bluuebunny/crossref_metadata_2025 huggingface dataset:
mRNA from the transforming segment of the adenovirus 2 genome in productively infected and transformed cells
Authors: Chinnadurai G , Rho H M , Horton R B , Green M
Published: January 2020

We have identified two mRNA species transcribed from the adenovirus 2 genome section (HindIII-G fragment) believed to harbor genes for initiation and maintenance of cell transformation. The HindIII-G fragment occupies the left 7.5% of the genome and is transcribed from left to right [poly(U:G) r strand]. Poly(A)-terminated labeled mRNA was isolated from polyribosomes of adenovirus 2 early infected KB cells and from the transformed cell line 8617, hybridization purified using the HindIII-G fragment, and electrophoresed on formamide-polyacrylamide gels. Viral mRNA's of 24S (1.2 X 10(6) daltons) and 14S (4.5 X 10(5) daltons) were isolated from early infected cells and of 22S (1.0 X 10(6) daltons) and 14S from 8617 cells. Hybridization competition indicated that HindIII-G-specific mRNA was present in the polysomes at one-sixth the concentration late after infection as compared with early, indicating that the proteins coded by the transforming segment may be synthesized at reduced amounts during late stages. Only 1/10 the amount of RNA labeled late annealed to the G fragment as compared with that labeled early (per weight of RNA). Thus, synthesis of transforming gene mRNA is probably "turned off" late after infection. Both 24S (22S) and 14S mRNA's from infected and 8617 cells were complementary to the Hpa I-E fragment (left 4.1% of genome). The Hpa I-E fragment is too small to encode 24S and 14S species, which implies that the 5'-terminal regions of both species are coded by the same DNA sequences.
Exonuclease associated with bacteriophage T5-Induced DNA polymerase
Authors: Das S K , Fujimura R K
Published: January 2020

T-5-induced DNA polymerase has been shown to possess a 3' leads to 5'-exonucleolytic activity. The exonuclease acts on both native and denatured DNA, but the apparent rate of degradation of denatured DNA is about five times faster than that for native DNA. The enzyme appears to act only on 3'-OH ends and produces mainly 5'-dNMP's. Like polymerase activity, exonuclease activity shows a pH optimum around 8.6. Mg2+, dithiothreitol, and N-ethylmaleimide had identical effects on both the activities. Nicked DNA was almost totally protected from exonuclease action under synthetic conditions, i.e., in the presence of 4dNTP's. Denatured DNA was partly degraded in the early phase of incubation with 4dNTP's, presumably due to unhybridized tails at the 3'-OH primer ends. However, the exonuclease activity was operative in both cases under synthetic conditions, as evidenced by template-dependent conversion of [3H]dTTP to [3H]dTMP.
Origin of polyoma virus-associated endonuclease
Authors: Rouget P , Parodi A , Blangy D , Cuzin F
Published: January 2020

Polyoma virus particles purified from infected cells, but not from the culture medium, exhibited an endonuclease activity distinct from the serum contaminant recently described. This endonuclease cleaved form I polyoma DNA once only per molecule, at one of three possible sites corresponding to the known adenosine-ribosylthymine-rich regions of the molecule.
Genome expression and mRNA maturation at late stages of productive adenovirus type 2 infection
Authors: Wold W S , Green M , Brackmann K H , Cartas M A , Devine C
Published: January 2020

RNA from adenovirus 2-infected KB cells was annealed in liquid with RNA in vast excess to viral heavy (l) and light (r) 32P-labeled DNA strands. Hybridization kinetics were analyzed by computer to estimate the number of viral RNA abundance classes, their relative concentrations, and the fraction of each DNA strand from which they originated. Early whole cell RNA extracted 5 h postinfection annealed rapidly to 10 to 15% of l and r strands and then slowly to final values of 60 and 40% of l and r strands. By 9 h postinfection the expression of late genes was apparent and whole cell RNA annealed to 20 and 75% of l and r strands. Whole cell RNA extracted between 12 and 36 h postinfection annealed to 7 to 15% and 75 to 90% of l and r strands. Late nuclear RNA hybridized to 10 and 90% of l and r strands, and late polyribosomal RNA hybridized to 20 and 75% of l and r strands. Based upon kinetic analyses, we estimate that mRNA synthesized exclusively during late stages arises from about 6 to 8% and 45 to 49% of l and r strands. This assumes that the early class I mRNA (in low concentration late) originates from 8 to 10% and 6 to 10% of l and r strands and that early class II mRNA (in high concentration late) is derived from 2% and 8 to 13% of l and r strands. Mixing experiments indicated that early mRNA is a subset of RNA extracted from polyribosomes late after infection and that late nuclear RNA contains sequences complementary to early l strand class I nRNA. RNA-RNA hybrids were isolated from late mRNA containing sequences from 60% of l and r strands, but it is not known when these were synthesized, and therefore whether complementary RNA transcripts are synthesized late after infection, as they are known to be synthesized early. These results demonstrate that portions of the genome are transcribed into RNA sequences that remain confined to the nucleus and are not exported to polyribosomes as mRNA.
RNA synthesis in cells infected with herpes simple virus. XIII. Differences in the methylation patterns of viral RNA during the reproductive cycle
Authors: Bartkoski M , Roizman B
Published: January 2020

