bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the Polyoma Viruses Induced from BHK-21 Cells Transformed by
A
Gene Mutants
Authors: Anderson D. M. , Folk W. R.
Published: January 2020

Polyoma viruses induced to replicate at 31°C in BHK-21 cells transformed by polyoma
A
gene mutants retain the thermosensitive growth characteristics and genome structure of the original mutants used for transformation.
DNA polymerase activities in growing cells infected with simian virus 40
Authors: Méchali M , Girard M , de Recondo A M
Published: January 2020

Growing CV1 cells were infected with simian virus 40 (SV40), and the levels of DNA polymerases-alpha, -beta, and -gamma were analyzed in the cytoplasm, nuclear Triton wash, and nucleus. In the cytoplasmic fraction, the amount of alpha-, beta-, or gamma-polymerase remained unaltered after SV40 infection. The activity of DNA polymerase-alpha increased five- to sixfold in the nuclear Triton wash and threefold in the nuclei and then remained enhanced only inside the nuclei. That of DNA polymerases-beta and gamma increased mostly in the nuclei after infection. These results suggest that DNA polymerase-alpha could be the major enzyme involved in SV40 DNA replication.
Polypeptide Synthesis in Simian Virus 5-Infected Cells
Authors: Peluso Richard W. , Lamb Robert A. , Choppin Purnell W.
Published: January 2020

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F
0
) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F
1
and F
2
. No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.
Properties of simian virus 40 transcriptional intermediates isolated from nuclei of permissive cells
Authors: Shani M , Birkenmeier E , May E , Salzman N P
Published: January 2020

A nucleoprotein complex that is an intermediate in viral transcription has been isolated from simian virus 40 (SV40)-infected BSC-1 cells after lysing infected nuclei with Sarkosyl. It contain DNA, DNA-dependent RNA polymerase II, and nascent RNA chains. RNA chain elongation continues for several hours in vitro and is dependent on exogenous ribonucleoside triphosphates. The complex sediments in neutral sucrose gradients with a main peak at about 24 to 26S. When the nascent RNA on the complex is treated with RNase A, a fraction of the RNA remains resistant to RNase and is hydrogen bonded to the DNA template. The pulse-labeled RNase-resistant RNA can be chased into RNase-sensitive RNA, indicating that it is located at the 3' terminus of the RNA chain. The rate of RNA displacement from the DNA template is consistent with an average rate of RNA chain elongation of 15 to 30 nucleotides per min. At least 70% of the RNA synthesized in this in vitro system is SV40 specific. Hybridization with the separated strands of SV40 DNA and with fragments of SV40 DNA generated with endonucleases HindII + III indicates that this RNA is complementary to all regions of the "late" SV40 DNA strand. Studies of SV40 RNA synthesis in this partially purified preparation at early and late times after infection should provide a way of locating promoter sites for transcription and identifying the form of SV40 DNA that serves as a template for late transcription.
Uniform representation of the human papovavirus BK genome in transformed hamster cells
Authors: Howley P M , Martin M A
Published: January 2020

The DNA of three cloned lines of hamster kidney cells transformed by human papovavirus BK DNA was examined by reassociation kinetics for viral sequences and found to contain 2.7 to 5.3 equivalents of viral DNA per diploid genome. In the one line examined with the four R-HindIII fragments of the human papovavirus BK genome, the entire viral genome was uniformly represented.
Reassociation of complementary strand-specific adenovirus type 2 DNA with viral DNA sequences of transformed cells
Authors: Johansson K , Pettersson U , Philipson L , Tibbetts C
Published: January 2020

Complementary strand-specific adenovirus DNA, either full length or from restriction enzyme cleavage fragments, was used to estimate the fractional representation and abundance of viral sequences in two adenovirus type 2 (Ad2)-transformed rat cell lines, A2F19 and A2T2C4. The reassociation method introduced is based on the linear relationship, after exhaustive hybridization, between the inverted fraction of hybrid DNA and the molar ratio of probe to cellular DNA in the reaction mixture. The amount of viral DNA in A2F19 cells represents 12 to 14% of the viral genome at a level of around seven copies per diploid cell equivalent. For the cell line A2T2C4, the pattern of integrated viral DNA sequences is more complex. With full-length Ad2 DNA strands as a probe, about 56% of the probe was represented in cellular DNA. When each of the four BamHI fragment strands of Ad2 DNA was used as a probe, the fraction of the viral DNA present also amounted to around 56% with one to five copies from different regions of the viral genome. The results demonstrate the advantage of using strand-specific viral DNA as a probe in reassociation analysis with denatured cell DNA. The method should be useful in any system in which complementary strand separation of viral DNA sequences can be achieved.
Two "early" mRNA species in adenovirus type 2-transformed rat cells
Authors: Flint S J
Published: January 2020

