bluuebunny/crossref_metadata_2025 huggingface dataset:
SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23
Authors: Markewych O , Boghosian A , Dosmar M , Ende D , Witmer H
Published: January 2020

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-10c coded for discrete classes of early and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.
Functions of gene C and gene D products of bacteriophage phi X 174
Authors: Fujisawa H , Hayashi M
Published: January 2020

Phage-related materials existing in cells infected with various mutants of bacteriophage phi chi 174 were investigated. A novel species of replicative-form (RF) DNA was found in cells infected with a phage mutant of gene B, C, D, F, or G. This species, called RFI, sedimented at a position between RFI and RFII in a neutral sucrose gradient. It was converted to RFI upon denaturation in alkali, denaturation in formamide and subsequent renaturation, or RNase treatment at low ionic strength. In cells infected with a phage mutant of gene C, RFI was derived from pulse-labeled RFII after a short chase. TLLS INFECTED WITH A MUTANT OF GENE B, D, or F. A possible function of the C gene product of phi chi 174 could be to prevent the conversion of RFII to RFI, thereby maintaining the availability of RFII to act as the template for single-stranded viral DNA synthesis. A protein complex containing no DNA, which sedimented with an S value of 108 in a sucrose gradient and contained virion proteins F, G, and H, and nonvirion protein D, was found in cells infected with the gene C mutant. A possible function of protein D was considered as a scaffolding protein for assembly of phage structural proteins.
Homology between double-stranded RNA and nuclear DNA of yeast
Authors: Vodkin M
Published: January 2020

The relationship between mycoviral double-stranded (ds) RNA and host cell DNA was investigated. Radiolabeled ds RNA was denatured and reannealed in the presence and absence of denatured DNA. RNA from killer strains of the yeast Saccharomyces cerevisiae and from nonkiller derivatives was utilized. The above-mentioned strains, as well as one that lacks all ds RNA, were sources for extracted DNA. Net hybridization of ds RNA to DNA occurred regardless of the strains from which the respective nucleic acids were prepared.
Variations in integration site of avian oncornaviruses in different hosts
Authors: Dastoor M N , Shoyab M , Baluda M A
Published: January 2020

We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.
Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4
Authors: Wood W J , Bernstein H
Published: January 2020

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.
Structure of hepatitis B Dane particle DNA before and after the Dane particle DNA polymerase reaction
Authors: Hruska J F , Clayton D A , Rubenstein J L , Robinson W S
Published: January 2020

DNA isolated from the hepatitis B antigen form known as the Dane particle was examined by electron microscopy before and after the endogenous Dane particle DNA polymerase reaction. The most frequently occurring form was an untwisted circular double-stranded DNA molecule approximately 1 mum in length. Less frequently occurring forms included circular DNA of approximately unit length and having one or more small single-stranded regions, similar circular molecules with one or more tails either shorter or longer than 1 mum in length, and very small circular molecules with tails. There was no increase in frequency or length of tails after a DNA polymerase reaction, suggesting that tails were not formed during this reaction. The mean length of circular molecules increased by 23% when DNA was spread in formamide compared with aqueous spreading, suggesting that single-stranded regions are present in most of the molecules. The mean length of circular molecules obtained from aqueous spreading increased by 27% after a Dane particle DNA polymerase reaction. This indicates that single-stranded regions were converted to double-stranded DNA during the reaction.
Replication process of the parvovirus H-1. VI. Characterization of a replication terminus of H-1 replicative-form DNA
Authors: Rhode S L
Published: January 2020

The linear duplex replicative form (RF) DNA of the parvovirus H-1 has been characterized with respect to cleavage by the bacterial restriction endonuclease of Escherichia coli, EcoRI. RF DNA has a single cleavage site 0.22 genome length from the left end of the molecule. The molecular weight of H-1 RF DNA determined by gel electrophoresis is 3.26 X 10(6). H-1 RF DNA has been found to dimerize by hydrogen-bounded linkage at the molecular left end, and in some molecules the viral strand is covalently linked to the complementary strand. Some 10% of monomeric RF DNA also has a covalent linkage between the viral and complementary strands at the left end. The EcoRI-B fragment, containing the left end of the RF molecule, appears to be a replication terminus by its labeling characteristics for both RF and progeny DNA synthesis. These findings suggest that the left end of H-1 RF DNA has some type of "turn-around" structure and that this end is not an origin for DNA synthesis.
Cyclic AMP regulation of mammary tumor virus production
Authors: Yang J , Nandi S
Published: January 2020

