bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular Sequences Related to Three New
onc
Genes of Avian Sarcoma Virus (
fps, yes
, and
ros
) and Their Expression in Normal and Transformed Cells
Authors: Shibuya M. , Hanafusa H. , Balduzzi P. C.
Published: January 2020

Two
onc
genes of avian sarcoma viruses unrelated to the
src
gene have recently been identified:
fps
of Fujinami sarcoma virus/PRCII/UR1 and
yes
of Y73/Esh sarcoma virus. In the first part of this study we demonstrated that UR2, the most recently isolated avian sarcoma virus, contains in its genome a unique sequence,
ros
, nonhomologous to
src, fps
, and
yes
sequences or to transforming genes of avian acute leukemia viruses. Using cDNAs specific to the inserts of avian sarcoma virus genomes, we examined the existence and the transcription of cellular nucleotide sequences related to the three new
onc
genes of avian sarcoma virus (
fps, yes
and
ros
) in various cells. The progenitor cellular sequences for these
onc
genes (c-
onc
) were present in uninfected chicken DNA in one or few copies per haploid genome. These c-
onc
sequences were detectable in cellular DNA of a wide variety of vertebrates, and the homology between viral and cellular
onc
was inversely related to the phylogenetic distance of animal species. The pattern of expression of these c-
onc
genes in different tissues of chickens was found to be unique to each gene. The expression of c-
fps
and c-
ros
genes was generally repressed in many tissues, but c-
fps
was expressed at higher levels in bone marrow (2.5 copies per cell) and lung (1.1 copies per cell), whereas c-
ros
was mainly transcribed in kidney (2.5 copies per cell). On the other hand, c-
yes
transcripts were easily detectable in all tissues analyzed and were found at high levels in kidney (26 copies per cell). These c-
onc
expressions were unaffected by infection with avian sarcoma viruses that contained other
onc
genes. In a few cultures of chicken and quail transformed cells derived from tumors induced by chemical carcinogens, we found that the levels of transcription of the four c-
onc
genes remained unaltered, compared with that in normal tissues.
Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1
Authors: Daksis J I , Priemer M M , Chan V L
Published: January 2020

A group of 43 phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus type 1 was isolated after the mutagenesis of infected cells with nitrosoguanidine. One of these mutants, designated PAA1rts1, was found to be temperature sensitive (ts), that is, unable to replicate at 39.5 degrees C, the nonpermissive temperature. Recombination analysis of PAA1rts1 indicated that the PAA1r mutation and the ts1 mutation are loosely linked and are located on two separate genes. PAA1rts1 showed a defect in viral DNA synthesis at 39.5 degrees C, which presumably can be attributed to the production of a PAA-resistant and thermolabile DNA polymerase. PAA1rts1 was also defective in the shutoff of host DNA synthesis at the restrictive temperature.
Simian virus 40-rabbit beta-globin recombinants lacking late mRNA splice sites express cytoplasmic RNAs with altered structures
Authors: White R T , Berg P , Villarreal L P
Published: January 2020

Deletions were introduced at exon-intron boundaries in the late region of a simian virus 40-beta-globin cDNA recombinant to study the role of splicing in the formation of simian virus 40 late cytoplasmic RNAs. The recombinant was used as a wild type because it allowed characterization of mutant RNAs expressed from defective genomes in the presence of comparable RNAs contributed by the coinfecting helper virus. Removal of a 17-base pair segment at map position 0.76, which included a portion of the leader sequence implicated in the splicing of the major 16S mRNA, prevented expression of 16S-type mRNA. The same mutant accumulated cytoplasmic 19S-type mRNA, but the assortment of the 5' ends of these mRNAs differed from the assortment of the wild-type counterparts. Another mutant that lacks nucleotide sequences implicated in the splicing of the major 16S mRNA and one of the principal 19S-type RNAs accumulated a 16S-type mRNA with a previously undetected leader splice, and assortment of 19S mRNAs with new or normally underrepresented splices, and even a species of unspliced cytoplasmic 19S mRNA.
Integrated adenovirus type 12 DNA in the transformed hamster cell line T637: sequence arrangements at the termini of viral DNA and mode of amplification
Authors: Eick D , Doerfler W
Published: January 2020

Approximately 20 to 22 copies of adenovirus type 12 (Ad12) DNA per cell were integrated into the genome of the cell line T637. Only a few of these copies seemed to remain intact and colinear with virion DNA. All other persisting viral genomes exhibited deletions or inversions or both in the right-hand part of Ad12 DNA. Spontaneously arising morphological revertants of T637 cells has lost viral DNA. In most of the revertant cell lines only the intact or a part of the intact viral genome was preserved; other revertant cell lines has lost all viral DNA. In three other Ad12-transformed hamster cell lines, HA12/7, A2497-3, and CLAC3 (Stabel et al., J. Virol. 36:22-40, 1980), major rearrangements at the right end of the integrated Ad12 DNA were not found. These studies were performed to investigate the phenomena of amplification, rearrangements, and deletions of Ad12 DNA in hamster cells.
Guanidine-resistant mutants of aphthovirus induce the synthesis of an altered nonstructural polypeptide, P34
Authors: Saunders K , King A M
Published: January 2020