mRNA isolated from adenovirus 2-infected HeLa cells at early times during the productive cycle and from two lines of adenovirus 2-transformed rat embryo cells (F17 and T2C4) was fractionated on sucrose gradients after disaggregation. Viral mRNA species were identified by hybridization across such gradients with the separated strands of restriction endonuclease fragments of 32P-labeled DNA known to be complementary to adeovirus 2 "early" and adenovirus 2-transformed cell mRNA. mRNA transcribed from the left-hand 14% of the adenovirus 2 genome was found to comprise two species, 16 to 17S and 20 to 21S: the same sized mRNA's were present both at early times during productive infection and in the two transformed rat cell lines. Direct comparison of the sequences present in these two mRNA species by additional saturation hybridizations suggests that they are not related to one another. Three additional regions of the adenovirus 2 genome, all of which are located in the right-hand 40% of the adenovirus 2 genome, are complementary to early mRNA sequences: each of these appears to specify one major mRNA species of about 22S. Thus, five major species of adenovirus type 2 early mRNA have been identified. Two of these, copied from the left-hand 14% of the viral genome, are also present in adenovirus 2-transformed rat cells.
Carbohydrates of Influenza Virus. I. Glycopeptides Derived from Viral Glycoproteins After Labeling with Radioactive Sugars
Authors: Schwarz Ralph T. , Schmidt Michael F. G. , Anwer Ursula , Klenk Hans-Dieter
Published: January 2020

The carbohydrate moiety of the influenza glycoproteins NA, HA
1
, and HA
2
were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA
1
, and HA
2
. The side chain of type II contains a high amount of mannose and is found only on NA and HA
2
. The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken embryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains.
Titration of murine leukemia viruses with rat cell line RFL
Authors: Koga M
Published: January 2020

Normal rat embryo cell (RFL) from syncytia after infection with murine leukemia virus. The assay for counting the number of syncytium foci produced in RFL cells is a sensitive method for a direct infectivity assay of murine leukemia virus.
Maintenance of bacteriophage P1 plasmid
Authors: Jaffé-Brachet A , D'Ari R
Published: January 2020

Three mutants of bacteriophage P1 affected in their ability to maintain the lysogenic state stably are described here. These mutants were normal in lytic growth, but lysogenic derivatives segregated nonlysogens at abnormally high rates (1 to 30% per division). Cells harboring these mutant prophages were elongated or filamentous. The mutations responsible for this prophage instability fell into two classes on the bases of their genetic location, their effect on the ability to lysogenize recA bacteria, and their suppressibility by ant mutations eliminating antirepressor activity. The two mutants that were able to form recA lysogens showed the same prophage instability and partial inhibition of cell division in recA as in rec+ lysogens. The fact that plasmid-linked mutations can cause prophage instability suggests that P1 codes for at least some of the functions determining its own autonomy and segregation.
Inhibition of Lytic Induction in Lysogenic Cyanophyces
Authors: Cocito C. , Goldstein D.
Published: January 2020

When the lysogenic strain SPI
cts
1 of the blue-green alga
Plectonema boryanum
carrying a temperature-sensitive mutation in the LPP2 prophage was heated at a nonpermissive temperature in the light, a lytic cycle occurred, with production of infectious viral particles. Inhibitors of transcription, translation, and photosynthetic functions interfered with this process and produced different effects when administered at different phases of the viral cycle. The presence of the inhibitors during the temperature shift did not allow a successful induction to take place; lysogens submitted to such a process produced a normal virus yield, however, when the drugs were removed and the temperature was shifted again. Incubation with the inhibitors during the early postinduction period reduced the virus yield; at later times, however, the inhibitory action rapidly declined. When cells were induced in the presence of chloramphenicol, incubated with actinomycin, and then grown in the dark, at either permissive or nonpermissive temperatures, virus multiplication was equally inhibited. These data indicate that: (i) provirus induction in lysogenic cyanophyces relies on the synthesis of early viral proteins; (ii) induction of mRNA is unstable and becomes rapidly inactivated when its translation is prevented; and (iii) inhibition of photosynthesis prevents the induction message from being expressed. It is suggested that the SPI
cts
1 prophage codes for a mutated repressor, which is reversibly inactivated at a nonpermissive temperature, and that the repressor must be inactivated at the same time that the message coded for by very early genes is translated, for a successful induction of the lytic cycle.
Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124
Authors: Dina Dino , Beemon Karen
Published: January 2020