Herpex simplex virus 1 (HSV-1) RNA labeled with with [methyl-3H] methionine at various times during the infectious cycle and purified by hybridization to viral DNA was analyzed for the presence of methylated nucleotides. The data indicate the following. (i) RNA labeled from 0 to 14 h postinfection and accumulating in the cytoplasm contained internal base-methylated nucleotides and terminal oligonucleotides consistent with the structure 7mG(5')ppp-(5')XmpYmpNp. Similar methylated nucleotides and oligonucleotides were also found in viral RNA accumulating in the cytoplasm of cells treated with cycloheximide from the time of infection. Previous studies (M. Kozak and B. Roizman, 1974) have shown that, whereas the RNA accumulating in the 14-h infected cells contains all of the sequences functioning as mRNA throughout infection, the RNA accumulating in the cytoplasm of cycloheximide-treated cells is associated with polyribosomes synthesizing the earliest (alpha) group of polypeptides specified by the virus. (ii) Cytoplasmic viral RNA from cells labeled 11 to 14 h postinfection as well as the total adenylated RNA in the cytoplasm and polyribosomes labeled in the same fashion contained the terminal oligonucleotide but not the internal base-methylated nucleotide.
Analysis of the envelope of Rauscher murine oncornavirus: in vitro labeling of glycopeptides
Authors: McLellan W L , August J T
Published: January 2020

The identity and localization of the oligosaccharides of Rauscher murine type C viral glycoproteins have been examined by techniques of in vitro labeling. Terminal sialic acid was labeled with tritium by borohydride reduction after selective periodate oxidation, and galactose was labeled by borohydride reduction after specific enzymatic oxidation of the nonreducing terminal of the sugar. The results were compared with those of protein surface labeling with pyridoxal phosphate or lactoperoxidase catalyzed radioiodination. Examination of the labeled reaction products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that in every case the major component labeled was a glycoprotein of about 70,000 daltons. The identity of this glycoprotein as the virion envelope component was confirmed by immunoprecipitation with mono-specific antiserum prepared against purified Rauscher virus glycopeptides of 69,000 and 71,000 daltons. No other protein or glycoprotein on the surface of the virion was detected, and disruption of virions-before labeling did not reveal additional distinctive glycoproteins. There was minor labeling of sugar residues of other components, but these remain to be characterized and are not now identified as other viral proteins. Studies of the structural organization of virion proteins using the cross-linking reagent methyl-4-mercaptobutyrimidate showed only linkage of the virion envelope or core proteins to themselves. These results indicate that most, if not all, of the oligosaccharides at the surface of Rauscher virus are entities of the 69,000- and 71,000-dalton glycopeptides and that they contain a terminal sialic acid and galactose and a subterminal galactose.
Increased length of DNA made by virions of murine leukemia virus at limiting magnesium ion concentration
Authors: Rothenberg E , Baltimore D
Published: January 2020

Conditions have been developed for reverse transcription by detergent-disrupted virions of Moloney murine leukemia virus which permit synthesis of molecules that appear to be complete transcripts of the 35S RNA subunits. At limiting Mg2+ concentration, DNA is synthesized in good yield, up to a maximum size of about 2.4 X 10(6) daltons. DNA larger than 2 X 10(6) daltons, taken from alkaline sucrose gradients, has no detectable self-complementarity and was protected from digestion by S1 nuclease to an extent of 90% by annealing to 70S RNA. All size classes of DNA made in these reactions are primed with RNA, because all are initiated with a pApdAjunction. To produce such long molecules, it is necessary to keep the concentration of Mg2+ in the reaction mixture below the total concentration of deoxyribonucleoside triphosphates. Under these conditions, degradation of the RNA template is minimized. The rate of DNA synthesis is also slowed by 30 to 50%, but products longer than 5,000 nucleotides, which are not found otherwise, are completed between 3 and 6h of reaction.
Cell-free synthesis of polyoma virus capsid proteins VP1 and VP2
Authors: Wheeler T , Bayley S T , Harvey R , Crawford L V , Smith A E
Published: January 2020

Polyadenylated RNA isolated from the cytoplasm of mouse 3T6 cells 28 h after infection with polyoma virus has been isolated and translated in vitro. Polyoma capsid proteins VP1 and VP2 have been identified in the cell-free product by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide fingerprinting. Polyoma mRNA species have been isolated by preparative hybridization to purified viral DNA immobilized on cellulose nitrate filters and shown to code for both VP1 and VP2. These experiments establish conditions for the isolation of late polyoma mRNA and the cell-free synthesis of polyoma capsid proteins and indicate that the active mRNA species are at least partially virus coded.
Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA
Authors: Plotch S J , Krug R M
Published: January 2020

