bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Structure of Rauscher Spleen Focus-Forming Virus
Authors: Bestwick Richard , Ruta Martin , Kiessling Ann , Faust Charles , Linemeyer David , Scolnick Edward , Kabat David
Published: January 2020

Rauscher spleen focus-forming virus contains functional
gag
and
pol
genes and a partially deleted
env
gene which is structurally related to the
env
genes of dual tropic murine leukemia viruses.
Nucleotide sequence of the region encompassing the JC virus origin of DNA replication
Authors: Frisque R J
Published: January 2020

The region of the JC virus (JCV) genome from 0.58 to 0.73 map units was sequenced by the Maxam-Gilbert technique. This segment of DNA specifies several regulatory elements and the amino-terminal portion of the early viral proteins. Comparisons with the analogous regions in the polyomaviruses simian virus 40 (SV40) and BK virus (BKV) confirm the close evolutionary relationship of these three viruses. Similarities include palindromic and symmetrical sequences near their origins of DNA replication, binding sites for their large T proteins, an AT-rich region (the Goldberg-Hogness, or TATA, box), and a large tandem duplication or triplication to the late side of their replication origins (however, these sequences differ). Homology between the sequences coding for the early proteins is also evident (79 and 93 of the first 110 amino acids are shared with SV40 and BKV, respectively). Of greater interest are features of the JCV genome which differ from those of other polyomaviruses. Absent in JCV and BKV are sequences which resemble the third T-antigen binding site of SV40. In addition, a set of sequences present in JCV and BKV DNAs (33 nucleotides in JCV and 22 nucleotides in BKV) and located near a 17-base-pair palindrome shared by all three viruses is missing in SV40 DNA. Another sequence, GGGXGGAG, which is repeated several times in many polyomaviruses and adenoviruses and which is thought to play a role in DNA replication or transcription or both, is not found near the JCV origin of replication. Finally, the tandem repeat of JCV, unlike those of BKV and SV40, includes the Goldberg-Hogness sequence.
Transcriptional regulation of three double-stranded RNA segments of bacteriophage phi 6 in vitro
Authors: Emori Y , Iba H , Okada Y
Published: January 2020

Three double-stranded RNA segments of bacteriophage phi 6 (L, M, and S) were transcribed in vitro by a virion-associated RNA polymerase. Regulation of L transcription was distinct from regulation of M and S transcription. Transcription of the L segment, which codes for early proteins, required manganous ion and high concentrations of all four ribonucleoside triphosphates and was inhibited by polyamines such as spermine. Transcription of the M and S segments, which code for late proteins, required manganous or magnesium ion and relatively low concentrations of all ribonucleoside triphosphates except GTP and was enhanced by polyamines. Optimal conditions for L transcription were more stringent than those for M and S transcription. These two apparently different patterns produced in in vitro transcription presumably reflect the two distinct in vivo transcription patterns; i.e., (i) similar amounts of three single-stranded RNA species were transcribed from the three corresponding segments of double-stranded RNA (early pattern) and (ii) a much larger amount of single-stranded RNA species was transcribed from M and S segments than from the L segment (late pattern). The early transcription pattern may be changed into the late pattern by a change of environment, such as substrate concentration. This suggests that the different enzymatic properties under the different environmental conditions of the virion-associated transcriptase are responsible for the transcriptional regulation throughout the infection cycle of bacteriophage phi 6.
Aberrant Distribution of Human Adenovirus Type 2 Late Proteins in Monkey Kidney Cells
Authors: Cepko Constance L. , Sharp Phillip A.
Published: January 2020

Monkey kidney cells (CV-C) infected with adenovirus type 2 displayed an aberrant distribution of 100K, 100K-hexon complex, hexon monomers, hexon trimers, penton base, and fiber proteins, relative to the patterns observed in adenovirus type 2-infected human cells. Human cell patterns were observed in CV-C cells when mutants selected for growth on monkey cells were used.
Alterations in the Structure of the Oligosaccharide of Vesicular Stomatitis Virus G Protein by Swainsonine
Authors: Kang M. S. , Elbein Alan D.
Published: January 2020

