bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Early and Late Simian Virus 40 Transcription: Overproduction of Early Viral RNA in the Absence of a Functional T-Antigen
Authors: Khoury George , May Evelyne
Published: January 2020

Virus-specific RNA synthesized in monkey cells after infection by both wild-type simian virus 40 (SV40) and the early SV40 temperature-sensitive mutant
tsA
58 has been analyzed. The fraction of SV40-specific RNA increased throughout infection with either wild-type SV40 or with
tsA
58 in direct proportion to the accumulation of progeny DNA molecules, suggesting their role in the late transcriptional process. Cytoplasmic fractions from cells infected at various temperatures (31.5 to 41°C) by wild-type virus and harvested 48 h later contained 4 to 8% virus-specific RNA, of which 5 to 10% was early SV40 RNA. In contrast, though 5 to 8% of the cytoplasmic RNA from
tsA
58-infected cells incubated at 31.5 to 37°C for 48 h was virus specific, the percentage of early virus-specific RNA ranged from 25 to 80% as the incubation temperature increased. In
tsA
58-infected cultures incubated for 48 h at 41°C (a temperature at which essentially no
tsA
58 DNA synthesis occurred), only 0.4% of the cytoplasmic RNA was virus specific, but at least 90% of this RNA was early. In experiments where cells were inoculated at 32°C and shifted at 48 h postinfection to 40°C for various times, the percentage of virus-specific pulse-labeled RNA varied from 3.5 to 10.0%. Of the virus-specific RNA, early SV40 RNA ranged from 14 to 65% in
tsA
58-infected cultures. Analogous studies with Sarkosyl-extracted viral transcription complexes to incorporate label into nascent (unprocessed) viral RNA yielded essentially identical results. This finding strongly suggests that the overproduction of early SV40 RNA occurs at the level of synthesis. While cytosine arabinoside effectively terminated most viral DNA replication in wild-type-infected cells, the ratio of early to late viral RNA remained less than 1:9. These results demonstrate that: (1) the amount of virus-specific RNA synthesized depends directly on the amount of viral DNA available for use as templates; once viral DNA replication has occurred, presumably providing progeny SV40 DNA molecules for templates, the level of transcription remains high; (ii) termination of viral DNA replication does not terminate late SV40 transcription; (iii) early SV40 RNA is overproduced by
tsA
58 at all temperatures, but especially at higher temperatures; and (iv) overproduction of early SV40 RNA appears to be correlated with defectiveness of the
tsA
mutant T-antigen. These results suggest that T-antigen may regulate its own production either by repressing the synthesis of early viral RNA or by stimulating the synthesis of late SV40 RNA or both.
RNase T1-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives
Authors: Faller D V , Hopkins N
Published: January 2020

We used two-dimensional gel electrophoresis to obtain fingerprints of RNase T1-resistant oligonucleotides of a B-tropic murine leukemia virus from BALB/c and five NB-tropic viruses independently derived from this B virus by passage through NIH Swiss mouse embryo cells in vitro. The fingerprints of the B- and NB-tropic viruses were very similar: approximately 33 of 35 large T1-resistant oligonucleotides appeared to be shared by these viruses. However, the five NB-tropic viruses possessed an apparently common alteration relative to their B virus progenitor. This change involved the acquisition of one oligonucleotide and, tentatively, the loss of one oligonucleotide. We do not know whether these changes represent an alteration responsible for the change from B- to NB-tropism. Fingerprints of B- and NB-tropic viruses were not affected when the viruses were grown in cells of different Fv-1 type.
Reovirus-Specific Enzyme(s) Associated with Subviral Particles Responds In Vitro to Polyribocytidylate to Yield Double-Stranded Polyribocytidylate·Polyriboguanylate
Authors: Gomatos Peter J. , Kuechenthal Ingrid
Published: January 2020

In reovirus-infected cells, virus-specific particles accumulate that have associated with them a polyribocytidylate [poly(C)]-dependent polymerase. This enzyme copies in vitro poly(C) to yield the double-stranded poly(C)·polyriboguanylate [poly(G)]. The particles with poly(C)-dependent polymerase were heterogeneous in size, with most sedimenting from 300
S
to 550
S
. Exponential increase in these particles began at 23 h, and maximal amounts were present by 31 h, the time of onset of exponential growth of virus at 30°C. Maximal amounts of particles with active transcriptase and replicase were present at 15 and 18 h after infection. Thereafter, there was a marked decrease in particles with active transcriptase and replicase until base line levels were reached at 31 h. Thus, the increase in poly(C)-responding particles occurred coincident with the decrease in particles with active transcriptase and replicase. The requirement for poly(C) as template was specific because no RNA was synthesized in vitro in response to any other homopolymer, including 2′-
O
-methyl-poly(C). Synthesis was optimal in the presence of Mn
2+
as the divalent cation, and no primer was necessary for synthesis. In contrast, the dinucleotide GpG markedly stimulated synthesis in the presence of 8 mM Mg
2+
. The size of the poly(C)·poly(G) synthesized in vitro was dependent on the size of the poly(C) used as template. This suggested that the whole template was copied into a complementary strand of similar size. The
T