Extracts of cells infected with guanidine-resistant mutants of aphthovirus were examined for differences in virus-induced polypeptides by using electrofocusing. Four of 1 independent spontaneous mutants induced the synthesis of an altered nonstructural polypeptide, P34. The precursor of P34, P52, and a previously unmapped polypeptide, P20c, also carried these charge-change mutations. No mutations in other regions of the genome were detected, and the remaining six guanidine-resistant mutants appeared entirely normal by electrofocusing. However, when the P34 of one of the latter mutants was examined by tryptic peptide fingerprinting, it too differed from that of the guanidine-sensitive parent. The frequency of P34 alterations among guanidine-resistant mutants suggests that P34 is functionally involved in the antiviral action of guanidine.
Colinearity of RNAs with the vaccinia virus genome: anomalies with two complementary early and late RNAs result from a small deletion or rearrangement within the inverted terminal repetition
Authors: Wittek R , Moss B
Published: January 2020

The colinearity of RNA transcripts with the vaccinia virus genome was investigated. Cytoplasmic RNA from infected cells was annealed to a cloned DNA segment that extended from 9 to 15.6 kilobase pairs from the left end of the genome and contained approximately 800 base pairs of the inverted terminal repetition (ITR). Remaining unhybridized single strands of DNA were digested with nuclease S1, and the lengths of the protected DNA fragments were determined by agarose gel electrophoresis under neutral and alkaline conditions. Uniformly 32P-labeled cloned DNA insert, separated recombinant DNA strands, and smaller restriction fragments, as well as 3' and 5' end-labeled DNA, were employed to map five early RNAs and one late RNA. One of the early RNAs hybridized to sequences within the ITR, and the other four hybridized to sequences proximal to the ITR. The late RNA was initiated proximal to the ITR but extended into it. Interestingly, the 3' portion of this late RNA was complementary to the early RNA transcribed from the opposite strand of the ITR. From a comparison of the lengths of the protected DNA fragments on neutral and alkaline gels, all except the complementary early and late RNAs appeared to be colinear with the genome. Although the anomalous nuclease S1 data obtained with the latter RNAs mimicked splicing, they were shown by DNA-DNA hybridization to result from a small deletion or rearrangement within the ITR. Thus far, no true examples of spliced vaccinia virus RNAs have been found.
Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells
Authors: Binger M H , Flint S J , Rekosh D M
Published: January 2020

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.
Biological and biochemical studies of African green monkey lymphotropic papovavirus
Authors: Takemoto K K , Furuno A , Kato K , Yoshiike K
Published: January 2020

The growth of African green monkey lymphotropic papovavirus (LPV) in human lymphoblastoid cell line BJA-B was found to be slow and inefficient due to the accumulation of defective particles. An analysis of molecularly cloned LPV DNAs showed that 3 of 19 clones had DNAs that were longer (5.1 kilobases) than the DNAs of the other clones. The 5.1-kilobase DNA was infectious for BJA-B cells, whereas the shorter (4.8-kilobase) molecules were defective. Unlike the wild-type virus, stocks of LPV made from cloned, infectious DNAs were homogeneous and had higher titers. Using stocks of nondefective LPV, we investigated other biological properties. LPV replication in another human B-lymphoblastoid cell line was observed. The virus did not cause tumors when it was inoculated into newborn hamsters. Serological surveys of human and nonhuman primate sera indicated that virtually all primates, including humans, show evidence of infection by viruses antigenically related to LPV.
Encapsidation and expression of the herpes thymidine kinase gene in polyoma virus
Authors: Dinces N B , Milman G
Published: January 2020

A recombinant DNA of 5,150 base pairs was prepared containing the intact early region of polyoma virus, including the viral origin of replication and the structural sequences of the herpes simplex virus type 1 thymidine kinase gene. Although no thymidine kinase activity was detected when herpes structural sequences alone were transfected into cells, activity was produced when the structural gene followed the polyoma early region. The recombinant DNA was encapsidated into polyoma virions when cotransfected into mouse 3T6 cells with helper DNA from an early polyoma virus mutant. Herpes thymidine kinase activity was detected by a rapid in situ autoradiographic assay in which [125]iododeoxycytidine was utilized as a substrate for the viral but not the cellular enzyme.
Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms
Authors: Lopez J , Webster R E
Published: January 2020

The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.
Herpesvirus infection alters the steady-state levels of cellular polyadenylated RNA in polyoma virus-transformed BHK cells
Authors: Nakai H , Maxwell I H , Pizer L I
Published: January 2020