The influenza virion transcriptase is capable of synthesizing in vitro complementary RNA (cRNA) that is similar in several characteristics to the cRNA synthesized in the infected cell, which is the viral mRNA. Most of the in vitro cRNA is large (approximately 2.5 X 10(5) to 10(6) daltons), similar in size to in vivo cRNA. The in vitro transcripts initiate in adenosine (A) or guanosine (G) at the 5' end, as also appears to be the case with in vivo cRNA (R.M. Krug et al., 1976). The in vitro transcripts contain covalently linked polyadenylate [poly(A)] sequences, which are longer and more heterogeneous than the poly(A) sequences found on in vivo cRNA. The synthesis in vitro of cRNA with these characteristics requires both the proper divalent cation, Mg2+, and a specific dinulceside monophosphage (DNMP), ApG or GpG. These DNMPs stimulate cRNA synthesis about 100-fold in the presence of Mg2+ and act as primers to initiate RNA chains, as demonstrated by the fact that the 5'-phosphorylated derivatives of these DNMP's, 32pApG or 32pGpG, are incroporated at the 5' end of the product RNA. The RNA synthesized in vitro differs from in vivo cRNA in that neither capping nor methylation of the in vitro transcripts has been detected. The virion does contain a methylase activity, as shown by its ability to methylate exogenous methyl-deficient Escherichia coli tRNA.
Identification of simian virus 40 protein A
Authors: Rundell K , Collins J K , Tegtmeyer P , Ozer H L , Lai C J , Nathans D
Published: January 2020

A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.
Identification of the site of interruption in relaxed circles producing during bacteriophage lambda DNA circle replication
Authors: Reuben R C , Skalka A
Published: January 2020

The DNA that accumulates in the lambda infection restricted to the early (circular) stage of replication consists of approximately two-thirds covalently closed circles and one-third relaxed circles bearing a single interruption in either strand of the duplex. The latter molecules are presumed to be a unique class in that the interruption is not repairable by DNA polymerase and ligase. Preferential radioisotopic labeling of the region immediately adjacent to the interruption, followed by hybridization to sheared fragments of the lambda chromosome with varying guanine plus cytosine content, suggested that the nick resides at the position of the mature molecular ends of the lambda chromosome. Digestion of the labeled molecules with restriction enzymes and reconstruction experiments in which Hershey circles were generated by annealing of interrupted strands isolated from the relaxed circles support this interpretation. The results indicate that the relaxed circles consist of a population containing one interruption in either of the two strands of the duplex jointly representing the two "nicks" contained in Hershey circles (in which the cohesive ends are annealed). These molecules could result from the inability of the maturation function to make the required staggered endonucleolytic cuts when the DNA substrate is a monomeric circle rather than a multimeric linear molecule. Alternatively, this interruption could be the result of an endonucleolytic cutting event critical to DNA replication.
Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus
Authors: Knipe D M , Baltimore D , Lodish H F
Published: January 2020

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.
Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168
Authors: Jacobson E D , Landman O E
Published: January 2020

Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168 is described. The number of binding sites on bacilli and protoplasts is determined for each phage. Bacilli and protoplasts possess roughly the same number of sites per unit area for phi 29, i.e., approximately 700 sites per bacillus. There are also approximately 700 sites per bacillus for 22a, but only about one-third as many sites per unit area on the protoplast surface. A model for phi 29 adsorption is proposed.
Isolation of a variant of human adenovirus serotype 2 that multiplies efficiently on monkey cells
Authors: Klessig D F
Published: January 2020

A variant of adenovirus serotype 2 (Ad2) that overcomes the block to multiplication of the wild-type Ad2 on monkey cells is described. The variant was selected, after nitrous acid mutagenesis, by sequential passage on monkey cells. This variant forms plaques with similar efficiency on human and monkey cells cells. The kinetics of its growth and its burst size on human and monkey cells are similar. These growth properties are similar to those found for wild-type Ad2 on monkey cells when the block is overcome by coinfection with simian virus 40.
Amount and distribution of virus-specific sequences in giant RNA molecules isolated from polyoma-infected mouse kidney cells
Authors: Lev Z , Manor H
Published: January 2020