Addition of dibutyryl cyclic AMP (cAMP) and agents (isoproterenol and epinephrine) that stimulate the activities of adenylate cyclase enhance the stimulation of mammary tumor virus (MTV) production two- to threefold by glucocorticoid in short-term primary cultures of mammary tumors. This cAMP potentiation seems to depend on the stimulated level of MTV production by glucocorticoid alone, which increases MTV production 5- to 10-fold over basal level but varies greatly in absolute terms. When the stimulated level by glucocorticoid alone is suboptimal, cAMP seems to restore the sensitivity of the cells to the stimulatory effect of glucocorticoid to its maximum.
Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis
Authors: Peters G , Harada F , Dahlberg J E , Panet A , Haseltine W A , Baltimore D
Published: January 2020

The small RNAs of Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis. The pattern of small RNAs is significantly different from that of Rous sarcoma virus. A subset of the virion small RNAs is associated with the genome RNA in the 70S complex. One of the associated molecules, a cellular tRNA, is tightly bound to the genome RNA and serves as the major primer for M-MuLV RNA-directed DNA synthesis in vitro.
Evidence for recombination between N- and B-tropic murine leukemia viruses
Authors: Hopkins N , Schindler J , Gottlieb P D
Published: January 2020

After co-infection of Sc-1 cells with N- and B-tropic murine leukemia viruses that differ in their XC plaque morphology, Hopkins et al. (1976) obtained viruses, designated XLP-N, that appeared to be recombinants, since they possess the N-tropism of one parent and the XC plaque morphology of the other (the B-tropic virus) parent. Here we present evidence, based on antigenicity and electrophoretic mobility, that some clonal isolates of XLP-N have inherited gp70 gene of their B-tropic virus parent. In addition to providing evidence that XLP-N viruses are recombinants, the fact that an N-tropic virus may apparently possess a gp70 derived from a B-tropic virus provides evidence, which is in agreement with the findings of others (Huang et al., 1973; Krontiris et al., 1973) that the N- or B-tropism of murine leukemia virus does not reside in gp70.
Characterization of components released by alkali disruption of simian virus 40
Authors: Christiansen G , Landers T , Griffith J , Berg P
Published: January 2020

Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 37 degrees C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and appeared by electron microscopy to be relaxed circular structures with an average of 21 beads joined by short, thin bridges. The major protein components in the complex were host cell histones, but SV40-specific proteins VP3 and VP2 were also present. The 7S protein particles were almost exclusively VP1 and, in negatively stained samples, resembled the capsomer structures of intact virions.
RNA synthesis of vesicular stomatitis virus. VII. Complete separation of the mRNA's of vesicular stomatitis virus by duplex formation
Authors: Freeman G J , Rose J K , Clinton G M , Huang A S
Published: January 2020

Full-length virion RNA and complementary mRNA's of vesicular stomatitis virus can be annealed to each other, digested with RNases, and then separated as five unique duplex RNA molecules on polyacrylamide slab gels. Similar RNA duplexes were detected whether mRNA or virion RNA was the radioactive component and whether the mRNA was synthesized in vitro or in vivo. The sharp banding pattern of these RNA molecules was dependent on treatment with RNase T2, suggesting that removal of poly(A) is necessary. Identification of the coding region contained in each RNA duplex was based on their previous identification as single-stranded mRNA on formamide-containing, polyacrylamide gels. Because the two smallest mRNA'S had not been previously separated, their identification was based on their in vitro transcriptional gene order. In the order of increasing mobilities on the slab gels, the RNA duplexes are identified as the hybrid of the region of the genome RNA hybridized to the complementary mRNA coding for the large protein, the glycoprotein, the nucleocapsid protein, the core-associated NS protein, and the matrix protein (L,G,N,NS, and M). Several lines of evidence support the presence of undegraded complete mRNA, excluding poly(A), in these RNA duplexes. Also, the two smallest mRNA's, separated by duplex formation, were denatured, and their individual oligonucleotide fingerprints were determined. From chemical length determinations, the molecular weights of the mRNA, minus poly(A), are 2.78 X 10(5) and 2.5 X 10(5), respectively, for the mRNA's of the NS and M proteins.
Localization of two cellular forms of the vesicular stomatitis viral glycoprotein
Authors: Knipe D M , Lodish H F , Baltimore D
Published: January 2020

