bluuebunny/crossref_metadata_2025 huggingface dataset:
Proposed replicative role of the NS polypeptide of vesicular stomatitis virus: structural analysis of an electrophoretic variant
Authors: Lesnaw J A , Dickson L R , Curry R H
Published: January 2020

The structural lesion in the temperature-sensitive mutant E1 of the New Jersey serotype of vesicular stomatitis virus has been assigned to the NS protein. Although the packaged wild-type and mutant NS proteins were similarly phosphorylated, the mutant NS protein migrated faster than the wild-type NS protein in polyacrylamide slab gels electrophoresed in the presence of sodium dodecyl sulfate. The resolution appears to be the result of conformational rather than size differences since the two proteins comigrated in polyacrylamide gels which contained 4 M urea in addition to sodium dodecyl sulfate. Peptide maps, obtained by limited proteolysis of 32P-labeled wild-type and mutant NS proteins with Staphylococcus aureus V8 protease and papain, revealed striking differences which suggested that the mutant alteration could involve an aspartic or glutamic acid residue. Since NS proteins obtained from naturally occurring revertants of E1 were indistinguishable from the wild-type protein in all of these analyses, the structural alteration in the mutant NS protein correlates with the functional lesion. Because E1 is defective in the RNA replication pathway at the restrictive temperature, a replicative role is proposed for the NS protein.
New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells
Authors: Bromley P A , Spahr P F , Darlix J L
Published: January 2020

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.
Heterogeneity of the 5' terminus of late mRNA induced by a viable simian virus 40 deletion mutant
Authors: Haegeman G , van Heuverswyn H , Gheysen D , Fiers W
Published: January 2020

d1-1811 is a viable simian virus 40 deletion mutant which lacks the DNA region corresponding to the major capping site of the late viral RNA. The exact size of the deletion (40 base pairs) was determined by comparison of the mutant DNA sequence with the wild-type simian virus 40 (strain 776) DNA sequence. Although d1-1811 forms somewhat smaller plaques, the amount of viral RNA late after infection was not significantly reduced compared with that of the wild type. Virus-specific, polyadenylate-containing, 32P-labeled late RNA was purified from the cytoplasm and enzymatically degraded to characterize the 5' terminus. The cap-containing oligonucleotides were isolated, and their structures were analyzed by further digestion. Instead of a single cap structure, we found a variety of capped 5' termini, with adenosine caps occurring much more frequently than guanosine caps. Nevertheless, there was a remarkable homology between both types of terminal sequences. Conceivably, the minor cap population present in wild-type simian virus 40 late mRNA may correspond to the collection of capped termini identified in the d1-1811 late mRNA . Cellular cytoplasmic RNA shows a similar pattern of cap structures, but the relative abundance is quite different.
Uptake of minute virus of mice into cultured rodent cells
Authors: Linser P , Bruning H , Armentrout R W
Published: January 2020

The uptake of minute virus of mice into cells in tissue culture was examined biochemically and by electron microscopy. Cell-virus complexes were formed at 4 degrees C, and uptake of virus was followed after the cells were shifted to 37 degrees C. The infectious particles appeared to enter cells at 37 degrees C by a two-step process. The first and rapid phase was measured by the resistance of cell-bound virus to elution by EDTA. The bulk of the bound virus particles became refractory to elution with EDTA within 30 min of incubation at 37 degrees C. The infectious particles became resistant to EDTA elution at the same rate. The second, slower phase of the uptake process was measured by the resistance of infectious particles to neutralization by antiserum. This process was complete within 2 h of incubation at 37 degrees C. During this 2-h period, labeled viral DNA became progressively associated with the nuclear fraction of disrupted cells. The uptake of infectious virus could occur during the G1 phase of the cell cycle and was not an S phase-specific event. The uptake process was not the cause of the S phase dependence of minute virus of mice replication. In electron micrographs, virus absorbed to any area of the cell surface appeared to be taken into the cell by pinocytosis.
Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: studies with simian virus 40 double mutants
Authors: Martin R G , Setlow V P , Edwards C A
Published: January 2020

