segawa K , Oda K , Yuasa Y , Shiroki K , Shimojo H
Published: January 2020

Endogenous protein phosphorylation in chromatin and ribosomes of monkey, mouse, and rat cells transformed by DNA, RNA tumor viruses, and a chemical carcinogen revealed the association of a protein of approximately 90,000 daltons (90K), which is highly phosphorylated in vitro. Peptide map analysis showed that the 90K proteins associated with these organelles of various transformed cells are similar irrespective of the species of cells and transforming agents. This species of protein could not be detected, or was scarcely detected, by phosphorylation in chromatin and ribosomes of untransformed cells and in revertants of transformed cells. These results suggest that the alteration in the pattern of endogenous protein phosphorylation in these organelles is closely related to the transformed state of cells.
Cell-free synthesis of simian virus 40 T-antigens
Authors: Paucha E , Harvey R , Smith A E
Published: January 2020

Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with simian virus 40 (SV40) anti-T serum demonstrated that both SV40 large-T and small-T antigens are synthesized in vitro in response to mRNA isolated from productively infected CV1 CELLS. Sucrose density centrifugation in gradients containing 85% formamide showed that the mRNA's for both forms of T-antigen sediment at about 17.5S, with the mRNA for small-t sedimenting marginally, but reproducibly, ahead of the mRNA for large-T. Hybridization experiments using restriction endonuclease fragments Hae III-E and Hind II/III-B showed that all fractions active in the cell-free synthesis of both forms of T-antigen hybridized equally to both fragments. This suggests that the mRNA's for SV40 T-antigens are at least partly virus coded and that the bulk of the early SV40 mRNA contains sequence information from both ends of the early region. The data are consistent with the suggestion that the large-T mRNA is spliced. SV40 complementary RNA (the product of transcription of SV40 DNA using Escherichia coli RNA polymerase) was also translated in the L-cell system and gave two families of polypeptides which specifically immunoprecipitate with anti-T serum. One family (the small-t family) includes a polypeptide indistinguishable by gel electrophoresis and tryptic peptide fingerprinting from small-t isolated from cells. The other family (the 60K family) has a major component with molecular weight approximately 60,000 and includes other polypeptides with molecular weights ranging from approximately 14,000 to about 70,000. The 60K family has petides in common with large-T but not with small-T. Together, the peptides of the small-t and 60K families account for virtually all of the methionine peptides of SV40 large-T. We conclude from these results (i) that small-t is probably entirely, and large-T at least predominantly, virus coded; (ii) that the small-t and 60K families represent the translation products of two different portions of the early region of SV40 DNA (approximately 0.65 to 0.55 map units and 0.54 to 0.17 map units); and (iii) that although most, if not all, of the large-T and small-t peptides are present in the cell-free product, some feature of sequence arrangement of SV40 complementary RNA prevents the translation of full-length large-T and results instead in the synthesis of fragments. We suggest that the absence of a splice in the complementary RNA is responsible for this result.
Physical mapping of herpes simplex virus type 1 mutations by marker rescue
Authors: Stow N D , Subak-Sharpe J H , Wilkie N M
Published: January 2020

A generally applicable technique which permits the rescue of selected genetic markers from fragments of herpes simplex virus DNA is described. Baby hamster kidney cells infected at the nonpermissive temperature with intact DNA from temperature-sensitive mutants or with fragmented wild-type DNA produce no, or little, infectious progeny. Coinfection results in an increased yield of virus, demonstrating the rescue of genetic information from the DNA fragments. This progeny virus consists of both wild-type and temperature-sensitive virus, demonstrating that both recombination and complementation can occur in coinfected cells. Rescue experiments using isolated fragments produced with various restriction endonucleases have enabled us to locate five temperature-sensitive mutations on the herpes simplex virus type 1 physical map. An adaptation of the technique has allowed the physical mapping of a mutation which affects the herpes simplex virus type 1 pyrimidine deoxyribonucleoside kinase gene. Comparison of the genetic and physical maps for these mutants reveals several anomalies which are discussed.
Synthesis of T4 DNA and bacteriophage in the absence of dCMP hydroxymethylase
Authors: Morton D , Kutter E M , Guttman B S
Published: January 2020

Several lines of research have suggested that the dCMP hydroxymethylase (HMase) coded by bacteriophage T4 is an essential protein in a DNA replication complex, as well as a supplier of hydroxymethyl dCMP for phage DNA synthesis. We show that a mutant [HMase, dCTPase, endonuclease II, endonuclease IV] which lacked this enzyme made cytosine-containing DNA at about two-thirds of the normal rate. When coupled with an alc mutation which permitted synthesis of late proteins, a small burst of phage was produced whose DNA contained no hydroxymethylcytosine. This pentuple mutant made both early and late proteins with abnormal kinetics, whereas the HMase+ parent showed normal kinetics. However, intracellular phage DNA showed no gross abnormalities in alkaline sucrose gradients. We conclude that HMase is not required for DNA synthesis when hydroxymethyl dCMP is not needed as a substrate; however, its absence still impairs both replication and transcription.
Evidence for an Adenovirus Type 2-Coded Early Glycoprotein
Authors: Jeng Yun-Hua , Wold William S. M. , Green Maurice
Published: January 2020