Two cell-associated forms of the glycoprotein (G) of vesicular stomatitis virus, termed G1 and G2, have been resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. G1 has the higher electrophoretic mobility, but both forms migrate more slowly than G protein synthesized in a wheat germ cell-free system (G0), which presumably is the unglycosylated form. G1 is a kinetic precursor of the G2 form, and the apparent cause of the electrophoretic difference between the two species is the presence of N-acetylneuraminic acid on the G2 form. Conversion of G1 to G2 occurs 10 to 20 min prior to the appearance of the G2 form of the protein on the cell surface. This suggests that the G protein may be completely glycosylated several minutes prior to its migration to the cell surface and that glycosylation is not the limiting step in its maturation. No glycoprotein comigrating with G0 can be detected in the infected cells, even after 5-min labeling periods; this suggests that partial clycosylation of G occurs concomitantly with or immediately after its synthesis.
Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus
Authors: Knipe D M , Baltimore D , Lodish H F
Published: January 2020

Cell fractionation and protein electrophoresis were used to study the intracellular sites of synthesis and intermediate structures in the assembly of the virion proteins of vesicular stomatitis virus. Each of the three major virion proteins assembled into virions through a separable pathway. The nucleocapsid (N) protein was first a soluble protein and later incorporated into free, cytoplasmic nucleocapsids. A small amount of N protein was bound to membranes at later times, presumably representing either nucleocapsids in the process of budding or completed virions attached to the cell surface. The matrix (M) protein also appeared to be synthesized as a soluble protein, but was then directly incorporated into membranous structures with the same density as whole virus. Very little M protein was ever found in membranes banding at the density of plasma membranes. The M protein entered extracellular virus very quickly, as though it moved directly from a soluble state into budding virus. In contrast, the glycoprotein (G) was always membrane bound; it appeared to be directly inserted into membranes during its synthesis. Glycosylation of the G protein was completed only in smooth membrane fractions, possibly in the Golgi apparatus. After a minimum time of 15 min following its synthesis, G protein was incorporated into the surface plasma membrane, from which it was slowly shed into virions. These multiple processing steps probably account for its delayed appearance in virus. From this work it appears that the three major structural proteins come into the surface budding structure through independent pathways and together they coalesce at the plasma membrane to form the mature virion.
Analysis of the defects of temperature-sensitive mutants of vesicular stomatitis virus: intracellular degradation of specific viral proteins
Authors: Knipe D , Lodish H F , Baltimore D
Published: January 2020

The metabolism of viral RNA and proteins has been studied in cells infected with temperature-sensitive mutant strains of vesicular stomatitis virus. Certain viral proteins encoded by the mutant strains, usually the putative mutant protein for the assigned complementation group, were shown to be degraded more rapidly at the nonpermissive temperature than were the wild-type proteins. Group III mutants (tsG33, tsM301) encode M proteins which are degraded three- to fourfold faster than the wild-type protein. This defect cannot be fully rescued by coinfection with wild-type virus, and thus the defect appears to be in the M protein itself. Mutants tsM601 (VI) and tsG41(IV) encode N proteins which are degraded much faster than the wild-type protein and also share the property of being defective in replication of viral RNA, suggesting a correlation between these phenotypic properties. Furthermore, the L proteins of tsG11(I) and tsG13(I) are more labile than the wild-type protein at the nonpermissive temperature. The G protein of tsM501(V) did not undergo the change in electrophoretic mobility previously shown to be the result of sialylation, suggesting that it is defective in maturation or glycosylation at the nonpermissive temperature. Three of the mutants previously isolated in this laboratory, tsM502(V), tsM601(VI), and tsM602(VI), were shown to be defective in viral RNA synthesis at the nonpermissive temperature. Mutant tsM601(VI) was defective mainly in viral RNA replication, whereas tsM502(V) appeared to be totally defective for viral RNA transcription and replication at the nonpermissive temperature.
Human papillomavirus DNA: physical mapping of the cleavage sites of Bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) endonucleases and evidence for partial heterogeneity
Authors: Favre M , Orth G , Croissant O , Yaniv M
Published: January 2020

The DNA of human papillomavirus (HPV) obtained from a pool of plantar warts is cleaved by bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) restriction endonucleases at one and four specific sites, respectively. These sites were localized on the previously established cleavage map of HPV DNA, using the Hind, HindIII, HpaI, and EcoRI endonuclease restriction sites as reference. The four HpaII sites were mapped, clockwise, at 1.4, 41.1, 44.3, and 52.8% of the genome length from the unique BamI cleavage site taken as point zero. The HpaII site mapped at 1.4% of the genome length was absent in 40 to 50% of the molecules, thus showing a genetic heterogeneity of HPV DNA.
Contact-Inhibited Revertant Cell Lines Isolated from Simian Virus 40-Transformed Cells. VII. Serum Detachment-Resistant Revertant Cells
Authors: Bradley W. E. C. , Culp L. A.
Published: January 2020