m

of the product was between 100 and 130°C. Hydrolysis of the product labeled in [
32
P]GMP with alkali or RNase T2 yielded GMP as the only labeled mononucleotide. This does indicate that the synthesis of the poly(G) strand in vitro did not proceed by end addition to the poly(C) template, but proceeded on a separate strand.
Species Identification and Genome Mapping of Cytoplasmic Adenovirus Type 2 RNAs Synthesized Late in Infection
Authors: McGrogan Michael , Raskas Heschel J.
Published: January 2020

Adenovirus type 2 cytoplasmic RNAs synthesized late in productive infection were resolved by electrophoresis on formamide gels. Regions of the adenovirus 2 genome specifying RNAs of distinct size were determined by hybridization to specific DNA fragments generated by cleavage with endo R·
Eco
RI and endo R·
Sma
I. From these studies 13 distinct viral RNA species were identified. A 26S to 28S size class and a 21S to 23S size class were each found to consist of four distinct RNA species. Three RNA species were identified in a 16S to 18S size class, and a fourth size class, 11S to 13S, was resolved into two components. The
Sma
I-D region (0.38 to 0.51 on the unit genome) and the
Eco
RI-F, D region (0.70 to 0.83) of the genome were found to code for multiple transcripts. Three RNAs (28S, 22S, and 18S) are specified by
Sma
I-D, and four components, 28S, 22S, 18S, and 16S, are encoded by EcoRI-F,D. The RNA represented by each set of multiple transcripts exceeds the coding capacity of the respective region, and the species within each set of RNAs appear to contain common sequences. The relationship between the cytoplasmic RNA species synthesized at late times and early cytoplasmic RNAs was determined by hybridization-inhibition experiments. The multiple transcripts encoded by the
Eco
RI-D fragment were found to contain sequences that are present in early cytoplasmic RNA. These studies enabled preparation of a map which accounts for transcription of approximately 67% of the
r
strand of the adenovirus 2 genome.
Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule
Authors: Scher B M , Dean D H , Garro A J
Published: January 2020

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.
Roles of the Early Genes of Bacteriophage T7 in Shutoff of Host Macromolecular Synthesis
Authors: McAllister William T. , Barrett Cornelia L.
Published: January 2020

Through the use of phage mutants in which various combinations of the early genes are active, and in which late gene expression is blocked, we have examined the roles of each of the five early gene products of bacteriophage T7 in regulating the synthesis of host RNA and proteins. At least two independent transcriptional controls operate during bacteriophage T7 development. The product of gene 0.7, acting alone, leads to a rapid (by 5 min) shutoff of host transcription. In the absence of gene 0.7 function, and in the absence of the phage-specified RNA polymerase, a delayed shutoff of host-dependent transcription begins at approximately 15 min after infection. This secondary control element requires either a functional gene 0.3 or gene 1.1. In the absence of any early gene products, host shutoff is not observed until much later in infection (>30 min). The delayed manner in which the products of genes 0.3 and 1.1 exert their effect suggests that their mode of action is indirect. Under conditions in which the late genes are transcribed (inefficiently) by the host RNA polymerase, gene 1.1 is observed to stimulate the synthesis of lysozyme (the product of a late phage gene). In contrast, when the late genes are transcribed by the phage-specified RNA polymerase (the product of gene 1), the kinetics of synthesis of the phage RNA polymerase itself, and of lysozyme, are not affected by the deletion of genes 0.3, 0.7, 1.1, and 1.3. We conclude that under these conditions, the products of these genes are required neither for regulation of expression of the late genes nor for the shutoff of early phage gene expression.
Mechanism of murine leukemia virus-induced fusion in rat myoblasts defective in differentiation
Authors: Wong P K , Yuen P H , Kaufman S J
Published: January 2020