Swainsonine, an inhibitor of glycoprotein processing, inhibits the formation of the normal oligosaccharide chain of the G protein of vesicular stomatitis virus. Thus, when vesicular stomatitis virus was grown in baby hamster kidney cells in the presence of swainsonine (15 to 500 ng/ml) and labeled with [2-
3
H]mannose, the oligosaccharide portion of the G protein was completely susceptible to the action of endoglucosaminidase H. However, the normal viral glycoprotein is not susceptible to this enzyme. Various enzymatic treatments and methylation studies of the mannose-labeled oligosaccharides suggest that swainsonine causes the formation of a hybrid-type oligosaccharide having an oligomannosyl core (Man
5
GlcNAc
2
-Asn) characteristic of neutral oligosaccharides plus the branch structure (NeuNAc-Gal-GlcNAc) characteristic of the complex oligosaccharides. A structure for this hybrid oligosaccharide is proposed. Swainsonine had no effect on the incorporation of [
14
C]leucine into viral proteins, nor did it change the number of PFU produced in these cultures. It did, however, slightly decrease the incorporation of [
3
H]glucosamine and increase the incorporation of [
3
H]mannose. Vesicular stomatitis virus raised in the presence of swainsonine bound much more tightly to columns of concanavalin A-Sepharose than did control virus. Swainsonine had to be added within the first 4 or 5 h of virus infection to be effective. Thus, when 100 ng of the alkaloid per ml was added at any time within the first 3 h of infection, essentially all of the glycoprotein was susceptible to digestion by endoglucosaminidase H. However, when swainsonine was added 4 h after the start of infection, 30% of the glycopeptides became resistant to endoglucosaminidase H; at 5 h, 70% were resistant. The effect of swainsonine was reversible since removal of the alkaloid allowed the cells to form the normal complex glycoproteins. However, the time of removal was critical in terms of oligosaccharide structure.
Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice
Authors: Herr W , Gilbert W
Published: January 2020

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.
"Endless" viral DNA in cells infected with channel catfish virus
Authors: Cebrian J , Bucchini D , Sheldrick P
Published: January 2020

The state of intracellular viral DNA in cells infected with channel catfish virus has been studied by the Hirt selective extraction procedure and by restriction endonuclease digestion. The sedimentation properties and restriction patterns of viral DNA in the Hirt supernatant fraction indicate that the majority, if not all, of the DNA is in the form of linear unit-length (Mr approximately equal to 85 x 10(6)) molecules. However, restriction digests of viral DNA in the pellet fraction lacked two fragments corresponding to the molecular ends of unit-length DNA. In addition, there appeared in HpaI digests of pellet DNA a new restriction fragment interpretable as the product of fusion between the ends of unit-length molecules. The size of the new fragment requires that fusion occur in such a way that one copy of the terminally repeated sequences (Mr approximately equal to 12.3 x 10(6)) of the unit-length DNA is lost in the process. In pulse-chase experiments, radioactivity flowed from the pellet fraction to the supernatant fraction, suggesting a precursor-product relationship for these DNA species. The results are easily understood if unit-length virion DNA is generated by excision from concatemeric structures.
Simian virus 40 sequences between 0.168 and 0.424 map units are not required for abortive transformation
Authors: Sompayrac L M , Danna K J
Published: January 2020

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding region for large T antigen. Despite this deletion, F8dl abortively transformed rat cells as efficiently as wild-type simian virus 40. From this result, we conclude that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for abortive transformation. Since abortive transformation requires the expression of the simian virus 40 maintenance functions, we also infer that the sequences deleted from F8dl are not required to maintain transformation.
The Parodi-Irgens feline sarcoma virus and simian sarcoma virus have homologous oncogenes, but in different contexts of the viral genomes
Authors: Besmer P , Snyder H W , Murphy J E , Hardy W D , Parodi A
Published: January 2020

We have identified the oncogene and the putative transforming protein of the Parodi-Irgens feline sarcoma virus (PI-FeSV). The PI-FeSV is defective and needs a helper virus for its replication. The v-onc sequences in the PI-FeSV were found to be related to the v-sis sequences of the simian sarcoma virus (SSV). PI-FeSV nonproducer cells express two viral RNAs, a 6.8-and a 3.3-kilobase RNA. The 6.8-kilobase RNA contains gag, sis, and env sequences but lacks the pol gene. The 3.3-kilobase RNA, on the other hand, contains only env sequences. We have detected one feline leukemia virus-related protein product in these cells, namely, a 76-kilodalton protein which contains determinants of the feline leukemia virus gag proteins p15 and p30. The v-sis sequences in the PI-FeSV have been located near the 5' end of the viral genome. Taken together, these results imply that the p76 protein contains both feline leukemia virus gag and sis sequences and probably is the transforming protein of this virus. In contrast, in SSV the sis sequences are located towards the 3' end of the viral genome, and the sis protein is thought to be expressed via a subgenomic RNA. PI-FeSV and SSV therefore use different schemes to express their onc-related sequences. The v-sis sequences in the PI-FeSV contain restriction sites which reflect the different origin of the v-sis sequences in the PI-FeSV and SSV. The homologous oncogenes of the PI-FeSV and SSV thus were transduced by two different retroviruses, feline leukemia virus and the simian sarcoma-associated virus, apparently from the genomes of different species.
The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration
Authors: Ruyechan W T
Published: January 2020

Properties of the major DNA-binding protein found in herpes simplex virus-infected cells were investigated by using a filter binding assay and electron microscopy. Filter binding indicated that the stoichiometry of binding of the protein with single-stranded DNA is approximately 40 nucleotides per protein molecule at saturation. Strong clustering of the protein in DNA-protein complexes, indicative of cooperative binding, was seen with the electron microscope. Measurements of single-stranded fd DNA molecules saturated with protein and spread for electron microscopy by using both the aqueous and formamide spreading techniques indicated that the DNA is held in an extended configuration with a base spacing of approximately 0.13 nm per base.
On the sequential packaging of bacteriophage P22 DNA
Authors: Adams M B , Hayden M , Casjens S
Published: January 2020