Flat revertant cells of simian virus 40-transformed mouse fibroblasts have been isolated on the basis of resistance to a selective detachment procedure. The revertants are generally similar to those isolated by other procedures.
Primer recognition by avian myeloblastosis virus RNA-directed DNA polymerase
Authors: Brown R D , Armentrout R W
Published: January 2020

Tryptophanyl-tRNA was specifically labeled at the 3' end with [3H]tryptophan and cleaved in half with RNase under denaturing conditions, and the 3' half was shown to hybridize exclusively at the 5' end of avian myeloblastosis virus RNA. The RNA-dependent DNA polymerase of avian myeloblastosis virus is capable of efficiently binding the 3' half of the primer molecule.
Stability of the carrier state in bacteriophage M13-infected cells
Authors: Merriam V
Published: January 2020

Bacteriophage M13-infected carrier cells were shown to be unstable to prolonged growth under all conditions. Carrier Hfr cells were transferred in dilute culture (10(3) to 10(4)/ml), where reinfection was impossible and the physiology of the cell was minimally altered. After an initial period of about 10 generations, during which all cells in the culture remained infected, there was exponential decay in the proportion of infected cells in the culture. Uninfected cells that appeared were M13 sensitive. Hfr and F' males were also transferred serially at high cell densities (10(7) to 10(9)/ml), where high levels of phage should permit reinfection. The proportion of phage-producing cells in the cultures remained constant for 7 to 15 generations and then dropped exponentially on further growth. Non-phage-producing cells appearing in the culture were refractory to infection by M13; in some cases cells scored as non-phage producers for 20 generations were observed to produce phage on further growth in liquid culture. F'trp+ males infected with M13 lost trp+ function almost immediately; this was not regained in these experiments. Infected cells grown in dilute culture or on plates remained infected longer, produced more PFU per cell for a longer period, and retained trp+ function in F'trp+ males for over 90 generations. Non-phage-producing cells that appeared were sometimes phage resistant, sometimes phage sensitive. The existence of a phage-related material accumulating at high cell densities and affecting expression of free episomes, episomal expression in Hfr males, and phage synthesis itself is suggested.
Quantitation of herpes simplex virus type 1 RNA in infected HeLa cells
Authors: Stringer J R , Holland L E , Swanstrom R I , Pivo K , Wagner E K
Published: January 2020

We have quantitatively analyzed the size and amount of herpes simplex virus (HSV)-specific RNA synthesized in HeLa cells using DNA and RNA excess hybridization. At 2 h after infection (early), transcripts from 20% of the total HSV DNA are present on polyribosomes as poly(A+) RNA. At this time, viral poly(A+) RNA comprises 60 to 75% of the newly synthesized poly(a+) mRNA on polyribosomes. By 6 h after infection (late), poly(A+) HSV RNA transcribed from 35 to 40% of the viral DNA is found on polyribosomes. These viral poly(A+) transcripts comprised as much as 90% of newly synthesized poly(A+) mRNA and are measurably larger than viral poly(A+) transcripts isolated early. Some but not all of this size difference is due to the fact that the poly(A) tails on early transcripts are shorter than those found on transcripts made late. Even late after infection, a small but readily measurable amount of cellular poly(A+) RNA is still being made and entering polyribosome complexes. In the nucleus, late after infection, poly(A+) HSV RNA is complementary to 50% of the total HSV DNA. Both early and late after infection, total nuclear viral transcripts are, on the average, larger than viral transcripts found on polyribosomes; however, nuclear HSV poly(A+) RNA is not measureably larger than the corresponding cytoplasmic viral poly(A+) sequences at either time. A major portion (30 to 40%) of the polyribosomal HSV RNA made either early or late after infection is not polyadenylated. This HSV poly (A-) RNA is transcribed from the same sequences as HSV poly(A+) RNA but, when labeled and isolated either early or late after infection, both nuclear and polyribosomal viral poly(A-) RNA molecules sediment faster in sucrose-formaldehyde gradients than their polyadenylated counterparts.
Effect of antibiotics on certain aspects of bacteriophage SP-15 development in Bacillus subtilis W23
Authors: Dosmar M , Markewych O , Witmer H
Published: January 2020