fu-1 cells, a line of rat myoblasts defective in differentiation, can be fused into multinucleate syncytia by Moloney murine leukemia virus. The effects of treating the virus with specific antibody, UV irradiation, and elevated temperature and the requirements for cellular RNA and protein synthesis have been studied as they relate to this virus-induced fusion. The results indicate that intact, but not necessarily infectious, virions are required to promote fusion of fu-1 cells. Neither actinomycin D nor cycloheximide altered the formation of syncytia; thus, neither viral nor cellular RNA or protein synthesis is required for fusion. fu-1 cells infected with the ts3 temperature-sensitive mutant of Moloney murine leukemia virus accumlate large amounts of budding virus on their cell membrane; however, this membrane-associated virus failed to induce syncytia. Upon release of the virus at the permissive temperature, fusion did occur. We conclude that contact or attachment of the immature virus to the cell membrane is not sufficient to promote murine leukemia virus-induced cell fusion; complete virions are required. From these data, we propose that adsorption and penetration of the virus may induce a change in the cell membrane that subsequently promotes the fusion of susceptible cells.
Physical and Genetic Characterization of Deletion Mutants of Simian Virus 40 Constructed In Vitro
Authors: Cole Charles N. , Landers Terry , Goff Stephen P. , Manteuil-Brutlag Simone , Berg Paul
Published: January 2020

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with
Hae
II or
Bam
HI endonuclease or partial cleavage with either
Hae
III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage λ 5′ -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the
Hae
II endonuclease site (map location 0.83), define a new genetic complementation group,
E
, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the
D
complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the
Eco
RI endonuclease site (0/1.0) and eleven with deletions at the
Bam
HI endonuclease site (0.15) fall into the
B/C
complementation group. Six additional mutants with deletions at the
Bam
HI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the
B/C
group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the
E, D
, and
B/C
genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the
A
complementation group. Thus, the
A
gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene
A
function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.
Further characterization of the complementation group B temperature-sensitive mutant of respiratory syncytial virus
Authors: Belshe R B , Richardson L S , Schnitzer T J , Prevar D A , Camargo E , Chanock R M
Published: January 2020

ts-2, a temperature-sensitive and plaque morphology mutant of respiratory syncytial virus and sole representative of complementation group B, was compared with members of the other complementation groups of respiratory syncytial virus (group A [ts-1] and group C [ts-7]). ts-2 was found to be 10- to 1,000-fold more restricted in growth and ability to spread at restrictive temperatures (37, 38, and 39 degrees C) than at the permissive temperature (32 degrees C). In temperature shift-up experiments, the ts defect of ts-1 and other members of complementation group A was found to effect a late function that was required for at least 13 h in the replicative cycle. The ts lesion of ts-7 affected a function early in the replication cycle. In contrast, ts-2 was not temperature sensitive when studied by the shift-up technique. The discrepancy between the ts plaque property and failure to detect temperature sensitivity during the shift-up experiment was resolved when it was shown that ts-2 had a defect in adsorption or penetration or both at the restrictive temperature. Clonal analysis of revertant ts-2 showed a coordinate restoration of ts+ phenotype ans syncytium-forming capacity. It appears that ts-2 has a defect in a protein that is involved in adsorption and/or penetration of virus and is also responsible for cell fusion activity.
Role of potassium in the synthesis and decay of two specific bacteriophage T4 messages
Authors: Walsh M L , Cohen P S , Ennis H L
Published: January 2020

The role of K+ in the in vivo metabolism of specific phage T4 messengers was studied. By using a mutant of Escherichia coli defective in its ability to accumulate K+ from the growth medium, it was possible to rapidly deplete cells of their intracellular K+ and in this way determine K+-dependent reactions in vivo. The rate constants for accumulation, synthesis, and decay of the early enzymes deoxynucleotide kinase and alpha-glucosyl transferase were determined. It was shown that there is a very close association between mRNA synthesis and its decay, indicating that a mechanism may be present in the cell that can regulate the concentration of these RNAs. Since the mRNA's for these enzymes are very stable in cells depleted of K+, K+ depletion may be a useful method for the isolation of functional T4 mRNA.
Physical organization of subgroup B human adenovirus genomes
Authors: Tibbetts C
Published: January 2020

Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved.
Changes in the capsid structure and stability of defective particles of bacteriophage R17
Authors: Iglewski W J
Published: January 2020