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3′ end (cDNA 3′) was prepared and shown to also be complementary to the 3′-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the “MSV-specific” portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3′ end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5′-terminal two-thirds, as well as to the 3′-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5′-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3′ portion of about 1,000 nucleotides, which is also present at the 3′ terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3′ terminus; and (iii) a second “common” region, again shared with MLV, which extends from 2,500 nucleotides to the 5′ terminus. This second common region appears to be located in the 5′ half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to
3
H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.
Transcription of the Genome of Bacteriophage φ29: Isolation and Mapping of the Major Early mRNA Synthesized In Vivo and In Vitro
Authors: Kawamura Fujio , Ito Junetsu
Published: January 2020

The φ29 early mRNA's synthesized in infected
Bacillus subtilis
were studied by using sedimentation velocity analysis, polyacrylamide gel electrophoresis, and hybridization of φ29 DNA fragments generated by the restriction endonuclease
Eco
RI. Viral RNAs synthesized in vivo in the resence of chloramphenicol were found to hybridize to
Eco
RI-A, -C, and -D fragments, but not to
Eco
RI-B and -E fragments, of the viral genome. Major early mRNA sedimenting as 16
S
material in neutral sucrose gradients was examined in detail. Radioactive φ29 RNA, purified by sucrose gradient centrifugation, was hybridized to either the
Eco
RI-A or
Eco
RI-C DNA fragment. The RNA was eluted from the hybrids and then tested for complementary hybrid formation with
Eco
RI-A and -C fragments. RNA eluted from the
Eco
RI-A fragment annealed only to the
Eco
RI-A fragment and not to the
Eco
RI-C fragment. Similarly, RNA eluted from the
Eco
RI-C fragment hybridized to the
Eco
RI-C and -D fragments. Viral RNAs synthesized in vitro using
B. subtilis
RNA polymerase hybridized to both
Eco
RI-A and -C DNA fragments. Furthermore, RNA initiated with [γ-
32
P]GTP also hybridized to both
Eco
RI-A and -C fragments. These results indicate that there are at least two efficient promotors for early transcription on the φ29 chromosome. In addition, a low-molecular-weight RNA initiated with [γ-
32
P]ATP was found to hybridize exclusively with the
Eco
RI-A fragment. Kinetic studies of φ29 mRNA synthesis during the lytic cycle have shown that viral RNAs hybridizable to the
Eco
RI-A and -C fragments are synthesized immediately after phage infection. On the other hand, mRNA specific for the
Eco
RI-B fragment was not synthesized for several minutes after phage infection. Based on the results of the in vivo and in vitro transcription studies, a transcription map of the φ29 chromosome is proposed.
Adenovirus Type 2 Early Nuclear and mRNA: Kinetic Estimation of
l
and
r
DNA Strand Fractions Complementary to Different Abundance Classes of Viral RNA
Authors: Wold William S. M. , Green Maurice , Brackmann Karl H. , Devine Catherine , Cartas Maria A.
Published: January 2020