We have identified an adenovirus type 2 (Ad2)-induced early glycopolypeptide with an apparent molecular weight of 20,000 to 21,000 (20/21K), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 20/21K polypeptide could be labeled in vivo with [
3
H]glucosamine. [
35
S]methionine- and [
3
H]-glucosamine-labeled 20/21K polypeptides bound to concanavalin A-Sepharose columns and were eluted with 0.2 M methyl-α-
d
-mannoside. The pulse-labeled polypeptide appeared as a sharp band with an apparent molecular weight of 21K, but after a chase it converted to multiple bands with an average molecular weight of 20K. This variability in electrophoretic mobility is consistent with glycosylation or deglycosylation of the 20/21K polypeptide. Analysis of the pulse and pulse-chase-labeled forms by using partial proteolysis indicated that the polypeptides were highly related chemically, but not identical. Most of the 20/21K polypeptide is localized in the cytoplasm fraction of infected cells lysed by Nonidet P-40. The 20/21K polypeptide and a 44K polypeptide, labeled with [
35
S]methionine or [
3
H]glucosamine in Ad2-infected human cells, were precipitated by a rat antiserum against an Ad2-transformed rat cell line (T2C4), but not by antisera against three other Ad2-transformed rat cell lines, or by serum from nonimmune rats. The partial proteolysis patterns of the 20/21K and the 44K polypeptides were indistinguishable, indicating that the two polypeptides are highly related, and suggesting that the 44K polypeptide might be a dimer of the 20/21K polypeptide. The 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results imply that the 20/21K polypeptide is synthesized in Ad2-infected human, monkey, and hamster cells, and in one but not all Ad2-transformed rat cells. Thus, the 20/21K polypeptide is probably viral coded rather than cell coded and viral induced.
Simian virus 40 production after viral uncoating in the CV-1 cell nucleus
Authors: Gershey E L , Diacumakos E G
Published: January 2020

A normal yield of infectious simian virus 40 was produced by a CV-1 cell culture after inoculation of a few cells with an average of one particle per nucleus by microinjection.
Recombinants between herpes simplex virus types 1 and 2: analyses of genome structures and expression of immediate early polypeptides
Authors: Preston V G , Davison A J , Marsden H S , Timbury M C , Subak-Sharpe J H , Wilkie N M
Published: January 2020

Recombinants between temperature-sensitive mutants of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) were constructed. Using restriction endonucleases, we analyzed the genome composition of 17 intertypic recombinants and detected crossovers in every region of the genome. The virion DNA of one recombinant appeared to be largely "frozen" in two of the four possible genome arrangements of HSV. Knowledge of the genome structures of recombinants enabled us to physically map immediate early polypeptides. We present evidence that the immediate early polypeptide Vmw IE 110 of HSV-1 and its functionally equivalent polypeptide, Vmw IE 118, of HSV-2 may map in the repetitive sequences bounding the long unique region of HSV.
Characterization of Rous sarcoma virus src gene products synthesized in vitro
Authors: Beemon K , Hunter T
Published: January 2020

The cell-free synthesis of three major proteins from virion RNA of nondefective Rous sarcoma virus (RSV), but not from RNA of transformation-defective deletion mutants, has been observed. The apparent molecular weights of these transformation-specific proteins are approximately 60,000 (60K), 25K, and 17K. Tryptic maps of methionine-containing peptides revealed the 17K, 25K, and 60K proteins to be overlapping in sequence. However, only partial homology was observed between the 17K, 25K and 60K proteins synthesized from Schmidt-Ruppin strain, subgroup D, RSV RNA and those synthesized from Prague strain, subgroup B, RSV, RNA. About half of the methionine peptides in the Schmidt-Ruppin strain, subgroup D, 60K protein were shared with the Prague strain, subgroup D, 60K protein, and the rest were distinct to each. The virion RNAs coding for the 60K, 25K, and 17K proteins were found to be polyadenylated and to sediment with maximal mRNA activity at about 23, 19 to 20, and 18S, respectively. In addition, transformation-specific proteins with molecular weights of 39K and 33K were observed by in vitro synthesis. These proteins are also related to the 60K, 25K, and 17K proteins and were synthesized from polyadenylated RSV RNA of approximately 21 to 22S. RNase T1-resistant oligonucleotides were analyzed in parallel, and the src-specific oligonucleotides were found to be first present in equimolar amounts in those gradient fractions sedimenting at 21 to 22S. Our data suggest that synthesis of the 60K protein is initiated near the 5' terminus of the src gene, whereas the 39K, 33K, 25K, and 17K proteins are initiated internally in the src gene. All of these proteins appear to be initiated independently, but they may have a common termination site.