A two-step hybridization with polyoma DNA was used to study the composition of giant RNA molecules synthesized in mouse kidney cells late in productive infection by polyoma virus. Giant molecules longer than a complete transcript of the polyoma genome were purified from cells that had been pulse-labeled for 30 min with [3H]uridine and annealed, under mild conditions (50% formamide, 37 degrees C), with polyoma DNA loaded on nitrocellulose filters. Hybridized RNA (6 to 7% of the entire population of 3H-labeled molecules and up to 15% of the molecules containing polyadenylic acid [poly(A)]] was eluted and annealed a second time with polyoma DNA under more stringent conditions. In this second step, 75% of the 3H-labeled RNA formed an RNase-resistant hybrid. Under the same conditions, complementary RNA hybridized with polyoma DNA to a maximal extent of 80%. Since the difference between 75 and 80% is within the experimental error of the hybridization assay, it is inferred that the giant molecules selected by the first hybridization may consist entirely of virus-specific sequences or contain, at the most, a minor fraction of nonviral sequences. To examine the possibility that such nonviral sequences are clustered at the 3'-terminus of these molecules, poly(A)+ giant RNA, which had not been preselected by hybridization with polyoma DNA, was fragmented by a limited alkaline hydrolysis. Fragments linked to the poly(A) segment were separated from the rest of the cleavage products. A one-step hybridization with polyoma DNA revealed that both fractions contain 8 to 10% of virus-specific sequences. These results indicate that the 3'-termini of the poly(A)+ polyoma-specific giant RNA molecules consist of viral rather than nonviral sequences.
Isolation and comparison of murine leukemia virus-related glycoproteins from AKR and New Zealand mice
Authors: Kennel S J
Published: January 2020

The major glycoprotein (gp70) of murine leukemia virus occurs free of virus in the serum and body fluids of certain strains of mice. These glycoproteins were isolated from New Zealand Black mouse (NZB) ascites fluid and from AKR and New Zealand White mouse (NZW) serum by immunoaffinity chromatography and were compared by immunological tests and peptide mapping. Glycoproteins gp70-NZB and gp70-NZW were indistinguishable by all criteria tested and were more closely related to gp70 from Moloney leukemia virus than was gp70-AKR.
Inhibition of bacteriophage M13 and phix174 duplex DNA replication and single-strand synthesis in temperature-sensitive dnaZ mutants of Escherichia coli
Authors: Haldenwang W G , Walker J R
Published: January 2020

A functional dnaZ product, known to be essential for host DNA polymerization and for the synthesis of M13 and phiX174 parental replicative-form (RF) DNA, is required also for RF replication and single-strand synthesis by both of these phages. All three stages of M13 and phiX174DNA replication (parental RF formation, RF replication, and single-strand synthesis) are inhibited in dnazts mutants at elevated temperatures. In addition, the thermolabile step in M13 parental RF formation appears to occur after RNA priming;i.e., the synthesis of M13 RF DNA proceeded when a dnaZts mutant, infected at a nonpermissive temperature, was transferred to a permissive temperature in the presence of rifampin.
Synthesis of Newcastle disease virus polypeptides in a wheat germ cell-free system
Authors: Clinkscales C W , Bratt M A , Morrison T G
Published: January 2020

We have isolated 18S RNA from cytoplasmic extracts of Newcastle disease virus-infected Chinese hamster ovary cells and tested its ability to direct protein synthesis in extracts derived from wheat germ. The products of the cell-free reaction directed by this RNA contain polypeptides that comigrate with NP, M,F, and 47K roteins from virions. In addition, the products contain a polypeptide (67K) that migrates on polyacrylamide gels slightly faster than the HN protein from virions. Tryptic peptide analysis of the cell-free products and proteins from virions confirms their identity.
Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue
Authors: Cooper G M , Castellot S B
Published: January 2020

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.
Circular Epstein-Barr virus genomes of reduced size in a human lymphoid cell line of infectious mononucleosis origin
Authors: Adams A , Bjursell G , Kaschka-Dierich C , Lindahl T
Published: January 2020

Circular Epstein-Barr virus (EBV) DNA molecules have been purified and characterized from a human lymphoid cell line derived from a case of heterophile antibody-positive, blood transfusion-induced infectious mononucleosis, 883L. The circular EBV DNA in three cell lines obtained by transformation of human umbilical cord blood leukocytes with a strain of EBV originally derived from 883L was also studied. As estimated from sedimentation velocity data and electron microscopy, the circular EBV DNA molecules are 10 to 15% smaller than either the circular EBV DNA previously found intracellularly in several other types of EBV-transformed cells or the linear EBV DNA present extracellularly in virus particles. In addition, the EBV-transformed cord blood cell lines studied here differed from other EBV-transformed cells in that integrated virus DNA sequences could not be detected.
Synthesis of Sindbis virus nonstructural polypeptides in chicken embryo fibroblasts
Authors: Brzeski H , Kennedy S I
Published: January 2020