RNA from unfractionated cells, nuclei, and polyribosomes was extracted from adenovirus 2 (Ad2)-infected KB cells early in infection and annealed in vast excess in liquid to purified Ad2
l
(heavy) and
r
(light) [
32
P]DNA strands (specific activity, 3 × 10
6
to 1.5 × 10
7
cpm/μg). The number of abundance classes of Ad2 RNA, their relative concentrations, and the strand fraction from which they arose were determined by a computer-assisted nonlinear regression analysis of hybridization kinetic data. Whole-cell RNA and nuclear RNA annealed to 60 and 40%, respectively, of
l
and
r
strands. Well-defined abundance (kinetic) classes were identified: abundant and scarce classes were complementary to 15 to 17 and 40 to 45%, respectively, of
l
strand, and to 11 to 16 and 17 to 23%, respectively, of
r
strand. In whole-cell RNA and nuclear RNA the abundant classes were 57 to 208 and 13 to 27 times more concentrated, respectively, than scarce classes. RNA-RNA hybrids were isolated that annealed to about 70% of both strands, indicating that whole-cell RNA and nuclear RNA hybridization values were minimal. Polyribosomal RNA appeared to anneal as three abundance classes to each DNA strand; abundant, scarce, and very scarce classes, respectively, hybridized to 6, 5, and about 10% of
l
strand and 7 (6 to 8), 10 (8 to 13), and about 19% of
r
strand. The abundant classes were 41 (11 to 67) times more concentrated than the scarce classes and 10
3
times more concentrated than the very scarce classes. Although the biological significance of these classes is not known, the very scarce classes probably represent nuclear RNA contaminants of polyribosomal RNA. The abundant and scarce classes may comprise mRNA, because together they are complementary to about the same fraction of each DNA strand (11% [10 to 12%] and 17% [14 to 20%] of
l
and
r
strands) known to be expressed as early mRNA. Thus, nuclear RNA contains Ad2 RNA sequences not found on polyribosomes; most or all of both DNA strands are transcribed, but only certain transcripts are processed into mRNA. It is not known whether “non-mRNA” transcripts are intermediates in the pathway of early mRNA production.
Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Authors: Schindler J , Hynes R , Hopkins N
Published: January 2020

We have sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the virion proteins of an N- and a B-tropic C-type virus derived from the BALB/c mouse and 21 putative recombinants, designated XLP-N viruses, obtained from seven crosses between these N- and B-tropic viruses. All the XLP-N viruses are N-tropic but posses the XC plaque morphology of their B-tropic virus parent. Three virion proteins, p15, p30, and gp70, of the parental viruses each differ in electrophoretic mobility. Two recombinants were found that possess a p15 that comigrates with p15 of the B virus; 19 possess a p15 that comigrates with N virus p15. Sixteen recombinants possess a gp70 that migrates like the gp70 of the B virus: four have gp70 with an electrophoretic mobility like that of the N virus gp70. All 21 recombinants possess a p30 that comigrates with p30 of their N virus parent. Given the origin and phenotype of XLP-N viruses, these results would seem to provide good evidence that these viruses are recombinants.
Localization of Single-Chain Interruptions in Bacteriophage T5 DNA. II. Electrophoretic Studies
Authors: Rhoades Marc
Published: January 2020

Upon denaturation, T5 DNA yields a large number of discrete, single-chain fragments that can be resolved by agarose gel electrophoresis. The positions of the more prominent of these fragments in the T5 duplex were determined by analyzing their sensitivity to digestion with λ exonuclease and their distribution among
Eco
RI fragments of T5 DNA. These experiments also provide firm evidence concerning the polarity of the strands in T5 DNA. An analogous study was carried out on the fragments produced by treating exonuclease III-degraded T5 DNA with the single-strand-specific SI endonuclease. This procedure yielded over 40 discrete duplex fragments that could be resolved with considerable precision by agarose gel electrophoresis. The positions of most of these fragments were determined by analyzing
Eco
RI fragments of T5
st
(+) and T5
st
(0) DNA. Over 20 sites where single-chain interruptions can occur in T5 DNA were identified, and the distribution of interruptions within the terminal repetition was shown to be identical at both ends of the molecule. A precise value for the size of the terminal repetition in T5 DNA was obtained by analyzing SI endonuclease digests of ligase-repaired, circular T5 DNA in agarose gels. The repeated segment represented 8.3% of the T5
st
(+) DNA. The results of this study also provide information concerning the properties of λ exonuclease. Hydrolysis by this enzyme was not terminated when single-chain interruptions were encountered either in the strand being degraded or in the complementary strand.
Antibody-independent detection of virus-specific glycoprotein synthesis is oncornavirus-infected cells
Authors: England J M , Dietzschold B , Halpern M S
Published: January 2020

A method is described for detecting the synthesis of avian and murine oncornavirus-specific glycoproteins without the use of antibodies raised against viral structural proteins. As applied to cells infected with avian tumor virus, the method served to resolve pr90, the precursor of the major envelope glycoprotein. A virus-specific glycoprotein of about 85,000 daltons, which has several properties expected to a precursor to gp69/71, was detected in cells infected with murine leukemia virus.
Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis
Authors: Fujisawa H , Hayashi M
Published: January 2020

A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174. The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles. Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle. The 108S material contained no DNA, and its formation occurred independently of DNA synthesis. The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant. Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles. A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described.
Temperature-sensitive mutants of human cytomegalovirus
Authors: Yamanishi K , Rapp F
Published: January 2020