Polyoma-transformed BHK cells are permissive for the replication of herpes simplex virus type 1. The effect of herpes infection on the steady-state levels of bulk mRNA sequences in these cells was studied by using cDNA to polyadenylated cytoplasmic RNA from uninfected cells. The principal findings were: (i) herpes simplex virus type 1 infection caused a pronounced reduction in the cytoplasmic levels of moderately abundant mRNAs' (ii) after infection, increased amounts of RNA complementary to the cDNA were isolated as part of the nonadenylated cytoplasmic RNA fraction.
Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus
Authors: Poirier F , Calothy G , Karess R E , Erikson E , Hanafusa H
Published: January 2020

Expression of the src gene of Rous sarcoma virus (RSV) in chicken embryo neuroretinal (NR) cells results in morphological transformation and sustained proliferation of a normally resting cell population. We have previously reported the isolation of mutants of RSV which retain full growth-promoting activity while displaying reduced transforming properties. Two such mutants, PA101 and PA104, were used to investigate whether the p60src-associated kinase activity is required for the mitogenic function of src. A comparison of the patterns of phosphorylation of wild-type and mutant p60src revealed that the phosphorylation of tyrosine residues of p60src of PA104 was markedly reduced, whereas the relative amount of phosphotyrosine in p60src of PA101 was comparable to that of the wild-type protein. In vitro kinase activity of p60src immunoprecipitated from NR cells infected with PA101 or PA104 as measured by phosphorylation of the heavy chains of specific immunoglobulin G molecules was 1/10 that of the wild-type molecule. Moreover, when NR cells infected with mutants temperature sensitive for mitogenic capacity were maintained at a temperature either permissive or restrictive for cell growth, quantitation of kinase activity indicated that proliferation of NR cells could not be linked to the absolute level of in vitro kinase activity of p60src. Transformation of NR cells by wild-type RSV resulted in a 10-fold increase in total cellular phosphotyrosine and in the phosphorylation of tyrosine residues of a 34K protein, a possible in vivo substrate for p60src. In contrast, phosphorylation of tyrosine residues of cellular targets was markedly reduced in NR cells infected with PA101 or PA104. These results indicate that the mitogenic capacity of RSV in NR cells does not require elevated levels of p60src kinase activity.
Evidence for an interaction between the membrane protein of a paramyxovirus and actin
Authors: Giuffre R M , Tovell D R , Kay C M , Tyrrell D L
Published: January 2020

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.
Capping of Viral RNA in Cultured
Spodoptera frugiperda
Cells Infected with
Autographa californica
Nuclear Polyhedrosis Virus
Authors: Jun-Chuan Qin , Weaver Robert F.
Published: January 2020

Viral RNA from fall armyworm (
Spodoptera frugiperda
) cells infected with
Autographa californica
nuclear polyhedrosis virus contains cap structures. Most of the cap labeled in vivo with [
3
H]methionine or
32
P
i
cochromatographed on DEAE-cellulose with the −5 charge marker; a minor component appeared at −4 net charge. The former is probably a cap 1 structure (m
7
GpppX
m
p
Yp), and the latter is probably a cap 0 (m
7
GpppXp). On the basis of relative labeling of the two caps with [
3
H]adenosine and [
3
H]guanosine, we concluded that each cap contained at least one adenosine residue in addition to guanosine and, therefore, that cap 0 contained m
7
GpppAp. Cleavage of [
3
H]methionine-labeled viral RNA with tobacco acid pyrophosphatase released a labeled component that cochromatographed on polyethyleneimine-cellulose with m
7
Gp, confirming the m
7
GpppX linkage in the cap. These results were also seen with host polyadenylated RNA. The caps appeared in nearly equal abundance in viral polyadenylated and non-polyadenylated RNAs. The ratio of
32
P
i
incorporated into the cap to that incorporated into mononucleotides suggested average lengths for the polyadenylated and non-polyadenylated RNAs of 1,800 and 1,200 nucleotides, respectively.
RNA polymerase-associated interactions near template promoter sequences of defective interfering particles of vesicular stomatitis virus
Authors: Isaac C L , Keene J D
Published: January 2020