Simian virus 40 mutants containing both a tsA mutation (rendering the 90,000 molecular weight [90K] T-antigen thermolabile) and a deletion between 0.54 and 0.59 map units (reducing the size and the amount of the 20K t-antigen) were used to transform Chinese hamster lung cells. The frequencies of transformation by the double mutants were comparable to that of the tsA mutant alone by both the focus and agar assays except when the cells were serum depleted before infection. Growth-arrested cells were transformed (using the agar assay) by the deletion mutants at less than 2% the frequency found when the 20K t-antigen was normal. Growth arrest had very little effect on the temperature sensitivity of the resultant transformed cell lines whether or not the deletion was present.
Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus
Authors: Shurtz R , Dolev S , Aboud M , Salzberg S
Published: January 2020

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.
Structural changes in simian virus 40 chromatin as probed by restriction endonucleases
Authors: Liggins G L , English M , Goldstein D A
Published: January 2020

The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes.
Incomplete genomes of the parvovirus minute virus of mice: selective conservation of genome termini, including the origin for DNA replication
Authors: Faust E A , Ward D C
Published: January 2020

Deletion mutants of minute virus of mice arising during a single high-multiplicity passage and after serial undiluted passage have been isolated, and the incomplete viral genomes contained therein have been analyzed. The DNA isolated from incomplete virions derived from a single high-multiplicity passage was heterogeneous, ranging in size from 15 to 70% of the intact viral genome, with an average molecular length of approximately, 2,000 nucleotides. Two distinct types of molecules, designated as type I D-DNA and type II D-DNA, could be distinguished on the basis of their degree of secondary structure, and these were present in roughly equal amounts. Type I D-DNAs were predominantly single-stranded, recombinant molecules in which the self-complementary sequences derived from both genomic termini were conserved. The 5' terminus was modified relative to the analogous wild-type structure. Although virtually all of the wild-type genome sequence was seen in the total type I D-DNA population, sequences which map between coordinates 47.3 and 87.1 were clearly underrepresented. However, the extent and position of the deletions in individual molecules varied significantly. The shortest molecules in the population lacked between 90 and 95% of the internal wild-type genome sequence and consisted of sequences derived almost exclusively from within 5.0 map units (250 nucleotides) at both ends of the viral genome. Moreover, these miniature recombinant molecules were selectively amplified during serial undiluted passage and were therefore believed to contain all of the critical recognition sites necessary for the replication of minute virus of mice viral DNA. Type II D-DNAs were virus-specific, double-stranded hairpin molecules whose complementary strands were covalently continuous at variable sites distal to the 5' end of the viral minus strand. In sharp contrast to the type I genomes, these hairpin molecules consisted of sequences which mapped entirely at the 5' end of the viral genome between positions 85.0 and 100. Furthermore, type II molecules were gradually lost from the total D-DNA population during serial undiluted passage, suggesting that these molecules are not competent for DNA Replication but arise as the result of fatal replication errors. Deletion mutants of the type described here for minute virus of mice should be valuable generally as aids to future studies on parvovirus DNA replication, transcription, and cell-virus interactions.
Secondary structures in polyoma DNA
Authors: Wu M , Manor H , Davidson N
Published: January 2020

Three reproducible secondary-structure features were observed on single strands of polyoma virus DNA mounted for electron microscopy by the T4 gene 32 protein technique: (i) a hairpin fold-back extending from 92.9 +/- 0.8 to 95.0 +/- 0.7 map units; (ii) a small loop extending from 63.2 +/- 3.1 to 68.5 +/- 2.8 map units; and (iii) a big loop extending from 51.9 +/- 2.3 to 68.9 +/- 2.1 map units. Both loops are bounded by inverted repeat stems of length 40 +/- 20 base pairs. The stem sequences around 68.5 and 68.9 of the large and small loops overlap, either partially or completely. Several lines of evidence indicate that the inverted repeat stems of the two secondary-structure loops lie in the regions of polyoma virus DNA flanking and probably very close to the sequences that are spliced out in the formation of the late 16S and 18S messages, whereas the hairpin fold-back appears to map at a splicing point of an early message. These structures may therefore be important for the processing of the primary transcripts to form the early and late messages.
Nucleotide sequences from the 3'-ends of vesicular stomatitis virus mRNA's as determined from cloned DNA
Authors: Rose J K , Iverson L
Published: January 2020