Serological and chemical methods were used to compare the capsid structure and stability of R17 phage and amA31 defective particles. Immunodiffusion analysis demonstrated identity between intact R17 and amA31 capside and between dissociated subunits of both R17 and amA31 and purified coat protein. Radioimmunoassays detected an antibody in R17 antisera that binds to intact R17 but could not be absorbed from R17 antisera with amA31. The R17 antibody remaining in amA31-absorbed sera did not neutralize infectivity of R17 phage. Differences between the surface composition of R17 and amA31 capsids were also detected by iodination. Capsids of R17 bound approximately four times more 125I than amA31, which was accounted for by a decreased 125I labeling of coat protein. Finally, amA31 capsids dissociated under milder conditions of sodium dodecyl sulfate treatment than R17 capsids. The sodium dodecyl sulfate dissociation of both R17 and amA31 capsids resulted in the formation of a transient 38,000-dalton intermediate, which subsequently dissociated to coat protein monomers. Preparations of dissociated R17 capsids also contained assembly protein was also found in preparations of dissociated amA31 capsids.
Superinfection exclusion by bacteriophage T7
Authors: McAllister W T , Barrett C L
Published: January 2020

Only two of the early genes of bacteriophage T7 were found to play a significant role in exclusion of superinfecting bacteriophage T3 particles; genes 0.3 and 1. Protein synthesis by the preinfecting phage particle was not required for efficient exclusion. These findings are discussed with regard to the known functions of these genes during T7 development.
Binding characteristics of Rauscher leukemia virus envelope glycoprotein gp71 to murine lymphoid cells
Authors: Fowler A K , Twardzik D R , Reed C D , Weislow O S , Hellman A
Published: January 2020

The major envelope glycoprotein (gp71) purified from Rauscher leukemia virus (R-MuLV) binds efficiently to murine lymphoid cells but not to either murine nonlymphoid cells or lymphoid cells from other species. Binding of 125I-labeled R-MuLV gp71 was competitively inhibited by unlabeled glycoprotein, as well as by whole R-MuLV, but not by murine xenotropic viruses, R-MuLV p30, and several unrelated proteins. Polyacrylamide gel electrophoresis profiles of iodinated gp71 after binding to lymphoid cells were similar to prebound profiles. Antibody to R-MuLV gp71 prevented binding, whereas normal serum had no effect. Adsorption of the glycoprotein to murine lymphoid cells occurs rapidly and is time and temperature dependent. The procedure described is sensitive for detecting the binding activity of approximately 10(4) cells. Binding was proportional up to 2.5 X 10(5) cells per ml and plateaued above 10(7) cells per ml. In the presence of excess R-MuLV gp71, BALB/c thymocytes bound approximately 2.4 X 10(4) molecules per cell.
Suppression of gene 49 mutations of bacteriophage T4 by a second mutation in gene X: structure of pseudorevertant DNA
Authors: Shah D B , DeLorenzo L
Published: January 2020

Mutations in gene 49 of bacteriophage T4 were suppressed by a second mutation in gene X. Mapping studies located gene X between genes 41 and 42. Complementation results indicated that mutations in FdsA gene (a suppressor of gene 49 mutants) were in gene X. The intracellular pseudorevertant DNA was examined for unusual properties which could explain its successful encapsidation. After the in vivo inactivation of a temperature-sensitive gene 32 (DNA unwinding) protein, the intracellular pseudorevertant DNA was converted into DNA pieces of approximately genome size. A similar conversion was observed after in vitro digestion of pseudorevertant DNA with single-strand-specific S1 endonuclease. Appreciable quantities of oligomeric intermediates were not produced during this conversion process. These data indicate that pseudorevertant DNA contains sizable single-stranded gaps and has a conformation similar to that of wild-type DNA. The results further suggest that the suppression of gene 49 mutant abnormal DNA phenotype and the encapsidation defect by a second mutation in gene X is associated with the formation of sizable single-stranded gaps. These studies raise the possibility that single-stranded gaps may be involved directly in the DNA encapsidation process, or may act indirectly as a consequence of their effect on the organization of intracellular DNA.
Replication of Epstein-Barr virus: ultrastructural and immunofluorescent studies of P3HR1-superinfected Raji cells
Authors: Seigneurin J M , Vuillaume M , Lenoir G , De-Thé G
Published: January 2020

We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (less than or equal to 50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (greater than or equal to 300 particles per cell), a productive cycle was the rule.
Mechanism for Control of Synthesis of Semliki Forest Virus 26S and 42S RNA
Authors: Sawicki Dorothea L. , Kaariainen Leevi , Lambek Caryl , Gomatos Peter J.
Published: January 2020