Methylation protection studies suggested that the NS protein component of the RNA polymerase of vesicular stomatitis virus contacts the RNA templates of defective interfering (DI) particles at the sequence 3'...GUCUAUUUUUAUUUUUGGUG...5',17 to 37 nucleotides downstream from the site of initiation of in vitro transcription. The data indicated that vesicular stomatitis virus and DI particle RNAs contain different polymerase binding sequences and that NS may function as a transcription initiator protein for template recognition at both sequences. These results are thus compatible with the hypothesis that differences in the rate of defective and nondefective viral particle replication and autointerference are due to higher-affinity binding sites for polymerase at the 3' end of DI particle RNAs. In addition, a unique DI particle (DI-LT2) RNA that contains a transcriptionally inactive vesicular stomatitis virus leader gene 72 to 118 nucleotides from its 3' end showed interactions with the viral polymerase similar to those reported previously for the 3'-terminal vesicular stomatitis virus leader gene (Keene et al., Proc. Natl. Acad. Sci, U.S.A. 78:6191--6195, 1981). The interaction of polymerase with the internal leader gene of DI-LT2 RNA suggested that the lack of leader RNA and mRNA production by this particle is not due to the inability of polymerase to bind to internal sites along the template. Instead, the initiation of transcription is more likely influenced by the position of the polymerase binding site relative to the 3' end or by requisite interactions between the catalytic polymerase component (L) and the proposed initiator protein (NS).
Differential effect of nucleoside analog triphosphates on ribonucleotide reductases from uninfected and herpes simplex virus-infected HeLa cells
Authors: Nakayama K , Ruth J L , Cheng Y C
Published: January 2020

Effects of the triphosphates of eight pyrimidine nucleoside analogs (5-substituted, 2'-fluoroara-, and acyclonucleosides) and acycloguanosine were examined on the ribonucleotide reductases prepared from uninfected and herpes simplex virus (types 1 and 2)-infected HeLa cells. Of the analogs tested, E-5-propenyl- and E-5-(2-bromovinyl)-dUTP were more potent inhibitors than dTTP of the enzymes from virus-infected cells, whereas only the former compound showed this effect on the uninfected HeLa enzyme.
Isolation and characterization of simian virus 40 early region deletion mutants
Authors: Sompayrac L M , Danna K J
Published: January 2020

We constructed a tsB4/dl884 double-mutant helper virus and used it to isolate two simian virus 40 early region deletion mutants that lack about half of the DNA sequences normally used to encode the large tumor antigen (T). Both mutants make a normal-sized small t antigen, but neither mutant can replicate its DNA in the absence of a T+ helper.
Homology between the glycoproteins of vesicular stomatitis virus and rabies virus
Authors: Rose J K , Doolittle R F , Anilionis A , Curtis P J , Wunner W H
Published: January 2020

We compared the predicted amino acid sequences of the vesicular stomatitis virus and rabies virus glycoproteins by using a computer program which provides an optimal alignment and a statistical significance for the match. Highly significant homology between these two proteins was detected, including identical positioning of one glycosylation site. A significant homology between the predicted amino acid sequences of vesicular stomatitis virus and influenza virus matrix proteins was also found.
Use of a hybrid infectivity assay to analyze primary transcription of temperature-sensitive mutants of the New Jersey serotype of vesicular stomatitis virus
Authors: Isle H D , Emerson S U
Published: January 2020

A hybrid infectivity assay specific for primary transcription was developed to analyze the production of functional mRNAs by vesicular stomatitis virus. A template prepared from wild-type virions of the New Jersey serotype of vesicular stomatitis virus was reconstituted with RNA polymerase proteins from the wild type or temperature-sensitive mutants, and the in vivo temperature sensitivity of the polymerase was determined by infectivity assay. The data demonstrate that the New Jersey temperature-sensitive mutants A1 and E1 have non-temperature-sensitive transcriptases, whereas the B1 and F1 mutants both have temperature-sensitive L proteins which are defective in primary transcription.
Transformation of NIH 3T3 cells with cloned fragments of human cytomegalovirus strain AD169
Authors: Nelson J A , Fleckenstein B , Galloway D A , McDougall J K
Published: January 2020

NIH 3T3 cells were transfected with restriction endonuclease and cloned human cytomegalovirus DNA fragments to identify the transforming region(s). Cleavage of human cytomegalovirus strain AD169 DNA with XbaI and HindIII left a transforming region intact whereas EcoRI inactivated this function. Transfection of cells with cosmids containing human cytomegalovirus DNA spanning the entire genome resulted in transformation by one cosmid, pCM1058, with the AD169 HindIII DNA fragments E, R, T, and a'. Cells were selected for their growth in 1.2% methylcellulose. The clones isolated had a significant replating efficiency and were oncogenic in BALB/c nu/nu mice. Transfection of cosmids and plasmids containing subsets of the viral sequences in pCM1058 identified a common region possessed by all of the transforming recombinant molecules. This region was in the HindIII E fragment with the left boundary defined by the EcoRI d-R junction and the right boundary defined by the HindIII E-T junction. Further mapping and transfection experiments determined that the transforming region was contained without a 2.9-kilobase fragment between map units 0.123 and 0.14 on the prototype molecule of the AD169 strain.
Analysis by microinjection of the biological effects of site-directed mutagenesis in cloned avian leukosis viral DNAs
Authors: Pugatsch T , Stacey D W
Published: January 2020