Molecular clones of vesicular stomatitis virus mRNA's were used to determine the 3'-terminal sequences of mRNA's encoding the N and NS proteins. This new approach to VSV mRNA sequencing allowed the first comparison of 3'-terminal sequences. The sequences showed a tetranucleotide homology, UAUG, immediately preceding the polyadenylic acid. In addition, both mRNA's had an AU-rich region including the tetranucleotide AUAU at positions 16 to 19 nucleotides from the polyadenylic acid. A possible secondary structure between the 3' end of N mRNA and the 5' end of the adjacent NS mRNA is noted. These structural features may serve as signals for termination (or cleavage) and polyadenylation of vesicular stomatitis virus mRNA's. Neither mRNA had the polyadenylic acidproximal hexanucleotide, AAUAAA, found in eucaryotic cellular and viral mRNA's transcribed from nuclear DNA. The probable location of the translation termination codon for the NS protein is only six nucleotides from polyadenylic acid in NS mRNA.
Isolation and characterization of polyoma virus genomes with deletions between the origin of viral DNA replication and the site of initiation of translation in the early region
Authors: Wells R D , Hutchinson M A , Eckhart W
Published: January 2020

We introduced deletions in the early region of the polyoma virus genome near the HaeII restriction enzyme cleavage site, between the origin of viral DNA replication and the site of initiation of translation of the polyoma T antigens. We analyzed the DNA of the deletion mutants by restriction enzyme digestion. Four of the mutants had deletions beginning very close to the HaeII site and extending clockwise toward the site of initiation of translation. The deletions near the HaeII site varied in size from about 10 base pairs to about 55 base pairs. The mutants containing deletions near the HaeII site were capable of lytic growth in mouse 3T6 cells and were capable of transforming rat F2408 cells, as judged by focus formation.
Differential adsorption of polyoma virions and capsids to mouse kidney cells and guinea pig erythrocytes
Authors: Bolen J B , Consigli R A
Published: January 2020

Adsorption of 125I-labeled polyoma virions and capsids to the surface of mouse kidney cells (MKC) and guinea pig erythrocytes was examined. Purified polyoma capsids lack the ability to compete with polyoma virions for specific binding sites on the surface of MKC. These same capsids were, however, able to block virion adsorption to guinea pig erythrocytes. UV-inactivated virions blocked cellular receptors on MKC and thus inhibited infectious virions from infecting the cells. Capsids were unable to inhibit virion infection of MKC. Adsorption of polyoma virions to MKC and infection of these cells were found to be independent of the ability of the virions to agglutinate guinea pig erythrocytes.
gag-Related polypeptides encoded by replication-defective avian oncoviruses
Authors: Rettenmier C W , Anderson S M , Riemen M W , Hanafusa H
Published: January 2020

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.
In Vivo Methylation of Bacteriophage φX174 DNA
Authors: Hattman Stanley , Gribbin Chris , Hutchison Clyde A.
Published: January 2020