Bacteriophage P22 is thought to package daughter chromosomes serially along concatemeric DNA. We present experiments which show that the average DNA packaging series length increases with time after infection, which supports this model. In addition, we have analyzed the effect on average series length of lowering the amount of the various individual proteins involved in DNA packaging. These results support the notion that the protein products of gene 2 and gene 3 are both more stringently required for initiation of sequential DNA packaging series than for their extension, and they are compatible with a model for the control of series length in which that length is determined, at least in part, by a competition between series initiation events and extension events.
Nuclease-sensitive sites in the two major intracellular simian virus 40 nucleoproteins
Authors: Hartmann J P , Scott W A
Published: January 2020

Simian virus 40 nucleoprotein isolated from the nuclei of infected cells contains a nuclease-sensitive site adjacent to the viral origin of replication (between 0.66 and 0.73 map unit). Nuclear extracts were subfractionated by sucrose gradient centrifugation to yield provirions (200S) and simian virus 40 chromatin (80S). The 80S fraction was cleaved either by DNase I or by an endonuclease endogenous to BSC-1 cells with high preference for the 0.66 to 0.73 region. The 200S fraction was treated to release core particles that were sensitive to nuclease cleavage; however, DNase I showed little or no preference for the 0.66 to 0.73 region of the provirion core nucleoprotein.
Alterations in Substrate Specificity and Physicochemical Properties of Deoxythymidine Kinase of a Drug-Resistant Herpes Simplex Virus Type 1 Mutant
Authors: Veerisetty V. , Gentry G. A.
Published: January 2020

The deoxythymidine kinase (dTK) activity of a 5-methoxymethyldeoxyuridine-resistant mutant (MMdU
r
-20) of herpes simplex virus type 1 was compared with that of the parental wild-type (WT) virus. The dTK activity induced by the mutant was consistently less than that induced by the WT virus, was inhibited by antibody specific for herpes simplex virus dTK, and was more thermostable than the WT dTK. Further, it was inhibited to a lesser degree than the WT dTK by the nucleoside analogs MMdU and arabinosylthymine (araT), which suggests that one of the effects of the mutation was a selective alteration in substrate recognition by the dTK. The loss of ability to inhibit the mutant dTK by
E
-(2)-5-bromovinyldeoxyuridine was not as great as that seen with araT and MMdU. This agrees well with our previous observation that the MMdU
r
-20 mutant of herpes simplex virus is only partially resistant to this analog, as compared with araT and MMdU (V. Veerisetty and G. A. Gentry, Virology
114
:576-579, 1981). [2-
14
C]araT was used to explore further the resistance to araT. Extracts of cells infected with the mutant, although producing a small amount of [
14
C]araTMP, were unable to produce [
14
C]araTTP, in contrast to extracts of cells infected with the WT virus. Both extracts, however, produced [
14
C]dTTP from [
14
C]deoxyribosylthymine. Finally, the ability of the extracts to phosphorylate [
14
C]dTMP was examined. It was found that this activity was greatly reduced relative to dTK activity in the case of the mutant. These findings suggest that a mutation in the dTK polypeptide has affected recognition not only of nucleoside substrates but of the nucleotide substrate dTMP as well, which agrees with the suggestion of Chen et al. that both activities are located on the same polypeptide (M. S. Chen and W. H. Prusoff, J. Biol. Chem.
253
:1325-1327, 1978; M. S. Chen, J. Walker, and W. H. Prusoff, J. Biol. Chem.
254
:10747-10753, 1979; M. S. Chen, W. P. Summers, J. Walker, W. C. Summers, and W. H. Prusoff, J. Virol.
30
:942-945, 1979).
Thermolabile in vivo DNA-binding activity associated with a protein encoded by mutants of herpes simplex virus type 1
Authors: Lee C K , Knipe D M
Published: January 2020

The major DNA-binding protein encoded by several temperature-sensitive mutants of herpes simplex virus type 1 was thermolabile for binding to intracellular viral DNA. The ability of DNase I to release this protein from isolated nuclei was used as a measure of the amount of protein bound to viral DNA. This assay was based upon our previous observation that the fraction of herpesviral DNA-binding protein which can be eluted from nuclei with DNase I represents proteins associated with progeny viral DNA (D. M. Knipe and A. E. Spang, J. Virol. 43:314-324, 1982). In this study, we found that several temperature-sensitive mutants encoded proteins which rapidly chased from a DNase I-sensitive to a DNase I-resistant nuclear form upon shift to the nonpermissive temperature. We interpret this change in DNase I sensitivity to represent the denaturation of the DNA-binding site at the nonpermissive temperature and the association with the nuclear framework via a second site on the protein. The DNA-binding activity measured by the DNase I sensitivity assay represents an important function of the protein in viral replication because three of five mutants tested were thermolabile for this activity. A fourth mutant encoded a protein which did not associate with the nucleus at the nonpermissive temperature and therefore would not be available for DNA binding in the nucleus. We also present supportive evidence for the binding of the wild-type protein to intracellular viral DNA by showing that a monoclonal antibody coprecipitated virus-specific DNA sequences with the major DNA-binding protein.
Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice
Authors: Tattersall P , Bratton J
Published: January 2020