Bacillus subtilis W23 was infected with bacteriophage SP-15. Two waves of phage-specific RNA synthesis were observed. Wave I was prereplicative, and wave II was coincident with replication of the viral genome. To determine the temporal appearance of general classes of phage-coded messengers and proteins, we studied the dependence of lysozyme synthesis, phage production, and DNA synthesis on time of addition of transcriptional and translational inhibitors. Lysozyme synthesis started to become refractile to a variety of transcriptional inhibitors (rifampin, streptolydigin, and actinomycin D) between 20 and 22 min postinfection and was completely refractile by 30 min. Nevertheless, functional enzyme did not appear until 45 to 47 min postinfection; lysozyme was maximal by 65 min. Rna isolated from SP-15 phage-infected cells was used to program the cell-free synthesis of lysozyme. The messenger was synthesized exclusively between 20 and 30 min postinfection. Lysozyme messengers were stable. The data imply that lysozyme messengers were present 52 min prior to their translation. Progeny virus formation remained sensitive to transcriptional inhibitors until 40 to 50 min postinfection, and sensitivity to chloramphenicol lasted 65 min. The first progeny viruses appeared at 75 min. Again, an unusually long lag between completion of functional messengers and their translation was evident. The aforementioned data indicated that transcription of lysozyme messengers and, at least, some messengers, whose products are essential for phage production, are uniquely associated with waves I and II of RNA synthesis, respectively. However, messengers whose products are essential for normal amounts of DNA synthesis were apparently synthesized during both waves; transcription of these messengers was transiently repressed (using the term broadly) between 30 and 40 min postinfection. Judging from the dependence of DNA synthesis on time of chloramphenicol addition, proteins essential for normal amounts of DNA synthesis were also synthesized in two discrete waves, each yielding sufficient protein for half-maximal levels of DNA synthesis. An hiatus in the synthesis of the proteins in question was evident between 45 and 65 min postinfection; evidence cited in this paper indicates that this hiatus did not result from messenger depletion, which, in turn, implied some type of translational-level control. This latter conclusion is substantiated by the lysozyme synthesis that occurred during the same interval when synthesis of certain proteins for DNA replication was transiently repressed.
Natural immunity in mice to the envelope glycoprotein of endogenous ecotropic type C viruses: neutralization of virus infectivity
Authors: Ihle J N , Lazar B
Published: January 2020

The ability of naturally immune mouse sera to neutralize ecotropic AKR murine leukemia virus (MuLV) was examined by using unfrozen virus preparations harvested for 1 h. In this assay several mouse sera significantly and consistently neutralized MuLV infectivity. The ability of these sera to neutralize was correlated with the presence of antibodies against MuLV detectable in a radioimmune precipitation assay using radioactively labeled intact virions. This neutralization was specific, in that either N- or B-tropic viruses, but not Friend MuLV, were neutralized. In addition, neutralization could be abrogated with purified AKR MuLV gp71 at concentrations that do not interfere with virus infectivity but could not be abrogated with Rauscher MuLV gp71. Neutralizing activity could be removed by absorption with intact AKR MuLV, but not by absorption with Friend MuLV, a BALB/c xenotropic virus, or with NZB xenotropic virus. All the neutralizing activity of (B6C3)F1 mouse sera was associated with the immunoglobulin G fraction.
Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein
Authors: Luka J , Siegert W , Klein G
Published: January 2020

The Eptstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solubilized from isolate nuclei of two EBV-transformed cell lines- Raji and AW-Ramos, by high-salt treatment. Its DNA-binding properties were studied by DNA-cellulose chromatography and a 51Cr release complement fixation assay. EBNA binds to both double-stranded and single-stranded calf thymus DNA, showing a higher affinity to double-stranded DNA. There was no detectable difference in the DNA binding of EBNA prepared from Raji and AW-Ramos cells.
Rabies virus protein synthesis in infected BHK-21 cells
Authors: Madore H P , England J M
Published: January 2020

Rabies virus specific polypeptide synthesis was examined under hypertonic conditions, which selectively inhibit cellular protein synthesis. The rabies virus proteins (L, G, N, M1, M2) were synthesized throughout the course of infection, with little change in their relative rates of synthesis. The rates of synthesis of the G and M1 polypeptides were more sensitive to increasing osmolarity than those of the L, N, and M2 polypeptides. Extrapolation to isotonicity of the results obtained under hypertonic conditions indicated that the molar ratios of the polypeptides synthesized under normal conditions were 0.4 (L), 64 (G), 100 (N), 75 (M1) and 35 (M2). A high-molecular-weight polypeptide (190,000), designated polypeptide L, was repeatedly detected both in infected cells and in extracellular virus. The estimated number of L polypeptide molecules per virion was 33. The synthesis of a viral glycoprotein precursor, designated gp78, , preceded the appearance of the mature viral glycoprotein in infected cells labeled with [3H]glucosamine under isotonic conditions. In cells labeled under hypertonic conditions, little or no mature viral glycoprotein was detected, but a virus-specific glycoprotein with an electrophoretic mobility similar to that of gp78 was observed. This glycoprotein could be chased into mature viral glycoprotein when the hypertonic conditions were made isotonic. These results suggest that a reversible block of viral glycoprotein synthesis occurs under hypertonic conditions.
Polyacrylamide gel electrophoretic analysis of herpes simplex virus type 1 immunoprecipitates obtained by quantitative immunoelectrophoresis in antibody-containing agarose gel
Authors: Norrild B , Vestergaard B F
Published: January 2020