A mutant (designated
mec
−
) has been isolated from
Escherichia coli
C which has lost DNA-cytosine methylase activity and the ability to protect phage λ against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an
E. coli
K-12
mec
−
mutant; thus, the
E. coli
C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C
mec
+
and C
mec
−
cells infected with bacteriophage φX174. φX·
mec
−
RFI is sensitive to in vitro cleavage by R·
Eco
RII and is cut twice to produce two fragments of almost equal size. In contrast, φX·
mec
+
RFI is relatively resistant to in vitro cleavage by R·
Eco
RII. R·
Bst
I, which cleaves
mec
+
/RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R·
Eco
RII. These results demonstrate that φX·
mec
+
RFI is methylated in vivo by the host
mec
+
enzyme and that this methylation protects the DNA against cleavage by R·
Eco
RII. This is consistent with the known location of two
mec
+
/ RII sequences (viz., [Formula: see text]) on the φX174 map. Mature singlestranded virion DNA was isolated from φX174 propagated in C
mec
+
or C
mec
−
in the presence of
l
-[
methyl
-
3
H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that φX·
mec
+
DNA had a 10-fold-higher ratio of [
3
H]5-methylcytosine to [
3
H]cytosine compared to φX·
mec
−
. Since φX·
mec
+
contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of φX174 is mediated by the host
mec
+
enzyme only. These results are not consistent with the conclusions of previous reports that φX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.
Restriction endonuclease mapping of unintegrated proviral DNA of Snyder-Theilen feline sarcoma virus: localization of sarcoma-specific sequences
Authors: Sherr C J , Fedele L A , Donner L , Turek L P
Published: January 2020

Extrachromosomal DNA purified from mink cells acutely infected with the Snyder-Theilen strain of feline sarcoma virus (FeSV) was digested with restriction endonucleases, and the DNA fragments were electrophoretically separated, transferred to a solid substrate, and hybridized with radiolabeled DNA transcripts complementary to different portions of the FeSV RNA genome. Major DNA species 8.4 and 5.0 kilobase pairs (kbp) long represent the linear, unintegrated proviruses of Snyder-Theilen feline leukemia virus and FeSV, respectively. Transfection experiments performed with electroeluted DNAs showed that the 8.4-kbp form led to the production of replicating nontransforming virus in mink and cat cells; in contrast, the 5.0-kbp DNA produced helper virus-independent foci of transformation in mouse NIH/3T3 cells and helper virus-dependent foci in mink cells at an efficiency comparable to that obtained with unfractionated extrachromosomal DNA. Sites of restriction endonuclease cleavage for six enzymes were oriented with respect to one another within the FeSV provirus. EcoRI recognized cleavage sites at 0.3 to 0.4 kbp from each terminus of FeSV DNA, reducing the 5.0-kbp DNA to molecules 4.3 kbp long; this enzyme excised a large internal proviral DNA fragment of corresponding size from the DNA of FeSV-transformed mink nonproducer cells. By using DNA transcripts complementary to different portions of the FeSV genome, sarcoma-specific sequences (the FeSV src gene) were positioned within 2.1 and 3.4 kbp from the 5' end of the proviral DNA with respect to the viral RNA genome. The src gene is flanked at both ends by sequences shared in common with feline leukemia virus. The localization of src sequences to this region suggests that a portion of an FeSV polyprotein which contains feline oncornavirus-associated cell membrane antigen (FOCMA-S) is the major product of this gene.
Characterization of a bacteriophage related to R-type pyocins
Authors: Kageyama M , Shinomiya T , Aihara Y , Kobayashi M
Published: January 2020

A bacteriophage with a contractile tail which shows very similar features to R-type pyocins was isolated and characterized. This phage, named PS17,was purified by DEAE-cellulose chromatography and CsCl density gradient centrifugation. It was a DNA-containing phage, and the density of the purified particles in CsCl was found to be 1.468. DNA from this phage had a density of 1.720 in CsCl, indicating its guanine plus cytosine content to be 61.2%. The head was polyhedral, 69 nm in diameter, and the tail was 150 nm in length. This phage was neutralized by antiserum preparations against five R-type pyocins, and the antiserum against this phage was active in neutralizing R-type pyocins. The properties of this phage, PS17, were compared with another similar phage, PS3, which was previously reported.
Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells
Authors: Seif R , Martin R G
Published: January 2020

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.
Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60
Authors: Siddell Stuart G. , Wege Helmut , Barthel Andrea , ter Meulen Volker
Published: January 2020

Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000- and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecular-weight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000- and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA.
Herpesvirus-Lymphoid Cell Interactions: Comparative Studies on the Biology of Herpes Simplex Virus-Induced Fc Receptors in B, T, and “Null” Lymphoid Cell Lines
Authors: Menezes José , Bourkas Angelo E.
Published: January 2020

We have investigated the induction of Fc receptor (FcR) in different types of lymphoid cell lines (LCL) infected with herpes simplex virus (HSV). Subpopulations of certain of these LCL normally express FcR unrelated to herpetic infection. Differentiation of virus-induced FcR from that related to normal cell function was therefore possible. FcR detection was carried out by means of a rosette assay using ox erythrocytes coated with 7S immunoglobulin G (EA rosettes). Both HSV types 1 and 2 were found to induce FcR in B, T, and “null” (i.e., non-B, non-T) type LCL; however, in all the LCL tested, this HSV-induced FcR expression appeared to be more restricted in the responding T LCL than in responding B and null type LCL. In addition, kinetic experiments revealed that the time course of HSV-induced FcR expression differed among these LCL types tested. Interestingly, a number of LCL were resistant to HSV infection or restricted HSV gene expression, including expression of the viral products responsible for FcR induction. In all the responding HSV-infected LCL, induction of FcR always paralleled the expression of HSV antigens. Synthesis of HSV-induced FcR was shown to be inhibited by phosphonoacetic acid, an inhibitor of herpesvirus DNA polymerase activity, whereas FcR of non-HSV origin was found to be resistant to inhibitor. This would infer that HSV codes for an FcR which can be differentiated from that of cellular origin by using phosphonoacetic acid. Therefore, two different mechanisms of FcR synthesis may be suggested, one virus mediated and the second probably under cellular control. In addition, the data obtained using Epstein-Barr virus producer as well as isogeneic monoclonal cell lines, with and without the Epstein-Barr virus genome, indicated that the resident Epstein-Barr virus genome in the target cell did not have a detectable effect in the induction of FcR by HSV.
Structure of murine sarcoma virus DNA replicative intermediates synthesized in vitro
Authors: Dina D , Benz E W
Published: January 2020

Moloney murine sarcoma virions synthesize discrete DNA products in vitro which closely resemble those found in vivo shortly after infection. These in vitro products have been isolated by electrophoresis and mapped with restriction endonucleases. In addition to the full-genome-length 6-kilobase pair linear DNA, a 5.4-kilobase pair circular DNA molecule, an incomplete linear DNA molecule, and a 600-base pair molecule were detected. The 6-kilobase pair DNA contained a 600-base pair direct terminal repeat which was missing from the circular form and was partially represented on the incomplete linear DNA molecule. The 600-base pair DNA contained sequences which were present in the 600-base pair direct repeat on the 6-kilobase pair DNA. The order of synthesis and the structure of these molecules detected in the in vitro reaction suggest that they are crucial intermediates in the formation of the final product of in vitro reverse transcription. A model which accounts for the synthesis of all of these molecules during the initial stages of viral replication is suggested.
Adenovirus type 12-induced rat tumor cells of neuroepithelial origin: persistence and expression of the viral genome
Authors: Ibelgaufts H , Doerfler W , Scheidtmann K H , Wechsler W
Published: January 2020

Four cell lines derived from adenovirus type 12-induced rat brain tumors were studied. The polyploid cells displayed neuroepithelial characteristics and were transplantable into syngeneic rats and nude mice. In tissue culture the cells grew in monolayers and multilayers. A very high saturation density was reached, and the cells plated in agar and were easily agglutinated with low concentrations of concanavalin A. Between 2 and 11 copies of the viral genome per diploid cellular genome were detected by reassociation kinetics analysis in the different lines. The patterns of distribution of viral DNA sequences in these lines, as revealed by blot analysis, suggest colinear integration of the intact viral genome into the cellular DNA. The patterns of integration were stable after more than 15 months of prolonged tissue culture and after animal reimplantation. Integration patterns were identical in three of the tumor lines and different in another line. Viral sequences were transcribed. The extent of homology found toward adenovirus type 12 DNA in polyadenylated polysome-associated mRNA isolated from the tumor lines suggests that the early and some of the late genes of adenovirus type 12 DNA are transcribed in these tumor cells. Infectious virus was not rescuable from these lines.
Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid
Authors: Sturman L S , Holmes K V , Behnke J
Published: January 2020