When cells infected with the Semliki Forest virus (SFV) mutant
ts
-4 were shifted to the nonpermissive temperature, synthesis of 26S RNA ceased, whereas synthesis of 42S RNA continued normally. These two single-stranded SFV RNAs are synthesized in two types of replicative intermediate (RI), 26S RNA in RI
b
and 42S RNA in RI
a
. Cessation of 26S RNA synthesis after shift up in temperature was accompanied by loss of RI
b
. When infected cells were shifted back down to 27°C, 26S RNA synthesis resumed, coincident with the reappearance of RI
b
. In both types of RI, the 42S minus-strand RNA is template for synthesis of plus-strand RNA. In pulse-chase experiments, we obtained RIs labeled only in their minus-strand RNA, and thus could follow the fate of RIs assembled at 27°C when they were shifted to 39°C. Our results show that, after shift up to 39°C, there was a quantitative conversion of RIs in which 26S RNA had been synthesized to RIs in which 42S RNA was synthesized. This conversion of RI
b
to RI
a
was reversible, since RIs in which 26S RNA was synthesized reappeared when the infected cultures were shifted back down to 27°C. We propose that, associated with RI
b
, in which 26S RNA is synthesized, there is a virus-specific protein that functions to promote initiation of 26S RNA transcription at an internal site on the 42S minus-strand RNA and to block transcription on the minus strand in this region by the SFV RNA polymerase that had bound and was copying the minus-strand RNA from its 3′ end. A ribonuclease-sensitive region would thus result in the sequence adjacent to the one that was complementary to 26S RNA. This virus-specific protein is not a component of the SFV RNA polymerase that continues to transcribe 42S RNA, and it is temperature sensitive in
ts
-4 mutant-infected cells. When this virus-specific protein is not present on RIs, the SFV polymerase transcribes the whole 42S minus-strand RNA and yields 42S plus-strand RNA.
Transcription of host-substituted simian virus 40 DNA in whole cells and extracts
Authors: Kuff E L , Ferdinand F J , Khoury G
Published: January 2020

Viral transcriptional complexes were extracted from the nuclei of monkey kidney cells infected with wild-type simian virus 40 (SV40) or a variant strain containing a high proportion of host-substituted DNA molecules. The RNAs synthesized by these complexes in an in vitro system were analyzed for their content of SV40 and host sequences by a technique of sequential hybridization to plaque-purified and substituted viral DNAs. The relative labeling of the two types of sequences was commensurate with their proportion in the viral DNA (about 20% host). The substituted virus contains both reiterated and unique types of cellular sequences, and both kinds appeared to be transcribed. Transcripts of the substituted sequences formed a much smaller proportion of the virus related RNA recovered from intact infected cells, suggesting that host sequence transcripts are synthesized but rapidly degraded in the whole cell. The alternative, that transcription of these sequences is artificially enhanced in the in vitro system, cannot be rigorously excluded. We compared the self-annealing of viral RNAs from nuclear extracts of cells infected with wild-type and substituted viruses; transcripts labeled both in vivo and in vitro showed a two- to threefold-higher level of self-annealing in the case of the variant than in the case of wild type SV40.
Herpesvirus ateles
DNA and Its Homology with
Herpesvirus saimiri
Nucleic Acid
Authors: Fleckenstein Bernhard , Bornkamm Georg W. , Mulder Carel , Werner Fred-Jochen , Daniel Muthiah D. , Falk Lawrence A. , Delius Hajo
Published: January 2020

Analysis of the structural organization of
Herpesvirus ateles
DNA shows that two types of viral DNA molecules are encapsidated in virions: (i) M-genomes, which contain 74% light sequences (L-DNA, 38% guanine plus cytosine) and 26% highly repetitive heavy sequences (H-DNA, 75% guanine plus cytosine), and (ii) defective H-genomes, which consist exclusively of repetitive H-DNA. The structure of M-genomes from
H. ateles
consists of an L-DNA region of about 70 × 10
6
daltons inserted between H-DNA termini of variable length. M-genomes with a shorter H-DNA region at one end of the molecule have a long stretch of H-DNA at the other end, resulting in a total molecular weight of 89.8 ± 8.5 × 10
6
. Thus it resembles the structure of M-genomes of
H. saimiri
. H-DNA of the two independent
H. ateles
isolates, strains 810 and 73, reveals different patterns after cleavage with restriction endonuclease
Sma
I. H-DNA of
H. ateles
810 appears to consist of identical tandem repeat units with a molecular weight of 1,035,000; the H-DNA repeat unit of strain 73 is shorter (930,000 molecular weight). Corresponding DNA sequences of the two
H. ateles
strains (810 and 73) are completely homologous in cross-hybridizations. However, a discrete nucleotide sequence divergence between these virus strains is detected by measuring melting temperatures (