Eight temperature-sensitive mutants of human cytomegalovirus have been isolated after mutagenesis with nitrosoguanidine. Three of these mutants have been classified into three separate complementation groups and are capable of synthesizing virus DNA at the nonpermissive temperature (39.5 degrees C). Two others appear unable to synthesize virus DNA at the elevated temperature.
Size and Genetic Content of Viral RNAs in Avian Oncovirus-Infected Cells
Authors: Hayward William S.
Published: January 2020

Viral complementary DNA (cDNA) sequences corresponding to the
gag, pol, env, src
, and
c
regions of the Rous sarcoma virus genome were selected by hybridizing viral cDNA to RNA from viruses that lack the
env
or
src
gene or to polyadenylic acid [poly(A)]-containing RNA fragments of different lengths and isolating either hybridized or unhybridized DNA. The specificities, genetic complexities, and map locations of the selected cDNA's were shown to be in good agreement with the size and map locations of the corresponding viral genes. Analyses of virus-specific RNA, using the specific cDNA's as molecular probes, demonstrated that oncovirus-infected cells contained genome-length (30-40S) RNA plus either one or two species of subgenome-length viral RNA. The size and genetic content of these RNAs varied, depending on the genetic makeup of the infecting virus, but in each case the smaller RNAs contained only sequences located near the 3′ end of the viral genome. Three RNA species were detected in Schmidt-Ruppin Rous sarcoma virus-infected cells: 39S (genome-length) RNA; 28S RNA, with an apparent sequence of
env-src-c
-poly(A); and 21S RNA, with an apparent sequence of
src-c
-poly(A). Cells infected with the Bryan high-titer strain of Rous sarcoma virus, which lacks the
env
gene, contained genome-length (35S) RNA and 21S
src
-specific RNA, but not the 28S RNA species. Leukosis virus-infected cells contained two detectable RNA species: 35S (genome-length) RNA and 21S RNA, with apparent sequence
env-c
-poly(A). Since
gag
and
pol
sequences were detected only in genome-length RNAs, it seems likely that the full-length transcripts function as mRNA for these two genes. The 28S and 21S RNAs could be the active messengers for the
env
and
src
genes. Analyses of sequence homologies among nucleic acids of different avian oncoviruses demonstrated substantial similarities within most of the genetic regions of these viruses. However, the “common” region of Rous-associated virus-0, an endogenous virus, was found to differ significantly from that of the other viruses tested.
Genetic Variation in the RNA Transcripts of Endogenous Virus Genes in Uninfected Chicken Cells
Authors: Wang San Y. , Hayward William S. , Hanafusa Hidesaburo
Published: January 2020

Uninfected cells from two different phenotypes of chicken embryos express significant amounts of endogenous viral information, though they do not produce virus particles. Cells of the phenotype gs
+
chf
+
are positive for both group-specific (gs) antigens and chicken helper factor (
chf
) activity, whereas cells of a second phenotype, gs
L
chf
+
(h
E
), demonstrate noncoordinate expression of these two viral activities (very low amounts of gs antigens, but extremely high helper activity). RNA from these cells was analyzed to determine the size, genetic content, and relative abundance of virus-specific RNAs in cells of each phenotype. Two major size classes of polyadenylic acid-containing RNA, homologous to the avian leukosis virus genome, were detectable in cells of both types. The larger RNA, which contained most of the sequences of the leukosis virus genome, was of different sizes in the two phenotypes, 31S in gs
+
chf
+
cells but 35S in the noncoordinate cell type. Analysis of the viral RNA with gene-specific complementary DNA probes revealed the following characteristics. (i) The 31S RNA appeared to lack portions of the
gag
and
pol
genes. (ii) A smaller RNA species, which sedimented at 21S in both cell types, was a transcript of the 3′-proximal portion of the viral genome, consisting of the
env
gene and the “common” sequences. (iii) The amount of
env
-specific RNA in the 21S region was more than six times higher in the noncoordinate cell type than in the gs
+
chf
+
cells; this difference was concordant with the 5- to 10-fold higher
chf
activity in the noncoordinate cells. (iv) The endogenous viral RNA in uninfected cells and the RNA from Rous-associated virus-0 virions hybridized only partially with DNA complementary to the common region of the Rous-associated virus-2 genome, whereas the RNA of all exogenous viruses tested hybridized almost completely to this complementary DNA. Small amounts of
src
-specific polyadenylated RNA were also present in uninfected chicken cells. This RNA sedimented as a single peak at 26S and was not covalently linked to any other identifiable virus-specific RNA sequences. The amount of
src
RNA was the same in the above two types of expression-positive cells and also in cells that were gs
−
chf
−
, indicating that the transcription of the cellular sequences homologous to the
src
gene is independent of the transcription of the other endogenous viral genes.
Interactions of Murine Leukemia Virus Core Components: Characterization of Reverse Transcriptase Packaged in the Absence of 70S Genomic RNA
Authors: Gerwin Brenda I. , Levin Judith G.
Published: January 2020