The identification of eight previously undescribed polypeptides in chicken embryo cells infected with Sindbis virus is reported. Seven of these polypeptides were distinguishable from the virus structural polypeptides and their precursors by their molecular weights and tryptic peptide maps. The eighth was closely related to pE2 (Schlesinger and Schlesinger, 1973), a precursor to one of the virus particle glycoproteins. Pulse-chase experiments and the use of an inhibitor of proteolytic cleavage allowed a division of the seven nonstructural (NS) polypeptides into three stable end products (NS p89, NS p82, and NS p60) and four precursors (p230, p215, p150, and p76). The labeling kinetics after synchronous initiation of translation indicated that synthesis of the NS polypeptides started at a single site and showed that the order of the genes coding for the NS polypeptides was (5' leads to 3') NS p60, NS p89, and NS p82. Short-pulse experiments under conditions of both synchronized and nonsynchronized translation suggested that cleavage of the primary translation product of the NS genes occurred only after its synthesis was completed and that the first cleavage removed the C-terminal polypeptide. From these and other experiments, we propose a detailed scheme for the synthesis and processing of Sindbis virus NS polypeptides.
Specificity of initiation of transcription of simian virus 40 DNA I by Escherichia coli RNA polymerase: identification and localization of five sites for initiation with [gamma-32P]ATP
Authors: Lebowitz P , Stern R , Ghosh P K , Weissman S M
Published: January 2020

Simian virus 40 (SV40) DNA I was transcribed with Escherichia coli RNA polymerase in the presence of gamma-32P-labeled ribonucleoside triphosphates in order to investigate the specificity of initiation of in vitro transcription. ATP and GTP served as predominant initiating nucleotides, the former being incorporated about twice as much as the latter. Cleavage of [gamma-32P]ATP-labeled SV40 complementary RNA (cRNA) with T1 RNase followed by homochromatographic analysis of the resultant 5' initiation fragments revealed the presence of four specific initiation fragments 6 to 9 nucleotides in length, designated AI, AII, AIIIa, and AIIIb. By means of hybridization of [gamma-32P]ATP-labeled SV40 cRNA to DNA from specific adenovirus 2-SV40 hybrids and specific restriction endonuclease fragments of SV40 DNA before chromatographic analysis, it was possible to identify and determine approximate localizations of five [gamma-32P]ATP initiation sites on the SV40 genome: one in Hin-G close to the Hin-G-B junction, giving rise to the AII fragment, two in the overalpping fragment Hin-A-Hae-A,giving rise to AI and AIII fragments, and two in the fragment Hin-A-Hae-E, also giving rise to AI and AIII fragments. All five sites either fall within or lie near regions of the genome that are cleaved by S1 nuclease and subject to partial alkaline denaturation. These five sites lie on the minus strand of SV40 DNA and initiate RNAs that are copied in a leftward direction. Cleavage of [gamma-32P]GTP-labeled cRNA with pancreatic RNase liberated three major 5' initiation fragments of short length, GI, GII, and GIII, suggesting the presence of three principal GTP initiation sites.
Distribution of "minor" nicks in bacteriophage T5 DNA
Authors: Johnston J V , Nichols B P , Donelson J E
Published: January 2020

Bacteriophage T5 DNA can be released from the phage particle in such a way that one end of 5 to 10% of the DNA molecules remains attached to either the phage head or tail. Under partial denaturation conditions, the DNA preferentially denatures in the vicinity of a nick so that the nicks can be located relative to the end that remains attached to the phage head or tail. Two classes of nicks were found. "Major" nicks were those found in more than 20% of the molecules and were located at the same points along the DNA molecule as reported by others. "Minor" nicks were found in 5 to 10% of the molecules and often occurred at specific locations near a "major" nick.
Further Studies on Bacteriophage T4 DNA Synthesis in Sucrose-Plasmolyzed Cells
Authors: Stafford Mary E. , Reddy G. Prem Veer , Mathews Christopher K.
Published: January 2020

This paper describes several technical improvements in the sucrose-plasmolyzed cell system used in earlier experiments on DNA synthesis in situ with
Escherichia coli
infected by DNA-defective mutants of bacteriophage T4 (W. L. Collinsworth and C. K. Mathews, J. Virol.
13
:908-915, 1974). Using this system, which is based primarily on that of M. G. Wovcha et al. (Proc. Natl. Acad. Sci. U.S.A.
70
:2196-2200, 1973), we reinvestigated the properties of mutants bearing lesions in genes 1, 41, and 62, and we resolved some disagreements with data reported from that laboratory. We also asked whether the DNA-delay phenotype of T4 mutants is related to possible early leakage of DNA precursors from infected cells. Such cells display defective DNA synthesis in situ, even when ample DNA precursors are made available. Thus, the lesions associated with these mutations seem to manifest themselves at the level of macromolecular metabolism. Similarly, we examined an
E. coli
mutant defective in its ability to support T4 production, apparently because of a lesion affecting DNA synthesis (L. Simon et al., Nature [London]
252
:451-455). In the plasmolyzed cell system, reduced nucleotide incorporation is seen, indicating also that the genetic defect does not involve DNA precursor synthesis. The plasmolyzed cell system incorporates deoxynucleotide 5′-monophosphates into DNA severalfold more rapidly than the corresponding 5′-triphosphates. This is consistent with the idea that DNA precursor-synthesizing enzymes are functionally organized to shuttle substrates to their sites of utilization.
Encephalomyocarditis virus RNA. II. Polyadenylic acid requirement for efficient translation
Authors: Hruby D E , Roberts W K
Published: January 2020