The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus at 37 degrees C resulted in formation of a complex between one of the viral glycoproteins, E1, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in E1, resulting in aggregation of E1 and interaction with the viral RNA in the nucleocapsid. E1 also bound rRNA. The E1-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein E1 from the peplomeric glycoprotein E2 permitted preparation of antisera against these isolated proteins. A model is proposed for the arrangement of the three major structural proteins in the coronavirus A59 virion in relation to the viral envelope and RNA.
Immunological relationships of OMC-1, an endogenous virus of owl monkeys, with mammalian and avian type C viruses
Authors: Barbacid M , Daniel M D , Aaronson S A
Published: January 2020

The relationships between OMC-1, an endogenous oncovirus of owl monkey, and representatives of the three oncoviral genera have been investigated by radioimmunological techniques. The major structural protein of OMC-1 was shown to share antigenic determinants with the corresponding proteins of certain type C viruses of rodent, feline, and cervine origin. It was not possible to demonstrate antigenic cross-reactivity between OMC-1 and endogenous type C viruses of baboons. These findings argue that OMC-1 and baboon endogenous viruses do not represent direct descendants of an ancestor virus that became integrated within primates prior to the divergence of New and Old World species. A close antigenic relationship was established between the major structural proteins of OMC-1, an endogenous virus of deer (deer kidney virus), and avian reticuloendotheliosis viruses. These findings establish OMC-1 and deer kidney virus in the evolutionary lineage that may have led to the generation of avian reticuloendotheliosis virus, a group of oncogenic viruses capable of crossing the interclass barrier between mammals and birds.
Characterization of RNA synthesized in isolated nuclei of herpes simplex virus type 1-infected KB cells
Authors: Miovic M L , Pizer L I
Published: January 2020

Nuclei isolated from herpes simplex virus (HSV)-infected KB cells actively synthesize HSV RNA in vitro; the RNA can be hybridized with HSV DNA or nuclear RNA from HSV-infected cells. Nascent RNA molecules labeled in vivo with 32PO4 were elongated, utilizing the nuclear system to incorporate Hg-CTP at their 3' ends, and then isolated on an affinity column. Hybridization of isolated nascent RNA molecules showed that greater than 50% of them were HSV specific and that more than 25% were self-complementary.
Influence of membrane (M) protein on influenza A virus virion transcriptase activity in vitro and its susceptibility to rimantadine
Authors: Zvonarjev A Y , Ghendon Y Z
Published: January 2020

The transcriptase activity of influenza A virus ribonucleoproteins was inhibited by 42 to 49% in vitro in the presence of membrane (M) protein. The addition of M protein to the system of ribonucleoprotein preparations isolated from rimantadine-sensitive or rimantadine-resistant influenza virus strains, as well as the addition of M protein isolated from a sensitive strain, in the presence of rimantadine further inhibited the transcriptase activity of such complexes by approximately 40%. In the system containing the same ribonucleoprotein preparations, but with M protein isolated from a rimantadine-resistant influenza virus strain, the transcriptase activity was not sensitive to rimantadine. The data show that M protein can influence the activity of influenza A virus virion transcriptase and that the susceptibility of influenza virus to rimantadine may be due to the peculiarities of M protein.
Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro
Authors: Hewick R M , Mellor A , Smith A E , Waterfield M D
Published: January 2020