Crossed immunoelectrophoresis was used to characterize herpes simplex virus type 1 (HSV-1) antigens produced by infected HEp-2 cells. We report on a method for analyzing the polypeptide content in individual antigen-antibody precipitates eluted from the second-dimensional agarose gel. Four glycoprotein antigens of HSV-1, Ag-8, Ag-11, Ag-6, and Ag-3, were isolated and analyzed for polypeptide content. The molecular weights of the polypeptides are presented.
Antigens of Pichinde virus I. Relationship of soluble antigens derived from infected BHK-21 cells to the structural components of the virion
Authors: Buchmeier M J , Gee S R , Rawls W E
Published: January 2020

Antigens detected by the complement-fixation (CF) test were prepared from BHK-21 cells infected with Pichinde virus.The preparations contained two antigens demonstrable by immunodiffusion. The antigen present in abundance was heat stable, Pronase resistant, and had a molecular weight of 20,000 to 30,000 as estimated by gel filtration. Polyacrylamide gel electrophoresis of purified antigen demonstrated two low-molecular-weight polypeptides. An identical antigenic determinant was found by disrupting purified virus with Nonidet P-40; however, none of the viral polypeptides co-migrated with the polypeptides derived from purified CF antigen. Pronase digestion of disrupted virus did not alter antigenicity but degraded the viral peptides to sizes similar to those associated with the major CF antigen. These observations suggest that the major CF antigen of Pichnide virus is a cleavage product of the structural proteins of the virus.
Autoradiographic method for detection of lactate dehydrogenase-elevating virus-infected cells in primary mouse macrophage cultures
Authors: Tong S L , Stueckemann J , Plagemann P G
Published: January 2020

Peritoneal cells from starch-injected Swiss mice were propagated in plastic petri dishes and on cover slips in a mouse L-cell-conditioned medium for 12 to 24 h and then infected with various multiplicities of lactate dehydrogenase-elevating virus (LDV). Over 95% of the cells in these cultures phagocytosed latex particles and were, therefore, considered macrophages. Infected and mock infected macrophage cultures were supplemented with [3H]uridine at various times after infection and with actinomycin D 30 min before addition of the [3H]uridine. After 1 or 2 h of further incubation, plate cultures were analyzed for LDV-specific RNA, and cover slip cultures were investigated by autoradiography. Other cultures were labeled in the absence of actinomycin D, and the culture fluid was analyzed for labeled LDV. There was a good correlation between the production of LDV-specific RNA and LDV and the number of heavily labeled cells in these cultures. The labeled cells in these cultures. The labeled cells, therefore, were equated with productively infected cells. Only a maximum of about 20% of the macrophages, however, became heavily labeled regardless of the multiplicity of infection or the time, after infection, at which the cells were exposed to [3H]uridine. Only background labeling was observed in the remainder of the cells and in mock-infected cells treated with actinomycin D. The highest proportion of labeled cells was observed when the cells were infected with a multiplicity of infection of about 2,000 mouse infectious units per cell and labeled from 6 to 8 h after infection. Thereafter, the proportion of productively infected cells decreased progressively, concomitant with a decrease in the amounts of viral specific RNA and of LDV produced by the cultures. The results indicate that the majority of the macrophages in primary macrophage cultures do not support LDV replication. Their nonpermissiveness may depend on the physiological state of the cells or reflect the presence of subpopulations of macrophages, but no morphological differences between productively infected an uninfected cells were detectable.
Virus-secific transcription in 3T3 cells transformed by the ts-a mutant of polyoma virus
Authors: Bacheler L T
Published: January 2020

Virus-specific RNA transcription has been measured in 3T3 cells transformed by the ts-a mutant of polyoma virus by RNA-excess hybridization to the separated strands of polyoma DNA. In two cloned sublines maintained at 39 degrees C, the nonpermissive temperature for the A gene function, RNA transcripts of a large fraction of the "early" strand are detected in both nuclear and cytoplasmic RNA fractions, but no "late" strand transcription is detected. Temperature shift to 31.5 degrees C, the permissive temperature, induces viral DNA replication and virus production accompanied by late strand transcription. In two independently derived noninducible cell lines, L strand transcription is never observed, even after cultivation at the permissive temperature. A smaller fraction of the E strand is transcribed in each noninducible cell than in its inducible parent, and this difference is further characterized as a lack of transcripts of portions of HpaII restriction endonuclease fragments 2 and 6.
Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides
Authors: Okasinski G F , Velicer L F
Published: January 2020