Differentially polyadenylated subpopulatons of encephalomyocarditis (EMC) viral RNA were isolated by affinity chromatography on oligodeoxythymidylic acid-cellulose. Translation of these RNA fractions in several in vitro protein-synthesizing systems, isolated from Ehrlich ascites tumor cells, demonstrated that poly(A)+EMC viral RNA was translated two to three times more efficiently than poly(A)-EMC viral RNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the polypetides synthesized by the in vitro system in response to the different RNAs showed no detectable differences in the size or relative amount- of the translational products. mRNA saturation curves indicated that the in vitro systems were stimulated maximally by equivalent amounts of RNA, wheter it be poly(A)-or poly(A)+ EMC viral RNA. Time course experiments showed that the differences in translatability were more pronounced late in the reaction when reinitiation was required, and that by eliminating reinitiation with high salt the apparent effect of poly(A) on translation was diminished. Together, these results suggest that poly(A) may be required for efficient initiation and reinitiation of protein synthesis in the cell-free systems. This interpretation is discussed relative to earlier data.
Heteroduplex study of the sequence relations between RD-114 and baboon viral RNAs
Authors: Hu S , Davidson N , Nicolson M O , McAllister R M
Published: January 2020

The regions of sequence homology and nonhomology between the RNA genomes of RD-114 and baboon endogenous type C viruses have been mapped by an electron microscope heteroduplex study. Short complementary DNA (cDNA) copies (approximately 150 to 200 nucleotides in length) of RD-114 RNA were prepared by an endogenous synthesis; labels of polydeoxythymidylic acid [poly(dT)] were attached to the 3' ends of the cDNA molecules by a reaction catalyzed by deoxynucleotidyl terminal transferase. The cDNA-poly(dT) was hybridized to RD-114 RNA and to baboon viral RNA dimer (50 to 70S) units, and the position- of the poly(dT) labels were observed by electron microscopy. With RD-114, labels were distributed uniformly along the genome. With baboon virus RNA (monomer length, 9.5 kilobases [kb]), the regions of high homology with RD-114 cDNA were observed to lie in the intervals from 1.5 to 2.5 kb and from 3.7 to 5.5 kb from the 5' end. The relations of these heteroduplex maps to the known antigenic similarities and differences among the several viral proteins and to the genetic maps of the viruses are discussed.
Transcriptional complexity of vaccinia virus in vivo and in vitro
Authors: Paoletti E , Grady L J
Published: January 2020

The transcriptional complexity of vaccinia virus both in vivo and in vitro has been measured by using DNA:RNA hybridization with RNA in excess. In vivo, "early" or prereplicative RNA was found to saturate at 25% or one-half of the viral genome. "Late" or postreplicative RNA from infected HeLa cells saturated at 52% or essentially the entire genome. This well-regulated transcriptional pattern of the virus in vivo was not maintained in vitro. In a number of experiments a range of saturation values from 40 to 50% was obtained for in vitro synthesized RNA. The complexity of polyadenylated and non-polyadenylated RNA, as well as total purified 8 to 12S RNA released from the virus, was indistinguishable from purified high-molecular-weight virion-associated RNA with a sedimentation value of greater than 20S and equivalent to total in vitro synthesized RNA. No additional hybrid formation was observed in experiments in which total in vitro RNA and late in vivo RNA from infected HeLa cells were combined, suggesting that the virus does not transcribe in vitro DNA sequences that are not also transcribed during productive infection. Approximately 15% complementary RNA was detected when radiolabeled total in vitro RNA was allowed to reanneal with late in vivo RNA, while as much as 8% of the in vitro synthesized RNA was found to be complementary.
Viruses of Entamoeba histolytica. VII. Novel beaded virus
Authors: Mattern C F , Keister D B , Daniel W A , Diamond L S , Kontonis A D
Published: January 2020

A third amoebal virus type was isolated from four different strains of Entamoeba histolytica. The virus was most frequently seen as a linear structure about 235 nm long and consisting of 14 beadlike structures about 19 nm in diameter. A "dimer" of twice the length and consisting of 28 beads was occationally encountered. The virus replicated in the nucleus, forming ordered arrays. Acridine orange staining of viral aggregates in infected nuclei suggested the presence of double-stranded nucleic acid.
In vitro synthesis of a unique RNA species by a T particle of vesicular stomatitis virus
Authors: Emerson S U , Dierks P M , Parsons J T
Published: January 2020

A T particle of vesicular stomatitis virus, containing most of the L-gene region, has been isolated. In vitro, these T particles synthesize exclusively a small adenine-rich RNA that is complementary to the T-particle genome. Partial sequence analysis of this small RNA indicates that it is an RNA of unique sequence with a length of approximately 45 nucleotides.
Stimulation of Cellular Thymidine Kinases by Human Cytomegalovirus
Authors: Estes John E. , Huang Eng-Shang
Published: January 2020