Virions produced by cells in the presence of actinomycin D (Act D virions) contain reverse transcriptase but are deficient in 70S genomic RNA. To assess the role of genomic RNA in encapsidation of a functional reverse transcriptase and to study the interaction of the enzyme and its template in the cores of intact virions, the reverse transcriptase enzymes of normal and Act D virions were compared. The enzymes were indistinguishable by column chromatography, sedimentation velocity, or template/primer preferences. In addition, these enzymes showed equal sensitivity to inactivation by antibodies directed against Rauscher murine leukemia virus DNA polymerase. The enzymes from Act D and normal virions had similar thermal decay rates and were both protected against heat denaturation by natural and synthetic template/primers. By these criteria, the DNA polymerase molecules synthesized and assembled into virions in the absence of genomic RNA are identical to those packaged under normal conditions. Additional studies designed to measure protection of reverse transcriptase by genomic RNA were carried out by comparing the thermal lability of the enzyme in intact Act D and normal virions. The thermal decay rate of reverse transcriptase in Act D virions was identical to that in control virions. In contrast to the lability of the virion-associated enzyme, however, genomic RNA in control virions was stable to heat treatment.
Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. II. Biochemical Evidence for Genetic Exchange
Authors: Vogel T. , Gluzman Y. , Winocour E.
Published: January 2020

The genome of the simian virus 40 (SV40) temperature-sensitive (
ts
) mutant
tsD202
rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol.
24
:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the
ts
phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R·
Hae
III cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the
tsD
lesion), is missing in the DNAs of the parental
tsD202
virus and of three independent revertants of
tsD202.
In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued
D202
populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R·
Hin
(II + III) cleavage patterns of SV40 strain 777 DNA and
tsD202
DNA revealed differences in the electrophoretic mobilities of
Hin
fragments A, B, and F. However, the corresponding
Hin
fragments from all four rescued
D202
genomes were identical in their mobilities to those of
tsD202
DNA, indicating that these regions of the rescued
D202
genome are characteristic of the
tsD202
parent. We conclude, therefore, that the genome of the rescued
D202
virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous
tsD202
mutant.
Recombination Between Temperature-Sensitive Mutants of the Arenavirus Pichinde
Authors: Vezza Annè C. , Bishop David H. L.
Published: January 2020

High-frequency recombination was obtained with temperature-sensitive, conditionally lethal mutants of the arenavirus Pichinde.
I protein: bacteriophage T7-coded inhibitor of Escherichia coli RNA polymerase
Authors: Hesselbach B A , Nakada D
Published: January 2020

Bacteriophage T7-coded inhibitor of Escherichia coli RNA polymerase, termed I protein, was purified from an inactive E. coli RNA polymerase-I protein complex isolated from phage T7-infected cells. A molecular weight of about 7,000 to 9,000 was assigned to the purified I protein by acrylamide-sodium dodecyl sulfate gel electrophoresis, Sephadex G-50 gel filtration, and glycerol gradient centrifugation analysis. I protein inhibits initiation of RNA synthesis by directly binding to the RNA polymerase holoenzyme and prevents the binding of the enzyme to the promoter sites on the template T7 DNA. However, once a highly stable transcriptional preinitiation complex between RNA polymerase holoenzyme and T7 DNA is formed at the promoter site on T7 DNA in the absence of nucleoside triphosphates, I protein does not inhibit the initiation of RNA synthesis by this preformed complex upon addition of nucleoside triphosphates. RNA synthesis by the core RNA polymerase and the binding of core RNA polymerase with template DNA are not inhibited by I protein, although a partial association between the core enzyme and I protein can be observed. I protein does not bind to sigma factor or T7 DNA. Therefore, binding of I protein with the RNA polymerase, which results in the inhibition of initiation of RNA synthesis, requires the presence of sigma factor in the RNA polymerase holoenzyme form.
Two methyltransferase activities in the purified virions of vesicular stomatitis virus
Authors: Testa D , Banerjee A K
Published: January 2020