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.
Characterization of an RNA-directed DNA polymerase found in association with murine intracytoplasmic A-particles
Authors: Kohno M , Tanaka H
Published: January 2020

An RNA-directed DNA polymerase was found to be associated with intracytoplasmic A-particles from DBA/2 mouse leukemia cells. The enzyme activity was detected after disrupting the purified particles with 2 M NaCl-20 mM dithiothreitol. The presence of a divalent cation and all four deoxyribonucleoside triphosphates was essential for this enzyme activity. The enzyme had a clear preference for Mg2+ over Mn2+. Cesium sulfate isopycnic gradient centrifugation of the DNA product synthesized in the actinomycin D-containing reaction revealed the presence of DNA-RNA hybrid. Furthermore, the purified DNA product was found to hybridize with RNA isolated from A-particles. These observations strongly indicate that the endogenous A-particle RNA serves as the template for the DNA polymerase.
Fidelity of adenovirus RNA transcription in isolated HeLa cell nuclei
Authors: Vennström B , Philipson L
Published: January 2020

An in vitro nuclear system from adenovirus type 2-infected cells was developed to study transcription of viral RNA. Nuclei isolated from adenovirus-infected HeLa cells late in the infectious cycle synthesized in vitro only RNA from the r-strand of adenovirus DNA. Around 15% of the virus-specific RNA in isolated nuclei was polyadenylated. Short pulse labeling of nascent RNA followbd by hybrization of size-fractionated RNA to specific restriction endonuclease fragments of the genome suggested that the origin(s) for transcription is located on the r-strand in the left 30% of the adenovirus 2 genome at late times in the infectious cycle. Pulse-chase experiments were used to estimate the elongation rate of adenovirus high-molecular-weight RNA in isolated nuclei. An elongation of a least six nucleotides per second was observed in vitro. Viral RNA synthesis in the vitro nuclei showed several similarities to the in vivo system late in the infectious cycle.
Effects of temperature and host cell genetic characteristics on the replication of the lipid-containing bacteriophage PR4 in Escherichia coli
Authors: Sands J A , Auperin D
Published: January 2020

The lipid-containing bacteriophage PR4 is of special intest because it can replicate in various gram-negative bacteria, including Escherichia coli, that carry one of a group of drug resistance plasmids. PR4 grown in E. coli strain PS2R contains about 10% lipid by weight, with the negatively charged phospholipid phosphatidylglycerol being the most abundant lipid in the virion. We now report the following. (i) PR4 attaches to E. coli with an attachment rate constant of Ka approximately 6.2 X 10(-10) ml/min, which is about twice that of the enveloped phage phi6 (to Pseudomonas phaseolicola), but a factor of 5 less than that of phage PM2 (to Pseudomonas BAL-31). (ii) Use of an E. coli glycerol auxotroph indicated that a normal amount of PR4 replication occurs only if glycerol starvation (inhibition of all phospholipid synthesis) begins no earlier than about halfway through the lytic cycle. (iii) Use of an E. coli fatty acid synthesis temperature-sensitive mutant and an E. coli phosphatidylethanolamine synthesis temperature-sensitive mutant indicate that PR4 replication can occur in the absence of either normal fatty acid synthesis or normal phospholipid synthesis if the infection takes place prior to the termination of overall cell growth and the onset of cell death, (iv) Whereas PR4 burst size in nutrient media at 30 degrees C to 42%C is about 40, the burst size at 20 degrees C is less than 3, Temperature-shift experiments show that the temperature late in infection determines the burst size.
Increase in lipid fluidity of cellular membranes induced by adsorption of RNA and DNA virions
Authors: Levanon A , Kohn A , Inbar M
Published: January 2020

Changes in the dynamic behavior of membrane lipids of mammalian cells induced by adsorption of animal viruses were quantitatively monitored by fluorescence polarization analysis with the aid of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene embedded in the surface membrane lipid core of intact cells. Adsorption of encephalomyocarditis, West Nile, and polyoma viruses to hamster (baby hamster kidney) and mouse (3T3) cells is accompanied by a rapid and significant increase in the degree of fluidity of membrane lipids of the infected cells. These changes in membrane fluidity, which are virus dose dependent, are inhibited by low temperature and by treatment of the cells before-hand with compounds known to block viral receptors on the cell surface. It is suggested that increase in membrane lipid fluidity, induced by the adsorption of virions, is an early event in the process of cell-virus interactions.
Adenovirus type 2 coded single-stranded DNA binding protein: in vivo phosphorylation and modification
Authors: Jeng Y H , Wold W S , Sugawara K , Gilead Z , Green M
Published: January 2020