Cloned avian leukosis viral DNAs were mutagenized in the long terminal repeat, in the leader sequence for env mRNA, and at the poly-env junction. The effect of these mutations in the viral DNA upon its ability to direct virus production or env mRNA synthesis was analyzed by microinjecting the mutant DNAs into chicken embryo fibroblasts and into chicken cells transformed by the env-deficient Bryan strain of Rous sarcoma virus, respectively. The results indicated that: (i) addition of up to 8 base pairs 19 nucleotides upstream of the Hogness box did not block transcription; (ii) deletion of 26 base pairs, including the tRNA primer binding site, allowed synthesis of all viral products and participation in recombination, but replication was blocked; (iii) deletion of fewer than 50 base pairs 250 bases downstream of the long terminal repeat depressed expression of all viral genes; and (iv) deletion of most of the gag and pol genes did not inhibit env mRNA synthesis, but virion packaging of the unspliced transcript was inefficient.
Effects of fowlpox virus infection on lipid metabolism in cultured chicken embryo cells
Authors: Buttke T M , Gafford L G
Published: January 2020

Lipid biosynthesis was measured in cultured chicken embryo cells after infection with fowlpox virus. Between 24 and 72 h postinfection, fowlpox virus-infected cells incorporated less [14C]acetate and 3H2O into fatty acids and sterols than did mock-infected cells, demonstrating a virus-dependent inhibition of general lipid metabolism. Two specific effects of fowlpox virus infection were an accumulation of C-4 alkylated sterol intermediates and inhibition of monounsaturated fatty acid biosynthesis.
Identification of ecotropic proviral sequences in high- and low-ecotropic-virus-producing mouse strains
Authors: Moore J L , Chan H W
Published: January 2020

The arrangement of endogenous ecotropic retroviruses in selected high- and low-ecotropic-virus-producing mouse strains was examined by Southern blot hybridization analysis, using an ecotropic retrovirus-specific DNA probe. High-ecotropic-virus-producing mouse strains of the AKR family displayed heterogeneity with respect to the number of copies and the sites of insertion of endogenous ecotropic specific DNA. This diversity was seen even among individuals of the same AKR subline. Contrastingly, individuals within the same low-ecotropic-retrovirus-producing mouse strain showed no evidence of variability in their endogenous ecotropic proviral sequences. These results favored the hypothesis that germ line proviral reinsertion was responsible for the proviral sequence heterogeneity observed in high-ecotropic-virus-producing mouse strains.
Phosphorylation pattern of large T antigens in mouse cells infected by simian virus 40 wild type or deletion mutants
Authors: Kress M , Resche-Rigon M , Feunteun J
Published: January 2020

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been extensively studied in productive infection of monkey cells. In this study, we analyzed the phosphorylation sites of large T antigen from SV40-infected nonpermissive mouse cells by partial proteolysis fingerprints and analysis of the phosphoamino acids present in the resulting fragments. The wild-type virus and deletion mutants (dl1263, dl1265, dl2194, and dl2198) were used for infection. On the basis of our results and published data (M. Schwyzer, R. Weil, and H. Zuber, J. Biol. Chem. 225:5627-5634, 1980), a cleavage map of large T antigen was established. It was reported that at least four sites of phosphorylation were present. The amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the prolinerich C-terminal end of the molecule at position 701 or 708. On the basis of the concensus as to the amino acid sequence surrounding the recognition sites for protein kinases, it was possible to more precisely locate this phosphothreonine at residue 701. Moreover, the C-terminal part of the molecule contained phosphoserine at a more internal position. In addition, this study firmly established the presence of a phosphothreonine in the N-terminal part of large T antigen. In conclusion, it was shown that the location of phosphorylation sites of large T antigen produced by nonpermissive mouse cells infected by SV40 is strikingly similar to that reported by other groups for large T antigen produced by SV40-infected permissive cells.
Cold-sensitive growth of simian virus 40 in semipermissive variants of CV1 cells
Authors: Fischer-Fantuzzi L , Vesco C
Published: January 2020

Two cell clones were isolated from the simian line CV1, permissive for simian virus 40 (SV40), by selection at low temperature with the tsA239 mutant of SV40. These clones exhibited cold-sensitive semipermissivity to both SV40 virions and SV40 DNA. On the basis of virus yields, their resistance to viral DNA was increased approximately 15 times over that of CV1 cells when the incubation temperature was lowered from 38.5 to 33.5 degrees C. A further 30- to 40-fold resistance increase was exhibited at both temperatures upon infection with SV40 virions. Partial characterization of these clones indicated that the cold sensitivity affected an early function in viral growth, between viral uncoating and the appearance of T-antigen positivity, with a burst-size decrease in all cells at the restricted temperature. This conditional defect appeared to be superimposed upon a temperature-independent uncoating defect, presumably carried in a CV1 subpopulation from which the two clones were ultimately selected.
Simian virus 40 encapsidation: characterization of early intermediates
Authors: Milavetz B , Hopkins T
Published: January 2020