Viral and cellular factors responsible for parvovirus target cell specificity have been examined for two serologically indistinguishable strains of the minute virus of mice which infect mouse cells of dissimilar differentiated phenotype. Both the prototype strain and the immunosuppressive strain grow in and form plaques on monolayers of simian virus 40-transformed human fibroblasts, a finding that has allowed the comparison of several aspects of their virus-host cell interactions. Although closely related by antigenic and genomic criteria, both the prototype strain and the immunosuppressive strain are restricted for lytic growth in each other's murine host cell, that is, in T cells and fibroblasts, respectively. The host range of each virus variant appears to be specified by a genetic determinant that is stably inherited in the absence of selection. In the restrictive virus-host interaction lytic growth is limited to a small or, in some cases, undetectable subset of the host cell population, and the majority of the infected cells remain viable, continuing to grow at the normal rate without expressing viral antigens. The susceptible host cell phenotype is dominant in T lymphocyte x fibroblast fusion hybrids, implying that different cell types express different developmentally regulated virus helper functions that can only be exploited by the virus variant that carries the appropriate strain-specific determinant.
Simian virus 40 mutants carrying extensive deletions in the 72-base-pair repeat region
Authors: Gheysen D , van de Voorde A , Contreras R , Vanderheyden J , Duerinck F , Fiers W
Published: January 2020

Simian virus 40 mutants were constructed with deletions at the late side of the origin of DNA replication by partial Bal 31 digestion at the SphI site or at the PvuII site. Some of these mutants lost virtually all of both 72-base-pair repeat segments ("enhancer" sequences) and exhibited a decrease in viability from 20-to 300-fold; one particular mutant, dl1852, even showed a reduction of almost 10(4)-fold. The very poorly growing deletion mutants were unstable and gave rise to DNA rearrangements upon further growth. An essential region for viability, at least in the absence of a 72-base-pair repeat, was revealed at the distal side of the 72-base-pair elements (L250 through L272). The effect of the deletions on T-antigen expression was measured, and the decreased viability of the mutants correlated with the impairment of T-antigen expression in all cases. The study of these mutants also revealed that the 72-base-pair repeats are not required for late transcription.
Characterization of a T-antigen-negative revertant isolated from a mouse cell line which undergoes rearrangement of integrated simian virus 40 DNA
Authors: Bender M A , Christensen J , Brockman W W
Published: January 2020

A transformation revertant has been isolated from an unusual line of simian virus 40 (SV40)-transformed BALB/c-3T3 cells in which rearrangements of integrated viral sequences are common. The revertant produces no SV40 T antigens, yields no virus on fusion with permissive cells, and can be retransformed by SV40 virions. SV40 DNA sequences are present within the cellular DNA, but interruption of the viral early transcription region by deletion and recombination with cellular sequences precludes the synthesis of T antigens. Analysis of this revertant lends further support to the notion that large T antigen plays an essential role in the maintenance of transformation in SV40-transformed BALB/c-3T3 cells. Examination of integration of SV40 DNA in this revertant, as well as in a temperature-sensitive A transformant, after retransformation by SV40 confirms that sequence homology plays little role in the insertion of SV40 DNA into cellular chromosomes.
Site-specific cleavage of bacteriophage T4 DNA associated with the absence of gene 46 product function
Authors: Albright L M , Geiduschek E P
Published: January 2020

A plasmid containing a copy of the late gene 23 was cleaved at two specific locations after bacteriophage T4 infection. Cleavage at the major site, which is at the 3' end of gene 23, was detected only in the absence of gene 46 product function and was independent of the state of modification of cytosine residues. Cutting of plasmid (cytosine-containing) DNA at this site was independent of phage DNA replication and late transcription functions. A second cleavage site, in vector DNA, was also mapped. The minor extent of cutting at this site was independent of gene 46 function. Gene 46 codes for, or controls, an exonuclease involved in T4 DNA recombination and in degradation of cytosine-containing DNA.
Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes
Authors: Rudolph K , Mann K
Published: January 2020

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.
Characterization of human papillomavirus type 13 from focal epithelial hyperplasia Heck lesions
Authors: Pfister H , Hettich I , Runne U , Gissmann L , Chilf G N
Published: January 2020