The adenovirus type 2-coded single-stranded DNA binding protein (DBP) was shown to be a phosphoprotein and to exist in at least two forms that differ in mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After a 30-min pulse with [35S]methionine or 32PO4, 35S- or 32P-labeled DBP had a nominal molecular weight of 74,000 whereas after a 30-min label followed by a 20-h chase, 35S- and 32P-labeled DBP had a nominal molecular weight of 77,000. Both large and small forms of 35S- and 32P-labeled DBP bound to single-stranded DNA-cellulose columns and were eluted by 0.4 to 0.6 M NaCl; both forms also were immunoprecipitated by antiserum against adenovirus type 1-simian virus 40-induced tumor cells (this antiserum contains antibodies against DBP) and by monospecific antiserum against 95 to 99% purified DBP. With highly purified 32P-DBP labeled 7 to 10 h postinfection, it was shown that the 32P radioactivity was firmly associated with protein material (i.e., not contaminating nucleic acids or phospholipids) and had properties expected of a phosphoester of an amino acid; paper electrophoresis of acid hydrolysates of this preparation identified phosphoserine but not phosphothreonine. Phosphoserine but not phosphothreonine was also identified in acid hydrolysates of another preparation of 32P-DBP labeled for 30 min, chased for 20 h, and then immunoprecipitated by adenovirus type 1-simian virus 40 antiserum.
Transcriptional termination at the end of the early region of bacteriophages T3 and T7 is not affected by polarity suppressors
Authors: Kiefer M , Neff N , Chamberlin M J
Published: January 2020

Bacterial mutations of known rho genotype (psu-1 through psu-4) were shown to have no effect on transcriptional termination at the termination site at the end of the early region of T3 and T7 DNAs. Transcriptional termination was assayed in these bacterial mutants by comparing the patterns of late protein production produced by T3 and T7 amber I bacteriophage infection. All the rho (psu) mutants tested showed the same pattern of late protein production as the wild-type strain (rho+ or psu+) after T3 or T7 amber I infection. The presence of the mutant rho allele during bacteriophage infection, therefore, did not allow the host RNA polymerase to read through the terminator located at approximately 20% of the T3 and T7 genomes. These results suggest that rho factor may not be involved in reading of the T3 and T7 20% terminators in vivo.
Regulation of herpesvirus macromolecular synthesis. VI. Synthesis and modification of viral polypeptides in enucleated cells
Authors: Fenwick M , Roizman B
Published: January 2020

Cells were enucleated with cytochalasin B after infection with herpes simplex virus 1. When protein synthesis was blocked by cycloheximide from the time of infection, mRNA for viral alpha-infected cell polypeptides (ICP) 4, 0, and 27 accumulated in the cytoplasm and was expressed after the removal of both drug and nucleus. A host protein, ICP 22, whose synthesis is stimulated in intact cells, was not made, and viral protein ICP4, which is normally modified to a form that migrates more slowly in polyacrylamide gels, was not modified in the absence of the nucleus. After enucleation at 2 h postinfection, a number of viral beta and gamma proteins continued to be made, starting at 20 to 25% of the normal rates and declining with a half-time of about 2 h. The synthesis of ICP 4 declined more rapidly, suggesting that it is switched off in the cytoplasm.
Kinetics of assembly of a parvovirus, minute virus of mice, in synchronized rat brain cells
Authors: Richards R , Linser P , Armentrout R W
Published: January 2020

The rates of assembly of the three classes of particles of minute virus of mice were examined in synchronized rat brain cells by a combination of electron microscopy and biochemical techniques. We observed a burst of virus assembly beginning about 8 h after the end of cellular S phase. Labeled thymidine incorporated into the 1.46 g/cm3 class of full virus particles was transferred almost quantitatively to the 1.42 g/cm3 class. The 1.46 g/cm3 virus appeared to be an immediate precursor to the 1.42 g/cm3 class. Conversion of the 1.46 density virus to the 1.42 density particles was observed at the time of virus assembly. The processing was rapid and occurred primarily in the nucleus. Infected cells did not contain significant pools of viral DNA in a form that could be encapsulated in the absence of DNA synthesis. The role of the empty virus capsids in the assembly process is discussed.