Simian virus 40 chromosomes were separated into various species by a two-step purification consisting of low-ionic-strength glycerol gradient sedimentation followed by low-ionic-strength agarose gel electrophoresis. For each species of simian virus 40 chromosome purified, the comigrating DNA and proteins were identified by agarose or polyacrylamide gel electrophoresis, respectively. Two species of chromosomes were identified which contained form I and form II DNA and large amounts of viral protein; they migrated more slowly than most of the free simian virus 40 chromosomes, which contained very little viral protein. The nuclease susceptibility of these chromosomes suggests to us that they are intermediates in encapsidation, and we describe an encapsidation model.
Seven infection-specific polypeptides in BHK cells infected with Bunyamwera virus
Authors: Short N J , Meek A D , Dalgarno L
Published: January 2020

Virus-specific polypeptide synthesis was examined in BHK cells and Vero cells infected with Bunyamwera virus. In BHK cells, in addition to the four previously reported virus-coded proteins (L, G1, G2, and N), three other infection-specific proteins were detected. These proteins, of nominal molecular weight 50,000 (p50), 16,000 (p16), and 13,000 (p13), were not labeled in mock-infected cells, were first synthesized between 4 and 8 h after infection, and were relatively prominent among the limited number of proteins generated late in infection. In preparations of purified Bunyamwera virus from BHK cell supernatants, p16 was detected but not p50 or p13. In Vero cells infected with Bunyamwera virus, both p50 and p13 were labeled strongly. Maprik virus, a member of the Mapputta group of arboviruses, is a member of the Bunyavirus genus (S.E. Newton, unpublished data). Maprik virus did not induce the synthesis of p50, p16, or p13; however, two smaller proteins (p17 and p15) which may correspond to p16 and p13 were labeled late in Maprik infection. Our data argue that p16 is a virus-coded component of the Bunyamwera virus particle and that p50 and p13 are virus-coded, nonstructural proteins.
Physical Mapping of Hybrid Bacteriophage T7/T3 RNA Polymerase Genes
Authors: Ryan Thecla , McConnell David J.
Published: January 2020

The late regions of the T7 and T3 bacteriophage genomes are transcribed by phage-specified RNA polymerases, the products of gene 1. Although these phage transcriptional systems share many characteristics and are obviously related, they have diverged to such an extent that neither of their respective RNA polymerases utilizes the promotor sites of the other phage at an appreciable rate. However, it is possible to construct viable T7/T3 hybrids which have hybrid gene 1 sequences; the resultant hybrid enzymes exhibit altered transcriptional patterns in that they are capable of transcribing both T7 and T3 DNA to various degrees. The aim of this study was to define more closely the region(s) of the gene 1 sequence which encodes the transcriptional selectivity determinant by correlating the genetic constitution of these hybrid gene 1 sequences with their transcriptional properties. The recombinant sites within the gene 1 regions of several T7/T3 hybrids were mapped by using restriction sites as genetic markers. The results indicated that forcing a crossover event within a particular region often results in the inadvertant selection of additional genetic rearrangements. Several of the hybrid gene 1 sequences were found to have resulted from multiple crossover events, even though only one was directly selected for. In some cases the predicted crossovers were not detected; instead, several hybrids contained recombination sites elsewhere in the gene 1 region. These findings suggest that only certain combinations of T7/T3 gene 1 sequences are compatible; it may be that active hybrid T7/T3 gene 1 sequences rarely result from single genetic rearrangements. Taken together, the results of this study suggest that more than one region of the gene 1 sequence is involved in transcriptional selectivity. More specifically, the region from approximately 25 to 59% (from the left of the gene), together with the carboxyl end, appears to play an important role.
Virus with a Multipartite Superhelical DNA Genome from the Ichneumonid Parasitoid
Campoletis sonorensis
Authors: Krell Peter J. , Summers Max D. , Vinson S. Bradleigh
Published: January 2020