T
m

) of DNA hybrid molecules. Some homology exists between
H. ateles
and
H. saimiri
DNA. Hybridization of L-DNA from
H. ateles
with L-DNA from
H. saimiri
shows about a 35% homology between the respective L-DNA sequences; the resulting heteroduplex molecules show a decrease of

T
m

by 13.5°C, corresponding to about a 9% mismatching in cross-hybridizing parts of L-regions. Very little homology is found between H-DNA of
H. ateles
and
H. saimiri
.
Encephalomyocarditis virus RNA. III. Presence of a genome-associated protein
Authors: Hruby D E , Roberts W K
Published: January 2020

A low-molecular-weight protein was found to be associated with intact 35S RNA isolated from purified encephalomyocarditis virus. This protein was positively charged at pH 3.5, sensitive to proteinase K treatment, and labeled with either 3H-amino acids or 32Pi.
5' Terminus of defective and nondefective Sendai viral genomes is ppp Ap
Authors: Leppert M , Kolakofsky D
Published: January 2020

Using RNase T2 digestion of 32P-labeled Sendai virus nondefective and defective-interfering RNAs, the 5' termini were isolated and demonstrated to be pppAp for both the plus and minus strands.
Procedure for purification of intact DNA from vaccinia virus
Authors: Cabrera C V , Esteban M
Published: January 2020

A procedure for the isolation of intact vaccinia DNA molecules by chromatography on hydroxyapatite in the presence of 6 M urea is described. When lysates of virions containing 0.5 to 10 microgram of DNA were employed, over 95% of the viral DNA could be recovered free of poteins. Vaccinia DNA molecules isolated in this manner sedimented at 68S in neutral sucrose gradients and had an average contour length of 62.3 micrometer when examined in an electron microscope, and the DNA could be cleaved with the restriction endonuclease EcoRI and BamHI. The results of these analyses showed that intact vaccinia DNA molecules of 120 X 10(6) to 130 X 10(6) molecular weight could be obtained by the procedures described.
In Vitro Translation of the Two RNAs of Nodamura Virus, a Novel Mammalian Virus with a Divided Genome
Authors: Newman J. F. E. , Matthews T. , Omilianowski D. R. , Salerno T. , Kaesberg P. , Rueckert R.
Published: January 2020

Nodamura virus is a small ribovirus containing two RNA molecules. Both RNAs were found to be active messengers for protein synthesis in cell-free extracts prepared from wheat embryo or HeLa cells. RNA 2 directed synthesis of a 43,000-dalton product, p43, whose tryptic fingerprint was similar to that of the major viral coat protein, vp40 (molecular weight, 40,000). Though p43 appears to be a precursor of vp40, processing did not occur in the cell-free extracts. RNA 1 directed synthesis of a 105,000-dalton protein, p105. Its tryptic fingerprint revealed no evidence of coat protein sequences. Hence, the two RNAs represent genes with few, if any, redundant coding sequences.
Deoxypyrimidine nucleoside metabolism in varicella-zoster virus-infected cells
Authors: Hackstadt T , Mallavia L P
Published: January 2020

Noninfected and varicella-zoster virus (VZV)-infected human foreskin fibroblasts were examined for thymidine kinase activity. The specific activity of VZV-infected cell extracts was approximately 7.5-fold greater than that of mock-infected cells and 3-fold greater than that of actively growing cells. The pH optimum of VZV-infected cell thymidine kinase activity was found to be 8.0, whereas thymidine kinase activity in noninfected cells exhibited a sharp pH optimum at 7.4. Electrophoretic analysis of cellular enzymes involved in pyrimidine nucleoside phosphorylation revealed at least three enzymes distinguishable by electrophoretic mobility and substrates used. These enzymes were presumed to be thymidine kinase, deoxycytidine kinase, and uridine kinase. The relative mobilities of these enzymes on 5% polyacrylamide gels were 0.18, 0.91, and 0.54, respectively. In VZV-infected cells, a single band of activity catalyzing the phosphorylation of thymidine, deoxyuridine, deoxycytidine, and cytidine was observed with a relative mobility of 0.48. Cellular pyrimidine-phosphorylating enzymes were not detected in VZV-infected cells. The molecular weight of the VZV-induced enzyme was determined to be 72,000 +/- 7%.
Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2
Authors: Mangel W F , Hewick R M , Bayley S T , Wheeler T , Harvey R , Waterfield M D , Smith A E
Published: January 2020