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.
Complex of Virus-Like Particles Containing Double-Stranded RNA from
Thielaviopsis basicola
Authors: Bozarth R. F. , Goenaga A.
Published: January 2020

Six randomly selected isolates of
Thielaviopsis basicola
were found to contain spherical virus-like particles (VLPs) approximately 40 nm in diameter. One isolate, ATCC 34114, selected for further analysis contained a complex of VLPs that sedimented as eight or more bands in sucrose density gradients and contained five size classes of double-stranded RNA. Five discrete precipitation lines were obtained in immunoelectrophoresis, which indicated that this isolate of
T. basicola
contains five distinct species of VLPs.
Virus-specific DNA in the cytoplasm of avian sarcoma virus-infected cells is a precursor to covalently closed circular viral DNA in the nucleus
Authors: Shank P R , Varmus H E
Published: January 2020

Three principal forms of viral DNA have been identified in cells infected with avian sarcoma virus: (i) a linear duplex molecule synthesized in the cytoplasm, (ii) a covalently closed circular molecule found in the nucleus, and (iii) proviral DNA covalently linked to high-molecular-weight cell DNA. To define precursor product relationships among these forms of viral DNA, we performed pulsechase experiments using 5-bromodeoxyuridine to label by density the linear species of viral DNA in the cytoplasm during the first 4 h after infection. After a 4-to 8-h chase with thymidine, a portion of the density-labeled viral DNA was transported to the nucleus and converted to a covalently closed circular form. We conclude that linear viral DNA, synthesized in the cytoplasm, is the precursor to closed circular DNA observed in the nucleus.
Endoribonuclease activity associated with animal RNA viruses
Authors: Kolakofsky D , Altman S
Published: January 2020

A specific endoribonucleolytic activity was detected when detergent-lysed vesicular stomatitis of Sendai virus was incubated with the precursor to Escherichia coli tRNA Tyr. The cleavage products produced and the characteristics of the reaction were similar to those previously reported for human KB cell RNase NU. Like RNase NU, the virus-associated reaction generates 5'-hydroxyl and 3'-phosphate groups at the cleavage sites. At protein concentrations similar to those used to test vesicular stomatitis and Sendai viruses, virions of Sindbis virus and poliovirus also exhibited endoribonucleolytic activity, but reovirus, simian virus 40, and minute virus of mice did not. This endoribonuclease may be of physiological relevance to some of the viruses we tested.
Binding and transcription of simian virus 40 DNA by DNA-dependent RNA polymerase from Escherichia coli
Authors: Hale P , Lebowitz J
Published: January 2020

Supercoiled simian virus 40 was transcribed more efficiently than nonsupercoiled DNA. The effect was increased from two- to fivefold by the addition of rifampin with triphosphates. The number and locations of polymerase binding sites with respect to Hin II-III restriction fragments were determined. The total number of binding sites was nine, as determined by UV difference spectroscopy. The locations of these binding sites were on the A, B, D, E, F, and G fragments, as determined by gel electrophoresis. The number of sites was the same for both supercoiled and relaxed or Hin II-III-digested DNA, and the point of saturation of supercoiled DNA by polymerase remained the same with increasing concentrations of rifampin from 0 to 8 microgram/ml.
Effect of chemical modification of supercoiled simian virus 40 DNA on the rate of in vitro transcription
Authors: Hale P , Lebowitz J
Published: January 2020

Superhelical simian virus 40 FI DNA could be modified with the single-strand-specific reagent N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC). A limited reaction, of less than 2% of the base pairs, resulted in almost total inhibition of in vitro transcription by DNA-dependent RNA polymerase from Escherichia coli. This effect was shown to be due to DNA modification and not to inhibition of polymerase activity by the reagent. Inhibition of enzyme activity occurred if the contaminating reagent was not absorbed with another protein before polymerase addition. No inhibition was observed when DNA and polymerase were incubated together to allow the formation of pre-initiation complexes before CMC was added. Studies of template saturation with polymerase showed that the inhibition of transcription by DNA modification was due to a loss of binding ability of the enzyme to the reacted, supercoiled DNA when reaction times of less than 2 h were used.
Comparison of two viable variants of simian virus 40
Authors: Kay A C , Rao G R , Singer M F
Published: January 2020

