bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, characterization, and physical mapping of a pseudorabies virus mutant containing antigenically altered gp50
Authors: Wathen M W , Wathen L M
Published: January 2020

A pseudorabies virus variant ( mar197 -1) containing a mutation in a viral glycoprotein with a molecular weight of 50,000 ( gp50 ) was isolated by selecting for resistance to a neurtralizing monoclonal antibody ( MCA50 -1) directed against gp50 . This mutant was completely resistant to neutralization with MCA50 -1 in the presence or absence of complement, and was therefore defined as a mar (monoclonal-antibody-resistant) mutant. The mutation did not affect neutralization with polyvalent immune serum. The mar197 -1 mutant synthesized and processed gp50 normally, but the mutation prevented the binding and immunoprecipitation of gp50 by MCA50 -1. Thus, the mutation was within the structural portion of the gp50 gene affecting the epitope of the monoclonal antibody. The mutation was mapped by marker rescue with cloned pseudorabies restriction enzyme fragments to the short region of the pseudorabies genome between 0.813 and 0.832 map units. This is equivalent to a 2.1-kilobase-pair region.
Leukemia induction by a new strain of Friend mink cell focus-inducing virus: synergistic effect of Friend ecotropic murine leukemia virus
Authors: Chesebro B , Wehrly K , Nishio J , Evans L
Published: January 2020

A new strain of Friend recombinant mink cell focus-inducing retrovirus, FMCF -1-E, was found to induce leukemias in NFS and IRW mice. Although the isolate was obtained from a stock of FMCF -1 ( Troxler et al., J. Exp. Med. 148:639-653, 1978), FMCF -1-E was distinguishable from FMCF -1 by oligonucleotide fingerprinting and antigenic analysis, using monoclonal antibodies. These analyses suggested that FMCF -1-E is a distinct FMCF isolate rather than a simple variant of FMCF -1. After neonatal inoculation, the latency for leukemia induction was 3 to 8 months. A similar long latency was also seen when Friend murine leukemia virus 57 was inoculated into adult (6-week-old) IRW mice. However, sequential inoculation of FMCF -1-E at birth followed by Friend murine leukemia 57 at 6 weeks of age led to a shortened latency period (2.5 to 4 months). Only neonatal inoculation of Friend murine leukemia virus 57 was able to induce a more rapid appearance of leukemia. The leukemia cell type in the majority of cases, regardless of virus inoculation protocol, was erythroid, but occasional myeloid, lymphoid, and mixed leukemias were also observed. In contrast to NFS and IRW mice, BALB/c mice were resistant to leukemia induction by FMCF -1-E and also showed some transient resistance to leukemia induction by Friend murine leukemia virus 57.
Detection of the v-abl gene product at cell-substratum contact sites in Abelson murine leukemia virus-transformed fibroblasts
Authors: Rohrschneider L R , Najita L M
Published: January 2020

Monoclonal antibodies to the p15 and p12 gag proteins were used to detect the P120gag-abl transforming protein of Abelson murine leukemia virus in nonproductively transformed normal rat kidney fibroblast cells. The results demonstrate that, in addition to the prominent plasma membrane location, P120gag-abl was associated with points of adhesion between the cell and the substratum. The localization of P120gag-abl was qualitatively similar to that reported for pp60src in the same normal rat kidney fibroblast cells and suggests that these transforming proteins may share some common transformation features.
Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells
Authors: Richardson W D , Anderson C W
Published: January 2020

Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 Mr virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus fiber mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus.
Role of a low-pH environment in adenovirus enhancement of the toxicity of a Pseudomonas exotoxin-epidermal growth factor conjugate
Authors: Seth P , Fitzgerald D J , Willingham M C , Pastan I
Published: January 2020

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.
Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells
Authors: Etchison D , Hansen J , Ehrenfeld E , Edery I , Sonenberg N , Milburn S , Hershey J W
Published: January 2020

Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex.
Indiscriminate degradation of RNAs in interferon-treated, vaccinia virus-infected mouse L cells
Authors: Benavente J , Paez E , Esteban M
Published: January 2020

In this report we used Northern blot hybridization analysis to characterize the fate of several species of viral RNA transcribed from internal and terminal regions of vaccinia DNA in interferon-treated, infected mouse L cells grown in suspension. All species of viral RNAs were expressed but were reduced in amount. Larger-sized RNAs were reduced more than smaller-sized RNAs. This reduction appears to be related to the activation of the interferon-mediated double-stranded RNA-dependent 2-5A synthetase-endoribonuclease system, as the rRNA cleavage pattern characteristic of this system was observed early in infection and in cell extracts in response to exogenous 2-5A. Thus, in interferon-treated, vaccinia-infected mouse L cells in suspension, there is indiscriminate degradation of viral and cellular RNAs, and this RNA breakdown might play a role in the interferon-mediated inhibition of protein synthesis.
Genome of an avian papillomavirus
Authors: Moreno-Lopez J , Ahola H , Stenlund A , Osterhaus A , Pettersson U
Published: January 2020