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.
Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNase H activity
Authors: Lai M H , Verma I M
Published: January 2020

Purified avian myeloblastosis virus reverse transcriptase contains two subunits that are structurally related. The large subunit, beta (molecular weight, 95,000), was converted in vitro by chymotrypsin into a polypeptide of molecular weight 63,000. This polypeptide was indistinguishable from the small subunit, alpha (molecular weight, 65,000), in its chromatographic behavior on the phosphocellulose column and its tryptic peptide composition. During this proteolytic conversion, a polypeptide of molecular weight 32,000 (fragment B) was obtained. It was composed of tryptic peptides unique to beta and appeared to be derived from the portion of the beta subunit that was cleaved off during the conversion of beta into alpha. Upon continued proteolysis, a smaller polypeptide of molecular weight 24,000 (fragment A) was generated. This polypeptide manifested only RNase H activity and shared common amino acid sequences with beta and alpha subunits. Fragment A did not share any amino acid sequence homology with fragment B.
Leukemogenic activity of thymotropic, ecotropic, and xenotropic radiation leukemia virus isolates
Authors: Haas M
Published: January 2020

Thymotropic, ecotropic, and xenotropic oncoviruses were isolated from the C57BL/6 mouse radiation leukemia system and were propagated in culture. The purified viruses were inoculated singly and in various combinations into groups of mice, and leukemia incidence was determined. Only the thymotropic virus was leukemogenic in vivo.
Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase
Authors: Cooper G M
Published: January 2020

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.
Primate retroviruses: immunological cross-reactivity between major structural proteins of new and old world primate virus isolates
Authors: Devare S G , Arthur L O , Fine D L , Stephenson J R
Published: January 2020

The major 35,000-molecular-weight internal antigen (p35) of the squirrel monkey retrovirus (SMRV) was isolated and partially characterized. Immunological analysis of SMRV p35 led to the demonstration of antigenic determinants common to SMRV and the Mason-Pfizer monkey virus (MPMV). A broadly reactive competition immunoassay was developed utilizing antiserum to MPMV to precipitate 125I-labeled SMRV p35. Although the major structural proteins of MPMV and SMRV competed with equal efficiency in this assay, type B and type C oncornavirus proteins lacked detectable reactivity. Antibodies reactive with the major structural proteins of both MPMV and SMRV were observed in sera of several normal rhesus monkeys with known prior exposure to MPMV-infected animals. These findings demonstrate the ability of sera from naturally immunized primates to recognize broadly reactive interspecies antigenic determinants shared by the major structural proteins of type D oncornaviruses, and they suggest possible horizontal transmission of MPMV among rhesus monkeys. Although sera from a number of squirrel monkeys contained antibody to SMRV p35, the possibility that this latter reactivity was due to endogenous virus activation rather than horizontal transmission cannot be ruled out.
Characterization of the 5'-terminal capped structures of late simian virus 40-specific mRNA
Authors: Haegeman G , Fiers W
Published: January 2020

32P-labeled, late simian virus 40-specific RNA was isoalted from infected CV1 cells and completely degraded with RNase T2 and bacterial alkaline phosphatase. The RNase-resistant material was fractionated two dimensionally and further characterized with Penicillium nuclease and nucleotide pyrophosphatase. Two major 5' termini were identified in late simian virus 40 RNA, namely, 7-methyl Gppp 2',6-dimethyl ApUp and 7-methyl Gppp 2',6-dimethyl Ap 2'-methyl, UpUp. Both 5' termini are present in unfractionated viral RNA as well as in the separated 16S and 19S species. As both caps differ only in secondary modification, it is possible that they are derived from the same site on the DNA. The relatively higher cap II content of the 16S mRNA may be related to its slower rate of turnover.
Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus
Authors: Brockman W W
Published: January 2020

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.
High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures
Authors: Bender W , Chien Y H , Chattopadhyay S , Vogt P K , Gardner M B , Davidson N
Published: January 2020

Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.
RNA tumor virus phosphoproteins: primary structural analysis and identification of phosphopeptides
Authors: Pal B K , Bryant M L , Roy-Burman P
Published: January 2020

Two-dimensional tryptic peptide mapping was used to compare the peptide sequences of the phosphoprotein (pp12) of cloned ecotropic and amphotropic wild mouse leukemia viruses, strains 1504 and 292. The maps of two ecotropic isolates were very similar to one another, as were the maps of two amphotropic isolates. There was also extensive similarity between the maps of this protein from ecotropic and amphotropic viruses, although characteristic peptide differences were readily recognized. These differences were consistent with the general type specificity of oncovirus phosphoproteins. The pp12 of the field isoalte of 292 virus contained five phosphopeptides, and the non-phosphorylated and variously phosphorylated species of this pp12 showed identical peptide maps, indicating differential phosphorylation of a single polypeptide.
Membrane-free scrapie activity
Authors: Malone T G , Marsh R F , Hanson R P , Semancik J S
Published: January 2020