Focal epithelial hyperplasia Heck lesions of a Turkish patient were shown to contain papillomavirus-specific DNA, which was molecularly cloned into bacteriophage lambda. It proved to be related to human papillomavirus (HPV) type 6 DNA and HPV type 11 DNA. Reassociation kinetics revealed a cross-hybridization of 4 and 3%, respectively. There was no cross-reactivity with HPV type 1, 2, 3, 4, 5, 8, or 10. This papillomavirus type will be referred to as HPV type 13. The DNA was characterized by cleavage with several restriction enzymes, and the cleavage sites were physically mapped. Papules from two additional cases of Morbus Heck contained HPV type 13 DNA as shown by Southern blot hybridization and by the characteristic cleavage patterns. This may indicate that HPV type 13 is more frequently associated with focal epithelial hyperplasia Heck than are other HPV types.
Structural proteins of Herpesvirus saimiri
Authors: Keil G , Fleckenstein B , Bodemer W
Published: January 2020

Herpesvirus saimiri particles were purified from productively infected owl monkey kidney cell cultures, and the virion polypeptides were analyzed by polyacrylamide gel electrophoresis. A total of 21 predominant proteins were found in lysates of H. saimiri 11 particles by Coomassie blue staining or by [35S]methionine labeling and autoradiography; all proteins were between 160,000 and 12,000 daltons in size. They are most probably virion constituents, as most of them were precipitated by immune sera, and no dominant proteins of equivalent sizes were found in mock-infected cultures. Four glycoproteins (gp 155/160, gp 128, gp 84/90, gp 55) and three polypeptides that appeared not to be glycosylated (p71, p35, p28) were assigned to the envelope or matrix of virions, whereas at least four phosphoproteins (pp132, pp118, pp55, pp13) and ten polypeptides without apparent secondary modification (p155/160, p106, p96, p67, p53, p36, p32, p15, p14, p12) were found in the nucleocapsid fraction. Analysis of virion proteins from different H. saimiri strains did not reveal appreciable differences in the migration behavior of most polypeptides, including all glycoproteins; however, determination of a strain-specific size pattern was possible for three of four phosphoproteins. The overall similarity in protein architecture of H. saimiri strains obviously does not reflect the variability in biology, such as oncogenic properties. In comparison, DNA sequence divergences appear to remain a better taxonomic criterion for strain distinction.
Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. II. A reduced level of ion concentrations causes the blockage of T7 maturation in K-12-M cells
Authors: Kuhn A H , Jütte H , Kellenberger E
Published: January 2020

Cellular leakage observed in Escherichia coli K-12-M shortly after T7 infection might be the cause of arrested phage morphogenesis. We observed in this strain, but not in the normal host, a drastic reduction of the intracellular concentration of potassium (60%), magnesium (40%), putrescine (90%), and spermidine (40%), whereas ATP was not significantly reduced. Leakage started about 1 min after the addition of phage and was arrested 3 to 5 min postinfection. Larger molecules such as o-nitrophenyl-beta-D-galactopyranoside could not enter the cells, showing that the permeability of the membrane was not generally affected. To prevent their leakage, we increased the outside concentrations of several small molecules and ions. The yield of progeny phage was substantially increased by the addition of 100 mM MgSO4.
Polyomavirus origin for DNA replication comprises multiple genetic elements
Authors: Muller W J , Mueller C R , Mes A M , Hassell J A
Published: January 2020

To define the minimal cis-acting sequences required for polyomavirus DNA replication (ori), we constructed a number of polyomavirus-plasmid recombinants and measured their replicative capacity after transfection of a permissive mouse cell line capable of providing polyomavirus large T antigen in trans (MOP cells). Recombinant plasmids containing a 251-base-pair fragment of noncoding viral DNA replicate efficiently in MOP cells. Mutational analyses of these viral sequences revealed that they can be physically separated into two genetic elements. One of these elements, termed the core, contains an adenine-thymine-rich area, a 32-base-pair guanine-cytosine-rich palindrome, and a large T antigen binding site, and likely includes the site from which bidirectional DNA replication initiates. The other, termed beta, is located adjacent to the core near the late region and is devoid of outstanding sequence features. Surprisingly, another sequence element named alpha, located adjacent to beta but outside the borders of the 251-base-pair fragment, can functionally substitute for beta. This sequence too contains no readily recognized sequence features and possesses no obvious homology to the beta element. The three elements together occupy a contiguous noncoding stretch of DNA no more than 345 base pairs in length in the order alpha, beta, and core. These results indicate that the polyomavirus origin for DNA replication comprises multiple genetic elements.
Mx gene control of interferon action: different kinetics of the antiviral state against influenza virus and vesicular stomatitis virus
Authors: Arnheiter H , Haller O
Published: January 2020