Virus was isolated from the lumen of the calyx region of ovaries in the parasitoid wasp
Campoletis sonorensis
(Hymenoptera: Ichneumonidae), and the nature of the viral DNA was analyzed. DNA purified from a homogeneous band of virus contained double-stranded superhelical molecules which were polydisperse in molecular weight. At least 25 different covalently closed circles were present, ranging in molecular weight from 4.0 × 10
6
to 13.6 × 10
6
. The virus DNA was analyzed with restriction enzymes, and the nature of the genetic complexity was evaluated by Southern blot hybridization of native superhelical and relaxed circular virus DNA and of
Sal
I- and
Hin
dIII-digested DNA. The data suggest that most of the variously sized covalently closed DNAs were composed primarily of nonhomologous sequences. The different size classes of covalently closed viral DNAs did not appear to exist in equimolar concentrations. However, there was no evidence from observation of virus particles in the electron microscope or from virus fractionation experiments that a mixture of viruses was present in the calyx fluid. The results from this study suggest' that the virus isolated from
C. sonorensis
, like those isolated from other endoparasitic hymenoptera, may belong to a new class of DNA viruses in which the genome is multipartite, with each DNA existing as a superhelical molecule.
Mouse Mammary Tumor Virus Proviral Sequences Congenital to C3H/Sm Mice Are Differentially Hypomethylated in Chemically Induced, Virus-Induced, and Spontaneous Mammary Tumors
Authors: Drohan W. N. , Benade L. E. , Graham D. E. , Smith G. H.
Published: January 2020

C3H/Sm mice have lost the exogenous milk-borne mouse mammary tumor virus (MMTV) characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors, yet they are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. Although the results clearly showed additional MMTV sequences in the virus-induced tumor which are not present in normal liver DNA, none of the spontaneous or chemically induced tumors could be shown to contain either newly acquired exogenous or amplified endogenous MMTV sequences. Interestingly, mammary tumors arising in C3H/Sm mice treated simultaneously with infectious MMTV (C3H) and dimethylbenz[
a
]anthracene (DMBA) possessed new exogenous MMTV DNA even though no quantitative change in tumor production was observed when these mice were compared with C3H/Sm mice treated with DMBA alone (Smith et al., Int. J. Cancer
26:
373-379, 1980). Our data indicate that the endogenous MMTV proviral units are extensively methylated in normal tissues, such as livers and normal nonlactating mammary glands. In the absence of MMTV (C3H), we found that in the rare, spontaneously occurring C3H/Sm mammary tumors, certain endogenous MMTV sequences were specifically hypomethylated. Hypomethylation of endogenous MMTV sequences was also noted in the chemically induced mammary tumors, even though radioimmune competition assays for MMTV gp52 and p28 are negative (Smith et al., Int. J. Cancer
27:
81-86, 1981). Our results support the conclusion that amplification of endogenous MMTV sequences is not intrinsic to C3H/Sm mouse mammary tumors arising spontaneously or after induction by chemicals. On the other hand, integration of exogenous MMTV DNA into the genome was a constant feature of mammary tumors developing in MMTV (C3H)-infected C3H/Sm mice, even when DMBA was used as the carcinogen. Hypomethylation of some endogenous MMTV sequences is characteristic of C3H/Sm mammary tumors, whether spontaneous or induced by chemicals, which suggests that these sequences are located in actively transcribing regions of the tumor cell genome.
Molecular cloning of a family of retroviral sequences found in chimpanzee but not human DNA
Authors: Bonner T I , Birkenmeier E H , Gonda M A , Mark G E , Searfoss G H , Todaro G J
Published: January 2020

A number of retrovirus-like sequences have been cloned from chimpanzee DNA which constitute the chimpanzee homologs of the endogenous colobus type C virus CPC-1. One of the clones contains a nearly complete viral genome, but others have sustained deletions of 1 to 2 kilobases in the polymerase gene. The pattern of related sequences detected in other primate species is consistent with the genetic transmission of these sequences for millions of years. However, the appropriately related sequences have not been detected in human, gibbon, or orangutan DNAs. These results suggest either that this family of sequences has been deleted from humans, gibbons, and orangutans, or that the genes were recently acquired in the chimpanzee and gorilla lineages.
Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA
Authors: Bosselman R A , van Straaten F , Van Beveren C , Verma I M , Vogt M
Published: January 2020

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.
Characterization of an infective molecular clone of the B-tropic, ecotropic BL/Ka(B) murine retrovirus genome
Authors: Kim J P , Kaplan H S , Fry K E
Published: January 2020

Using molecular cloning techniques, we amplified the unintegrated, linear proviral DNA of the BL/Ka(B) virus, a non-leukemogenic retrovirus of mouse strain C57BL/Ka. Two independent clones in lambda phage vector 607 and one subclone in pBR322 were infective when transfected into mouse fibroblasts. Analysis of the progeny virus revealed biological properties and a restriction map identical to those of the parental viral shock. Comparison of the restriction map with the maps of other ecotropic murine viruses reveals many similarities. Particularly interesting is the comparison of the N-tropic Akv virus and the B-tropic BL/Ka(B) virus. The long terminal repeats of the two viruses are virtually identical, as are 22 of 23 restriction sites located outside of the region which spans from 1.8 to 3.8 kilobases from the left end of the genome. Within this region, however, only three of nine sites examined are shared. This suggests that the BL/Ka(B) virus was derived from an endogenous N-tropic virus closely related to Akv by recombinational events which altered the sequence in the last half of the gag gene and the first third of the pol gene. This change is probably responsible for the observed difference in the Fv-1 tropism of the two viruses.
Characterization of a temperature-sensitive defect of enterovirus type 70
Authors: Takeda N , Miyamura K , Kono R , Yamazaki S
Published: January 2020