Determinations of scrapie activity in subcellular fractions from infected hamster brains through the asymptomatic and symptomatic course of infection revealed the presence of substantial amounts of scrapie infectivity in the 100,000 X g supernatant fractions, indicating that association with physically discernible membrane structures is not necessary for the transmission of the scrapie agent. An increase of scrapie infectivity in the 100,000 X g supernatant fractions after vigorous homogenization of infected membrane-rich fractions suggests that the agent is identical in membrane-rich and 100,000 X g supernatant fractions.
Antiviral Action of a Rifamycin Derivative: Formation of Rous Sarcoma Virus Particles Deficient in 60 to 70S RNA
Authors: Szabo Charles , Bissell Mina J.
Published: January 2020

Growth of Rous sarcoma virus-transformed cells in the presence of rifazone-8
2
, a rifamycin derivative, results in the formation of noninfectious virus particles lacking 60 to 70S RNA.
Altered E2 glycoprotein of Sindbis virus and its use in complementation studies
Authors: Bracha M , Schlesinger M J
Published: January 2020

We have detected a Sindbis virus variant that contains a smaller-molecular-weight form of the viral glycoprotein E2. The molecular weight of the PE2 precursor and the glycosylation pattern of the smaller E2 are normal, thus indicating that this E2 is formed by an aberrant proteolytic cleavage. The altered E2 was detected in an RNA+ temperature-sensitive mutant that was defective in proteolytic cleavage, but the abnormal PE2-to-E2 reaction could be separated from the ts mutation and is not itself a temperature-sensitive defect. We used the variant E2 as a marker to monitor the complementation reaction between an RNA+ and an RNA- mutant and discovered that complementation was not reciprocal; the RNA defect was corrected by the RNA+ mutant gene products but the RNA+ defect was not complemented by any RNA- gene products. Other studies have shown that the smaller E2 is not preferentially selected during viral maturation and budding. No significant changes have been detected in the biological activity of virions with this altered E2 protein. Comparison of the electrophoretic migration of the E1 and E2 Sindbis viral glycoproteins in a two-dimensional polyacrylamide slab gel system that was first run in the absence of sulfhydryl-reducing reagent and then with beta-mercaptoethanol indicated that the mobility of E1, but not that of E2, was significantly altered by reduction.
Mapping host range-specific oligonucleotides within genomes of the ecotropic and mink cell focus-inducing strains of Moloney murine leukemia virus
Authors: Shih T Y , Weeks M O , Troxler D H , Coffin J M , Scolnick E M
Published: January 2020

The site of recombination of a mink cell focus-inducing strain (Mo-MuLV83) derived from an ecotropic Moloney murine leukemia virus (Mo-MuLV) was mapped by fingerprint analysis of the large RNase T1-resistant oligonucleotides, employing a two-dimensional gel electrophoresis method. Mo-MuLV83, in contrast to the ecotropic Mo-MuLV, demonstrated a broadened host range, i.e., growth not only on mouse cells but also on mink cells, and recombination involved the env gene function. The genomic RNA of these two viruses shared 42 out of a total of 51 to 53 large T1 oligonucleotides (81%) and possessed a similar subunit size of 36S. Most of these T1 oligonucleotides were mapped in their relative order to the 3' polyadenylic acid end of the viral RNA molecules. There were 10 common oligonucleotides immediately next to the 3' termini. A cluster of 7 (in Mo-MuLV83) or 10 (in Mo-MuLV) unique T1 oligonucleotides were mapped next to the common sequences at the 3' end, and they all appeared concomitantly in a polyadenylic acid-containing RNA fraction with a sedimentation coefficient slightly larger than 18S. Therefore, the env gene of Mo-MuLV was situated at a location approximately 2,000 to 4,000 nucleotides from the 3' end of the genomic RNA, and the gene order of Mo-MuLV appeared to be similar to that of the more rigorously determined avian oncornaviruses. cDNA(SFFV) specific for the xenotropic sequences in the spleen focus-forming virus RNA hybridized to the cluster of unique oligonucleotides of Mo-MuLV83 RNA. This suggests that the loci of recombination involve the homologous env gene region of a xenotropic virus.