Thymidine kinase (TK) activity in WI-38 and MRC-5 human fibroblasts was analyzed by discontinuous polyacrylamide gel electrophoresis (disc-PAGE) and discontinuous glycerol gradient electrophoresis (disc-GEP) after subculture or human cytomegalovirus (HCMV) infection. Two peaks of TK activity with different relative fraction-of-migration (

R
f

) values were resolved by disc-PAGE or disc-GEP in extracts from log-phase and infected cells. Growing WI-38 cells expressed a slowly migrating (

R
f

= 0.14 PAGE,

R
f

= 0.4 GEP) peak of TK activity, which was partially inhibited by 1.0 mM dCTP, but which retained little activity at pH 4.5. Growing MRC cells also displayed a slowly migrating peak (

R
f

= 0.10 PAGE) with similar properties. Both cell types expressed a faster-migrating TK activity (

R
f

= 0.45 PAGE,

R
f

= 0.7 GEP) in the growing and resting state that was strongly inhibited by 1 mM dCTP but retained 50% activity at pH 4.5. When either cell type was infected with HCMV, there was a rapid and high-level stimulation of the slowly migrating form of TK and a slight stimulation of the faster-migrating form. Two strains of HCMV (AD169 and Town) failed to produce an electrophoretically distinct virus TK in either cell type after infection. TK enzymes were partially purified by disc-GEP from extracts of log-phase WI-38 or AD169-infected WI-38 cells. Characterization of these enzymes with respect to phosphate donor specificity, pH optima, thermostability, and salt inhibition showed the HCMV-stimulated TKs to be of cellular origin.
Anatomy of Herpes Simplex Virus DNA. IX. Apparent Exclusion of Some Parental DNA Arrangements in the Generation of Intertypic (HSV-1 × HSV-2) Recombinants
Authors: Morse Lawrence S. , Buchman Timothy G. , Roizman Bernard , Schaffer Priscilla A.
Published: January 2020

We are reporting the physical location of parental DNA sequences in 28 recombinants produced by crossing herpes simplex viruses (HSV) 1 and 2. The parental crosses were of two kinds. In the first, temperature-sensitive mutants of HSV-1 and HSV-2 were crossed to produce wild-type recombinants. In the second, temperature-sensitive mutants of HSV-1 rendered resistant to phosphonoacetic acid were crossed with wild-type HSV-2, and recombinants that multiplied at nonpermissive temperature and were resistant to the drug were selected. The DNAs of the recombinants were mapped with
Xba
I,
Eco
RI,
Hpa
I,
Hsu
I,
Bgl
II, and, in some instances,
Kpn
I restriction endonucleases. The results were as follows. (i) We established the colinear arrangements of HSV-1 and HSV-2 DNAs. (ii) There was extensive interchange of genomic regions, ranging from the exchange or the entire L of S component of HSV DNA to substitutions of regions within the same component. In some recombinants, the reiterated sequences
ab
and
ac
bracketing the L and S components of HSV DNA were heterotypic. Most recombinants grew well and showed no obvious defects. (iii) The number of crossover events ranged from one to as many as six. Although crossover events occurred throughout the DNA, some clustering of crossover events was observed. (iv) Analysis of recombinants permitted localization of several markers used in this study and appears to be a useful technique for marker mapping. (v) As previously reported, HSV DNA consists of four populations, differing in relative orientation of the L and S components. All recombinants could be displayed in one arrangement of L and S such that the number of crossover events was minimized. The data are consistent with the hypothesis that only one arrangement of the parental DNA participates in the generation of recombinants.
Structure and formation of circular dimers of simian virus 40 DNA
Authors: Goff S P , Berg P
Published: January 2020

Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.
Ultrastructural studies of H-1 parvovirus replication. V. Immunocytochemical demonstration of separate chromatin-associated and inclusion-associated antigens
Authors: Singer I I , Rhode S L
Published: January 2020

Electron microscopy and immunocytochrome c staining were used to define the phenotypes of several temperature-sensitive (ts) H-1 mutants. They were classified into three separate groups based on the properties of their capsids at the restrictive temperature (rT): (class 1) ts2 did not assemble capsids but produced spherical and irregular amorphous inclusions; (class 2) ts1 and ts7 exclusively synthesized empty particles which all aggregated and crystallized; and (class 3) ts8 and ts10 formed noncrystalline aggregates of empty virions, but many individual full, as well as empty, capsids were associated with euchromatin. Synthesis of progeny DNA and hemagglutinin at rT were normal for class 3 mutants, but defective for those in classes 1 and 2. The immunospecific staining patterns of these mutants indicated that the H-1 capsid proteins probably form two separate intranuclear antigens: (i) a thermostable chromatin-associated antigen present in proteins that have not formed capsids and are concentrated on heterochromatin and nucleolar-associated chromatin and (ii) a thermolabile inclusion-associated antigen found in the proteins of assembled empty capsids that compose H-1 inclusions.
Bunyamwera virus-induced polypeptide synthesis
Authors: Pennington T H , Pringle C R , McCrae M A
Published: January 2020