The allele Mx regulates the extent to which interferon alpha/beta inhibits the growth of influenza viruses in mouse cells such as peritoneal macrophages. The time course of induction of the antiviral state against an influenza A virus is comparable in macrophages with and without Mx and is similar to that found with vesicular stomatitis virus. In contrast, the decay of the antiviral state against influenza virus is markedly slower in Mx-positive cells and slower than that against vesicular stomatitis virus observed in either Mx-positive or Mx-negative cells. Thus, after removal of interferon alpha/beta, Mx-positive cells remain protected against influenza virus at times when they have lost protection against vesicular stomatitis virus. These results suggest that interferon alpha/beta treatment activates different antiviral mechanisms, each acting against distinct groups of viruses and each independently controlled by host genes.
In vitro and in vivo studies of bovine parvovirus proteins
Authors: Lederman M , Bates R C , Stout E R
Published: January 2020

Total cytoplasmic RNA from bovine parvovirus (BPV)-infected cells or BPV-specific RNA selected by hybridization to cloned BPV genomic sequences were translated in a message-dependent rabbit reticulocyte lysate. Immunoprecipitation, using immunoglobulin G from rabbits injected with purified BPV, resulted in the detection of [35S]methionine-labeled polypeptides with MrS of 80,000, 72,000, 62,000, and 60,000. These in vitro translation products had the same mobility on sodium dodecyl sulfate-polyacrylamide gels as that of the four proteins found in purified virions. The three largest polypeptides had amino acid sequence homology, as judged by serological methods and partial proteolysis with Staphylococcus aureus V8 protease. Additional noncapsid proteins with MrS of 25,000, 27,000, and 31,000 were also detected as translation products of these RNAs. All of the above species were immunoprecipitated by immunoglobulin G from a calf which was naturally infected with BPV. All four capsid proteins but only one of the lower-molecular-weight polypeptides were detected after the immunoprecipitation of BPV-infected cells. The results presented here indicate that the BPV genome codes for four capsid proteins and a noncapsid protein which may be structurally related to the capsid proteins.
Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region
Authors: Laub O , Rall L B , Truett M , Shaul Y , Standring D N , Valenzuela P , Rutter W J
Published: January 2020

We have constructed two simian virus 40 early replacement recombinants that have the coding sequences for hepatitis B virus surface antigen (HBsAg). One construction, LSV-HBsAg, has the coding region for HBsAg but not the portion encoding the putative pre-surface antigen leader. Transformed monkey kidney cells (COS) infected with this recombinant express large quantities of the characteristic partially glycosylated HBsAg molecule, which are assembled into 22-nm particles that appear similar to those produced by human liver cells infected with hepatitis B virus. This result indicates that the pre-surface antigen sequences are not required for the synthesis of HBsAg or its assembly into particulate structures. The second recombinant, LSV-HBpresAg, has the entire surface antigen gene, including the putative promoter and pre-surface antigen region. COS cells infected with this recombinant plasmid produce 40- to 50-fold less HBsAg than those infected with the LSV-HBsAg recombinant plasmid. RNA mapping studies suggest that the transcription of the HBsAg gene is initiated at more than one site, or alternatively, that RNA splicing of transcripts occurs in the pre-surface antigen region.
Suppression of a VP1 mutant of simian virus 40 by missense mutations in serine codons of the viral agnogene
Authors: Margolskee R F , Nathans D
Published: January 2020

We isolated second-site revertants of a partially defective VP1 mutant of simian virus 40. The suppressing mutation in each of these pseudorevertants was mapped to the viral agnogene. Of six independently isolated pseudorevertants, all had a missense mutation in a serine codon, near the beginning of the agnogene, that would cause replacement of serine at position 7, 11, or 17 in the agnoprotein by a hydrophobic amino acid. Our results suggest that the agnoprotein interacts in a specific way with VP1 during the late stages of viral development.
Monoclonal antibodies to newcastle disease virus: delineation of four epitopes on the HN glycoprotein
Authors: Iorio R M , Bratt M A
Published: January 2020

Eighteen independent hybridomas producing monoclonal antibodies to Newcastle disease virus have been prepared by fusion of SP2 cells with spleen lymphocytes from a BALB/c mouse immunized with intact UV-inactivated Newcastle disease virus strain Australia-Victoria. They have been divided into three groups on the basis of radioimmunoprecipitation, infected cell surface and cytoplasmic fluorescence, and isotype. The anti-HN group is made up of nine antibodies which give surface fluorescence on infected cells and immunoprecipitate the HN glycoprotein. These antibodies bind to HN in nitrocellulose transfers of sodium dodecyl sulfate gels, but only if it has been neither reduced nor boiled. To varying degrees, all of these HN antibodies neutralize infectivity. These results suggest that they recognize exposed determinants of a conformational nature on the native HN molecule. They have been used in competition antibody-binding radioimmunoassays and additive neutralization assays, and on the basis of these studies the epitopes they recognize have been subdivided into four domains, two of which are overlapping, on the HN glycoprotein. The relatively weaker neutralizing activity observed with some of these antibodies cannot be explained by lower avidities for their epitopes because there is not an inverse correlation between estimated binding constant and neutralizing activity. The four antibodies in the second group all give a predominantly cytoplasmic fluorescence pattern, immunoprecipitate the nucleocapsid protein, and bind to nucleocapsid protein in nitrocellulose transfers of reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gels. All five of the antibodies in the third group are of the immunoglobulin M class, unlike the others which are all immunoglobulin G antibodies. Members of this group show variable fluorescence patterns, but none is able to immunoprecipitate or bind to a specific viral antigen transferred to nitrocellulose paper from sodium dodecyl sulfate-polyacrylamide gels.
Comparison of the DNA sequence of the Crawford small-plaque variant of polyomavirus with those of polyomaviruses A2 and strain 3
Authors: Rothwell V M , Folk W R
Published: January 2020