The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5.
Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells
Authors: Collins Carolyn J. , Boettiger David , Green Todd L. , Burgess Mary B. , Devlin Blythe H. , Parsons J. Thomas
Published: January 2020

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.
Effect of a tsA mutation of simian virus 40 late gene expression: variations between host cell lines
Authors: Alwine J C , Khoury G
Published: January 2020

Infection of AGMK or CV-1 cells by the early simian virus 40 mutant tsA58 at the permissive temperature (32 degrees C) followed by a shift to the nonpermissive temperature (41 degrees C) caused a substantial decrease in the levels of late viral RNA in the cytoplasm of AGMK cells but not CV-1 cells. At the translational level, this depression of late viral RNA levels was reflected by a decrease in late viral protein synthesis. Thus, in AGMK cells, an early region gene product (presumably large T-antigen) appeared to be continuously required for efficient expression of the late viral genes. In contrast, late simian virus 40 gene expression, once it is initiated in CV-1 cells, continued efficiently regardless of the tsA mutation. The difference in expression of the late simian virus 40 genes in these tsA mutant-infected monkey kidney cell lines may reflect a difference in host cell proteins which regulate viral gene expression in conjunction with early viral proteins.
Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells
Authors: Graessmann A , Graessmann M , Tjian R , Topp W C
Published: January 2020

The ability of the two early simian virus 40 (SV40) coded proteins, the large and small T-antigens, to abortively induce the disappearance of cytoplasmic actin-containing networks in cultured cells has been studied in rat embryo fibroblasts after microinjection of intact SV40 DNA, DNA fragments from the early region of SV40, and a purified SV40 large T-antigen related protein (the D2 hybrid protein) isolated from cells infected with the adenovirus-SV40 hybrid virus Ad2+D2. Injection of either the 107,000-dalton D2 hybrid protein or SV40 DNA from the deletion mutant dl 884 SV40, which lacks part of the region (0.54 to 0.59) encoding small t-antigen, failed to cause any detectable change in the structure of actin cables in recipient cells over a period of 72 h. By contrast, injection of wild-type SV40 DNA or a DNA fragment containing the entire region coding for a small-t antigen leads to the disruption of actin cable networks within 24 h of injection. It appears likely that the SV40 small-t protein is necessary for the abortive loss of actin cables in injected cells. Epidermal growth factor also causes loss of actin cables in rat embryo fibroblasts or Rat 1 cells (an established rat embryo line), but only after exposure of the cells to epidermal growth factor in the culture medium and not after injection of epidermal growth factor into the cells.
Viable deletion mutant in the medium and large T-antigen-coding sequences of the polyoma virus genome
Authors: Bendig M M , Thomas T , Folk W R
Published: January 2020

A polyoma virus mutant that maps in the early region between the known hr-t and ts-a mutants has been isolated. Its 66-base-pair deletion results in structural changes in both medium and large T-antigens but causes no substantial alterations in viral replication or cell transformation.
Location and cloning of the herpes simplex virus type 2 thymidine kinase gene
Authors: McDougall J K , Masse T H , Galloway D A
Published: January 2020

The herpes simplex virus type 2 thymidine kinase gene has been mapped to a position colinear with the herpes simplex virus type 1 thymidine kinase gene and cloned within a 4.0-kilobase fragment in pBR 322.
Murine Leukemia Virus Proteins Expressed on the Surface of Infected Cells in Culture
Authors: Buetti Elena , Diggelmann Heidi
Published: January 2020