Bunyamwera virus-induced polypeptide synthesis in BSC-1 cell has been studied using polyacrylamide gel electrophoresis and autoradiography. Four virus-induced polypeptides were identified. Their molecular weights were 200 X 10(6) (L), 128 X 10(6) (G1), 31 X 10(6) (G2), and 23 X 10(6) (N). Pulse-chase experiments, short labeling experiments, and experiments using amino acid analogs failed to show evidence of polypeptides processing by proteolytic cleavage. Analysis of the kinetics of synthesis of these polypeptides showed that a clear division into early and late categories could be made, the onset of synthesis of polypeptide N and L rapidly reached a peak and then declined. Polypeptides G1 and G2 were made for several hours; their rate of synthesis then declined. All four polypeptides then continued to be made in relatively small amounts for many hours.
Genome of infectious bronchitis virus
Authors: Lomniczi B , Kennedy I
Published: January 2020

Techniques are described for the growth and rapid purification of the avian coronavirus infectious bronchitis virus (IBV). Purified IBV has a sedimentation coefficient of 320S and a buoyant density of 1.22 g/ml in sucrose-deuterium oxide equilibrium gradients. IBV RNA extracted by proteinase K in the presence of sodium dodecyl sulfate and further purified by phenol extraction and gradient centrifugation is single stranded and has a sedimentation coefficient of 64S, as determined by isokinetic gradient centrifugation. Analysis on sucrose gradients under both aqueous and denaturing conditions together with agarose gel electrophoresis in the presence of the chaotropic agent methylmercuric hydroxide gave a value of 8 X 10(6) for the moleclar weight of IBV RNA. This value was confirmed by RNase T1 fingerprinting, which also indicated that IBV RNA is haploid. No evidence was found of subunit structure in IBV RNA. From these results together with the recently reported observation that IBV RNA is infectious and contains a tract of polyadenylic acid (Lomniczi, J. Gen. Virol., in press), we conclude that the genome of the coronaviruses is a single continuous chain of about 23,000 mononucleotides that is of messenger polarity.
Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin
Authors: Wright P J , Bowden D S , Westaway E G
Published: January 2020

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.
Synthesis and Glycosylation of Polyprotein Precursors to the Internal Core Proteins of Friend Murine Leukemia Virus
Authors: Evans Leonard H. , Dresler Steven , Kabat David
Published: January 2020

Synthesis and post-translational processing of murine leukemia virus proteins were analyzed in a murine cell line (Eveline) that produces large amounts of Friend lymphatic leukemia virus. Immunoprecipitation of
l
-[
35
S]methionine-labeled cell extracts demonstrated that several different virus-specific proteins antigenically related to the virion core (
gag
) proteins p12 and p30 become radioactive within 1 min of labeling and exhibit labeling kinetics characteristic of primary translation products. The most abundant of these were proteins with molecular weights of 75,000 and 65,000. There were, in addition, two large glycosylated polyproteins with apparent molecular weights of 220,000 and 230,000, which were precipitated by antisera to p30 or p12 but not by antiserum to the major envelope glycoproteins gp69/71. Several lines of evidence, including labeling with
d
-[
3
H]glucosamine and binding to insolubilized lectins, suggested that the 75,000-dalton internal core polyprotein is slowly processed to form a glycoprotein with an apparent molecular weight of 93,000. On the contrary, the 65,000-dalton protein appeared to be an immediate precursor to the virion core proteins. Its processing can involve intermediates containing p30 and p12 antigens with molecular weights of 50,000 and 40,000; however, the latter did not appear to be obligatory intermediates. The detection of the 40,000-dalton protein suggested that the genes for p30 and p12 are adjacent on the viral genome. These results indicated that there are several pathways of synthesis and post-translational processing of polyprotein precursors to the
gag
proteins and that several of these polyproteins are glycosylated. A comparison of
gag
precursor processing in rapidly growing, slowly growing, and stationary cells indicated that different pathways are favored under different conditions of cell growth. Our analysis of envelope glycoprotein synthesis has confirmed the existence of two rapidly labeled 90,000-dalton glycoproteins, which appear to be precursors to the envelope glycoproteins gp69/71.
Transient association of Semliki Forest virus capsid protein with ribosomes
Authors: Söderlund H , Ulmanen I
Published: January 2020

HeLa cells infected with Semliki Forest virus were exposed to [35S]methionine for 1 min and chased for various periods. The analysis of labeled ribonucleoproteins showed that the viral capsid protein associated first with the large ribosomal subunit in polysomes, from which it was chased to assembling nucleocapsids and to free monosomes.