The DNA sequence of two wild-type strains of polyomavirus (A2 and strain 3) are known. We have determined the majority of the DNA sequence of a third strain, the Crawford small-plaque virus. This virus has been noted for its capacity to induce readily detected tumor-specific transplantation antigen in hamster cells, a property that is most likely attributable to an altered middle T-antigen. A comparison of its DNA sequence with those of the A2 and strain 3 viruses reveals numerous nucleotide substitutions, insertions, and deletions throughout the genome. Most sequence changes in coding regions are silent mutations; however, variability in proteins can be predicted from these sequence data at 5 locations in middle T-antigen, 10 in large T-antigen, and 10 in VP1. The Crawford small-plaque virus noncoding regulatory region contains, in addition to nucleotide substitutions, a 44-base-pair tandem repeat of sequences on the late side of the origin of DNA replication.
Leupeptin inhibits retrovirus infection in mouse fibroblasts
Authors: Andersen K B
Published: January 2020

The protease inhibitor leupeptin was shown to inhibit retrovirus infection in mouse fibroblasts at a step shortly after internalization of the virus particles. The inhibited step most likely was the passage of virus particles through the lysosomes or other acid vesicles. Leupeptin was also shown to inhibit degradation of virus particles in the lysosomes. The results are discussed with respect to the involvement of proteases in the infectious route.
Transcriptional mapping of the DNA polymerase gene of vaccinia virus
Authors: Traktman P , Sridhar P , Condit R C , Roberts B E
Published: January 2020

Vaccinia virus DNA polymerase, a single-subunit enzyme of 110,000 molecular weight, is induced early after infection. Genetic analysis suggests that the gene encoding the enzyme maps within a 15-kilobase HindIII fragment located 45 kilobases from the left-hand end of the genome. We identified the in vitro translation product with these properties and mapped the transcript by hybrid selection, RNA filter hybridization, and S1 nuclease mapping. Two mRNAs from this region, 3.4 and 3.9 kilobases in size, could be translated in vitro to yield a 110K polypeptide. The two RNAs shared a common 5' terminus and had staggered 3' ends. Sequences mapping entirely within this gene were shown to be biologically active in rescuing mutants with temperature-sensitive or drug-resistant polymerase activity to the wild-type phenotype.
Isolation of transformation-defective, replication-nondefective early region 1B mutants of adenovirus 12
Authors: Fukui Y , Saito I , Shiroki K , Shimojo H
Published: January 2020

We isolated three adenovirus 12 early region 1B mutants (in205B, in205C, and dl205) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. These mutants could replicate efficiently in human embryo kidney or KB cells but showed markedly reduced transforming capacities both in vitro and in vivo. In cells infected with the mutants, the early region 1B gene was transcribed efficiently. In cells infected with in205B, the products corresponding to the early region 1B-coded 19,000-molecular-weight polypeptide was detected by in vitro translation but not immunoprecipitated extract of labeled cells. In cells infected with in205C or dl205, the products corresponding to the same polypeptide were not detected by either in vitro translation or immunoprecipitation of labeled cell extracts. The results suggest that the 19,000-molecular-weight polypeptide encoded by early region 1B is required for cell transformation but not for viral propagation.
Mouse mammary tumor virus: specific methylation patterns of proviral DNA in normal mouse tissues
Authors: Hu W S , Fanning T G , Cardiff R D
Published: January 2020

The methylation state of endogenous mouse mammary tumor virus (MuMTV) proviral DNA was examined in normal mouse tissues. DNAs from various tissues were cleaved with the methylation-sensitive enzymes HhaI and HpaII and analyzed by Southern blotting. Tissue-specific MuMTV proviral DNA methylation patterns were found in the BALB/c, C3H, C57BL, GR/A, and GR-Mtv-2- mouse strains. MuMTV proviral DNA was hypomethylated in DNAs from the spleens and testes of all strains examined. The GR/A mouse strain, which was most thoroughly studied, also showed hypomethylation of MuMTV proviral DNA in bone marrow and placental tissues. Analysis of RNAs extracted from GR/A liver, mammary tumor, testes, placenta, and spleen tissues demonstrated that MuMTV proviral hypomethylation need not reflect significant proviral transcription.
DNA methyltransferase induced by frog virus 3
Authors: Willis D B , Goorha R , Granoff A
Published: January 2020