Infection of JLS-V9 cells in culture with Rauscher murine leukemia virus induced the appearance on the cell surface of two classes of viral proteins: Rauscher murine leukemia virus gp70, and glycoproteins related to the viral core (
gag
) proteins with apparent molecular weights in sodium dodecyl sulfate polyacrylamide gels of 80 × 10
3
and 95 × 10
3
. The latter proteins were identified by lactoperoxidase-catalyzed iodination of the cell surface and by metabolic labeling with [
3
H]mannose followed by immunoprecipitation with an antiserum directed against the major viral core protein, p30. Tryptic peptide maps of chloramine T-iodinated proteins indicated that 80 × 10
3
- and 95 × 10
3
-molecular-weight proteins were closely related. The 95 × 10
3
-molecular-weight protein from Rauscher murine leukemia virus-infected cells had a tyrosine fingerprint which was identical to that of the 95 × 10
3
-molecular-weight
gag
surface polyprotein of endogenous virus-producing AKR-A cells, suggesting that expression on the cell surface of glycosylated forms of
gag
precursor polyproteins may not be an exclusive property of leukemic thymocytes, but a more general phenomenon in murine leukemia virus infection. Tryptic fingerprint analysis of iodinated viral and cell-bound gp70's before and after desialylation indicated a lower level of glycosylation in the cell-bound gp70 population than in virions. Analysis of only surface-iodinated gp70 showed a simple pattern of exposed tryptic peptides which was very similar in Rauscher murine leukemia virus-infected cells and in AKR-A cells.
Avian Retroviruses That Cause Carcinoma and Leukemia: Identification of Nucleotide Sequences Associated with Pathogenicity
Authors: Sheiness Diana , Bister Klaus , Moscovici Carlo , Fanshier Lois , Gonda Thomas , Bishop J. Michael
Published: January 2020

Avian myelocytomatosis virus (MC29V) is a retrovirus that transforms both fibroblasts and macrophages in culture and induces myelocytomatosis, carcinomas, and sarcomas in birds. Previous work identified a sequence of about 1,500 nucleotides (here denoted onc
MCV
) that apparently derived from a normal cellular sequence and that may encode the oncogenic capacity of MC29V. In an effort to further implicate onc
MCV
in tumorigenesis, we used molecular hybridization to examine the distribution of nucleotide sequences related to onc
MCV
among the genomes of various avian retroviruses. In addition, we characterized further the genetic composition of the remainder of the MC29V genome. Our work exploited the availability of radioactive DNAs (cDNA's) complementary to onc
MCV
(cDNA
MCV
) or to specific portions of the genome of avian sarcoma virus (ASV). We showed that genomic RNAs of avian erythroblastosis virus (AEV) and avian myeloblastosis virus (AMV) could not hybridize appreciably with cDNA
MCV
. By contrast, cDNA
MCV
hybridized extensively (about 75%) and with essentially complete fidelity to the genome of Mill Hill 2 virus (MH2V), whose pathogenicity is very similar to that of MC29V, but different from that of AEV or AMV. Hybridization with the ASV cDNA's demonstrated that the MC29V genome includes about half of the ASV envelope protein gene and that the remainder of the MC29V genome is closely related to nucleotide sequences that are shared among the genomes of many avian leukosis and sarcoma viruses. We conclude that onc
MCV
probably specifies the unique set of pathogenicities displayed by MC29V and MH2V, whereas the oncogenic potentials of AEV and AMV are presumably encoded by a distinct nucleotide sequence unrelated to onc
MCV
. The genomes of ASV, MC29V, and other avian oncoviruses thus share a set of common sequences, but apparently owe their various oncogenic potentials to unrelated transforming genes.
Possible involvement of a calcium-stimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process
Authors: Karnik S S , Gopinathan K P
Published: January 2020

Ca2+ ions are necessary for the successful propagation of mycobacteriophage I3. An assay for the phage DNA release in the presence of an isolated cell wall preparation from the host was established, and in this system Ca2+ ions also stimulated the release of DNA. The inhibition of phage DNA injection caused by Tween 80 (polyoxyethylene sorbitan monooleate), a nonionic detergent routinely used in mycobacterial cultures, was reversed by Ca2+. The presence of a phage-associated ATP-hydrolyzing activity was demonstrated. This enzyme was stimulated by Ca2+ ions and inhibited by Tween 80. From this and the behavior of the two agents at the level of DNA injection, as well as the fact that phage I3 has a contractile tail structure, we conclude that the phage-associated ATPase is involved in the DNA injection process.