The mechanism of the failure of enterovirus type 70 to replicate at a nonpermissive temperature (39 degrees C) was investigated, and the following results were obtained. (i) Viral RNA synthesis was not observed at 39 degrees C in LLC-MK2 cells, in accordance with our previous findings with primary monkey kidney cells (Miyamura et al., Intervirology 9:206-213, 1978). (ii) Shutoff of host cell macromolecular synthesis by virus infection was as efficient at 39 degrees C as at a permissive temperature (33 degrees C). This inhibitory effect similarly occurred even in the presence of guanidine hydrochloride. (iii) Viral protein synthesis proceeded in vivo at the nonpermissive temperature, and the rate of the protein synthesis was higher than that at the permissive temperature under the conditions in which sufficient viral mRNA had been accumulated. This was also confirmed by analyzing the intracellular proteins synthesized at the nonpermissive temperature by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which identified them as virus-specific proteins. (iv) When infected cells were incubated at 39 degrees C and then transferred to 33 degrees C, viral RNA synthesis took place even in the presence of cycloheximide. (v) Furthermore, in experiments performed with an in vitro cell-free assay system, viral polymerase activity was found in the membrane-bound preparation extracted from infected cells which had been incubated at 39 degrees C in the presence or absence of guanidine hydrochloride. These results indicate that early translation of mRNA proceeds normally at the nonpermissive temperature and that the temperature-sensitive defect resides in the transcriptional stage.
Evidence that infectious pancreatic necrosis virus has a genome-linked protein
Authors: Persson R H , Macdonald R D
Published: January 2020

The double-stranded RNA segments of infectious pancreatic necrosis virus were extracted from virions by a method which avoids proteinase. In contrast to proteinase-treated RNA, such segments (i) exhibited a lower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and agarose gels, (ii) had a slightly lower buoyant density, and (iii) demonstrated a marked tendency toward aggregation as observed by electron microscopy. A small amount of protein tightly bound to the RNA could account for the above properties, and a 110,000-dalton protein was liberated from purified virion RNA by sequential digestion with RNase III and RNase A. The amount of radioactivity associated with RNA from virions labeled in vivo with [35S]methionine suggested that an average of 1.4 molecules was bound per RNA segment. Interactions between RNA segments seen in electron micrographs appeared to occur only among the ends of the segments, suggesting these were the exclusive sites of protein attachment.
Organization of chicken DNA sequences homologous to the transforming gene of avian myeloblastosis virus. I. Restriction enzyme analysis of total DNA from normal and leukemic cells
Authors: Perbal B , Baluda M A
Published: January 2020

Hybridization probes consisting of cloned DNA recombinants which represent different regions of the leukemogenic sequence (amv) from avian myeloblastosis virus were used to carry out a more detailed restriction endonuclease analysis of the homologous sequences (proto-amv) present in normal and leukemic chicken DNA. The results show that four large introns interrupt the normal cellular proto-amv sequences and that there is no major rearrangement of these sequences in leukemic myeloblasts.
Assembly of vesicular stomatitis virus: distribution of the glycoprotein on the surface of infected cells
Authors: Jacobs B L , Penhoet E E
Published: January 2020

This study demonstrates that the glycoprotein of vesicular stomatitis virus clusters in the plasma membrane of infected Chinese hamster lung cells during morphogenesis and suggests that viral nucleocapsids are required for this clustering. A mutant virus (ts E-1) which is temperature sensitive for the synthesis of viral nucleocapsids but not viral membrane proteins was used. The surface distribution of the viral glycoprotein in cells infected by this virus was determined by a specific indirect immunoferritin stain. Early in infection at permissive temperatures, the glycoprotein was randomly distributed on membrane ghosts. Later, clusters of ferritin the size and shape of virus particles were seen. In contrast, ghosts prepared from virus-infected cells maintained at a restrictive temperature always had a random distribution of viral glycoprotein.
Adenovirus-induced alterations of the cell growth cycle: effects of mutations in early regions E2A and E2B
Authors: Murray J D , Braithwaite A W , Taylor I W , Bellett A J
Published: January 2020

Mutants ts125 (E2A) and ts36 (E2B) of adenovirus type 5 induced alterations to cell cycle progression at the nonpermissive temperature which were detectable by flow cytometry. Thus neither E2A, nor gene N in E2B, is required for these effects. Whereas the wild-type virus induced cells with aneuploid (between 4n and 8n) DNA contents, as did ts125 at the permissive temperature, ts125 induced peaks of cells with 8n, 16n, and 32n DNA contents at the nonpermissive temperature. This was probably due to the failure of regulation of E1A by E2.