Over 20% of the cytosine bases in frog virus 3 DNA are methylated at the 5-carbon position. To determine whether this high degree of methylation is the result of a virus-specific enzyme, we examined the kinetics of induction and the substrate specificity of a DNA methyltransferase from frog virus 3-infected fathead minnow cells. A novel DNA methyltransferase activity appeared in the cytoplasm of infected cells at 3 h postinfection. This activity was induced in the absence of viral DNA replication and was therefore probably an early viral enzyme. In contrast to the methyltransferase activity extracted from uninfected cell nuclei, the cytoplasmic enzyme showed a strong template preference for double-stranded over single-stranded and for unmethylated over hemimethylated DNA. The dinucleotide sequence dCpdG was a necessary and sufficient exogenous substrate for methylation in vitro. A mutant of frog virus 3, isolated as resistant to 5-azacytidine and having unmethylated virion DNA, did not induce cytoplasmic DNA methyltransferase, leading to the conclusion that this activity is coded for by the virus.
Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins
Authors: Oker-Blom C , Ulmanen I , Kääriäinen L , Pettersson R F
Published: January 2020

We have analyzed the structure of the rubella virus genome RNA and the virus-specific RNA species synthesized in B-Vero cells infected with rubella virus. A single-stranded, capped, and polyadenylated RNA species sedimenting at 40S in a sucrose gradient was released from purified virions treated with sodium dodecyl sulfate. This RNA species migrated with an Mr of about 3.8 X 10(6) in an agarose gel after denaturation with glyoxal and dimethyl sulfoxide. Infected cells labeled with [3H]uridine in the presence of actinomycin D contained, in addition to the 40S RNA, a single-stranded polyadenylated 24S RNA species as shown by sucrose gradient analysis. In a Northern blot analysis, this RNA hybridized to a cDNA probe derived from the 3' portion of the genomic 40S RNA. In vitro translation of the 24S RNA species yielded a 110,000-dalton polypeptide, in addition to some smaller products which were immunoprecipitated with an antiserum prepared against the structural proteins E1, E2a, E2b, and C. Since the sum of the molecular weights of the nonglycosylated envelope proteins and the capsid protein has been estimated to be about 116,000 (C. Oker-Blom et al., J. Virol. 46:964-973, 1983), these results suggest that the 24S RNA species represents a subgenomic mRNA coding for a precursor (p110) to the structural proteins of rubella virus. Thus, the strategy of gene expression of rubella virus appears to be similar to that of the alphaviruses.
Identification of the viral sequence required for the generation of recovered avian sarcoma viruses and characterization of a series of replication-defective recovered avian sarcoma viruses
Authors: Wang L H , Beckson M , Anderson S M , Hanafusa H
Published: January 2020

The ability of transformation-defective deletion mutants of Schmidt-Ruppin Rous sarcoma virus to induce tumors and generate recovered sarcoma viruses (rASVs) was correlated with the partial src sequences retained in the transformation-defective viral genomes. Since all the transformation-defective viruses that were capable of generating rASVs retained a portion of the 3' src sequence, regardless of the extent of the 5' src deletion, and those lacking the 3' src were unable to generate rASVs, it appears that the 3', but most likely not the 5', src sequence retained in the transformation-defective viral genome is essential for rASV formation. However, rASVs derived from a particular mutant, td109, which retained a portion of the 3' src sequence, but lacked most (if not all) of the 5' src sequence, were all found to be defective in replication. Analyses of the genomic sequences of 13 isolates of td109-derived rASVs revealed that they contained various deletions in viral envelope (env), polymerase (pol), and structural protein (gag) genes. Ten isolates of rASVs contained env deletions. One isolate (rASV3812) contained a deletion of env and the 3' half of pol, and one isolate (rASV398) contained a deletion of env and pol. The one with the most extensive deletion (rASV374) had a deletion from the p12-coding sequence through pol and env. In addition, the 5' src region of td109-derived rASVs were heterogeneous. Among the 7 isolates analyzed in detail, one isolate of rASV had a small deletion of the 5' src sequence, whereas three other isolates contained extra new sequences upstream from src. Both env- and env- pol- rASVs were capable of directing the synthesis of precursor and mature gag proteins in the infected nonproducer cells. We attribute the deletions in the replication-defective rASVs to the possibility that the 5' recombination site between the td109 and c-src sequence, involved regions of only partial homology due to lack of sufficient 5' src sequence in the td109 genome for homologous recombination. A model of recombination between the viral genome and the c-src sequence is proposed to account for the requirement of the 3' src sequence and the basis for the generation of deletions in td109-derived rASVs.
Characterization of Bluetongue Virus Ribonucleic Acid
Authors: Verwoerd D. W. , Louw H. , Oellermann R. A.
Published: January 2020

An improved purification procedure yielded bluetongue virus free from any single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 × 10
6
to 2.8 × 10
6
daltons, with a total molecular weight estimate of about 1.5 × 10
7
for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.