The DNAs of two viable strains of simian virus 40, 776 and 777, have been compared by using restriction endonucleases. Differences between the two strains were detected at five separate points on the simian virus 40 genome. One of these differences, in the region of DNA coding for the major viral coat protein, was confirmed by tryptic peptide analysis of coat proteins from the two strains. Some physiological differences between the two strains were examined and can, in general, be explained by differences observed between the DNAs of the two strains. In addition, defective variants derived from strain 777 interfere more efficiently with the replication of strain 777 than with the replication of strain 776.
Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation
Authors: Singer Irwin I. , Rhode Solon L.
Published: January 2020

The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and
ts
1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [
3
H]thymidine uptake in cultures infected with wild-type H-1 versus
ts
14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome
c
-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [
3
H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in
ts
1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [
3
H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis.
Purification and structures of recombining and replicating bacteriophage T7 DNA
Authors: Langman L , Paetkau V
Published: January 2020

During the infection of Escherichia coli by bacteriophage T7, there is a gradual conversion of host DNA to T7 DNA. Recombination and replication occur during this time. We have devised a new way of examining the physical structures of the intermediates of these processes. It is based on the observation that there are no sites in T7 DNA susceptible to cleavage by the restriction endonuclease EcoRI. E. coli DNA, on the other hand, is susceptible to degradation by EcoRI. Thus, phage and host DNA can be separated by sucrose gradient centrifugation after treatment with EcoRI. Concatemeric T7 DNA contains a high proportion of branched, gapped, and whiskered structures. These appear to be intermediates of replication and recombination. This approach also monitors the conversion process from host to T7 DNA.
Biological properties and physical map of the genome of a new papovavirus, HD virus
Authors: Bosslet K , Sauer G
Published: January 2020

The superhelical DNA of the HD papovavirus is heterogeneous and consists of two discrete size classes with molecular weights of 3.45 X 10(6) and 3.25 X 10(6). Both size classes of DNA are encapsidated into HD virion particles. Their relative intracellular amounts differ, depending on the cell system. Vero-76 carrier cultures in which HD virus was detected contain both size classes of DNA, with the larger molecules prevailing by a factor of 10. Five clonal lines derived from Vero-76 cell cultures contain exclusively the larger DNA. On the other hand, after cocultivation of Vero-76 with CV-1 cells for several passages, minicircular DNA is accumulated such that both size classes are synthesized in equal amounts. Any of the originally viral DNA-producing cell lines may, upon subcultivation, cease yielding virus. The RITA cell line of Cercopithecus aethiops origin is the only cell line among numerous ones tested which upon infection permits the establishment of a one-step growth cycle. However, between 6 and 8 days after infection, viral DNA synthesis is discontinued, and a persistent viral infection cannot be established. Physical maps of the genomes were constructed, and it could be shown that the smaller, minicircular DNA had originated from the larger DNA as the result of a deletion. The sequences missing in the minicircular DNA are confined to the relative map position 0.15 to 0.21.
Isolation and characterization of cytoplasmic inclusions from influenza A virus-infected cells
Authors: Shaw M W , Compans R W
Published: January 2020

Influenza A viruses induce the accumulation of electron-dense inclusions in the cytoplasm of infected cells during the latter stages of the replication cycle. Cell fractionation studies showed that these inclusions could be recovered in subcellular fractions containing ribosomes and polysomes. Isolation of these inclusions was accomplished by procedures involving RNase treatment of these fractions followed by repurification, or by fluorocarbon extraction and gradient centrifugation. Electron microscopy indicated that the isolated inclusions exhibited a major periodicity of approximately 8 nm with minor periodicities of approximately 4 nm. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the influenza virus coded nonstructural protein was the only protein component present in isolated inclusions.
In vitro termination of adenovirus DNA synthesis by a soluble replication complex
Authors: Arens M , Yamashita T
Published: January 2020

The double-stranded DNA from a soluble DNA replication complex that was labeled with deoxyribonucleoside triphosphates and completed in vitro was digested with EcoRI, Sma I, and Hpa I restriction endonucleases. All regions of the adenovirus type 2 genome were labeled in vitro, but restriction fragments derived from the ends of the DNA molecules were relatively more highly labeled than those derived from internal regions. The in vitro endogenous DNA polymerase reaction also exhibited strand-specific labeling near the molecular ends, in that restriciton fragments from the left end were labeled predominantly in the r strand and fragments from the right end were labeled predominantly in the l strand.