A papillomavirus which we designate FPV was isolated from chaffinches (Fringilla coelebs). A physical map of the FPV genome was constructed, and selected regions of this genome were studied by nucleotide sequence analysis. The results make it possible to align the FPV genome with the genome of bovine papillomavirus type 1 and to show, moreover, that avian and mammalian papillomaviruses have a similar genome organization.
Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene
Authors: Dowbenko D J , Lasky L A
Published: January 2020

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.
Monoclonal antibodies to a monkeypox virus polypeptide determinant
Authors: Roumillat L F , Patton J L , Davis M L
Published: January 2020

Three monkeypox virus (MPV) antibody-secreting murine monoclones were characterized as being of the immunoglobulin G1 isotype, gave a 4+ reaction in the indirect fluorescent-antibody test, gave a positive reaction in the enzyme immunoassay, and did not neutralize MPV. These monoclonal antibodies were determined by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis transblot method to react to a 15,500-molecular-weight MPV polypeptide. This reactivity could not be removed by adsorption to a vaccinia virus-infected cell suspension. The three monoclonal antibodies were specific for MPV when tested against epidemiologically unrelated isolates of cowpox virus, variola virus, vaccinia virus, and MPV.
Mapping of the amino terminus of the H-1 parvovirus major capsid protein
Authors: Paradiso P R , Williams K R , Costantino R L
Published: January 2020

The amino terminus of VP2', the major capsid protein of the parvovirus H-1, was identified and mapped to the H-1 genome. The protein initiates at the start codon at nucleotide 2797 and is translated uninterrupted to the stop codon at nucleotide 4582. The primary sequence predicts a protein of 593 amino acids (65,500 daltons) with an amino acid composition which very closely matches the experimentally determined composition of the pure protein. The data suggest that the VP2' mRNA has a 5' leader sequence of ca. 650 bases and that protein translation initiates downstream from the sole splice junction.
The major site of tyrosine phosphorylation in polyomavirus middle T antigen is not required for transformation
Authors: Mes-Masson A M , Schaffhausen B , Hassell J A
Published: January 2020

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.
Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection
Authors: O'Hare P , Hayward G S
Published: January 2020

The promoter-regulatory regions from the herpes simplex virus type 1 (HSV-1) gene for the immediate-early, 175,000-molecular-weight (175K) protein and the HSV-2 delayed-early gene for a 38K protein were linked to the readily assayable bacterial gene for the enzyme chloramphenicol acetyltransferase (CAT). Unexpectedly, in measurements of the constitutive expression of the recombinant genes 40 to 50 h after transfection of Vero cells, enzyme levels expressed from the delayed-early 38K-promoter-CAT construct (p38KCAT) were at least as high as those from the immediate-early 175K-promoter-CAT construct (p175KCAT). In contrast, enzyme levels expressed after transfection of a similar recombinant gene containing a second delayed-early promoter region, that of the HSV-1 thymidine kinase gene, were ca. 20-fold lower. The amounts of enzyme expressed from both p38KCAT and p175KCAT could be increased by up to 20- to 40-fold after infection of the transfected cells with HSV. In comparison, virus infection had no significant effect on enzyme levels expressed from recombinant CAT genes containing the simian virus 40 early promoter region, with or without the 72-base-pair enhancer element. Experiments with the temperature-sensitive mutants HSV-1 tsB7 and HSV-1 tsK indicate that induction of expression from p175KCAT was mediated by components of the infecting virus particle, whereas that from p38KCAT required de novo expression of virus immediate-early proteins. In addition, we show that functions required to induce expression from both p175KCAT and p38KCAT could also be provided by infection with pseudorabies virus and cytomegalovirus.
Characterization of the envelope proteins of pseudorabies virus
Authors: Hampl H , Ben-Porat T , Ehrlicher L , Habermehl K O , Kaplan A S
Published: January 2020

Previously we have reported that among the proteins of purified pseudorabies virions there are four major glycoproteins (T. Ben-Porat and A. S. Kaplan, Virology 41:265-273, 1970). Several minor glycoproteins can also be identified by two-dimensional gel electrophoresis. Removal of the viral envelope with Triton X-100 selectively removes from the virions all of the glycoproteins as well as several non-glycosylated proteins. Sedimentation analysis or chromatography of these proteins reveals that several are complexed with one another, some being covalently linked via disulfide bridges. Analysis of the proteins by immunoprecipitation with monoclonal antibodies reactive with the membrane proteins showed also that three of the four major virus glycoproteins (125K, 74K, and 58K; gIIa, gIIb, and gIIc, respectively) are linked covalently by disulfide bridges. Furthermore, all three share extensive sequence homology as indicated by the identity of their antigenic determinants and by partial peptide mapping; they probably originate from a single protein precursor. The fourth major glycoprotein (98K; gIII) is not complexed to any other protein. Three minor glycoproteins (130K [gI], 98K [gIV], and 62K [gV]), which form a noncovalently linked complex with a 115K nonglycosylated protein, have also been identified. Of the monoclonal antibodies used in this study, only those reactive with the major 98K glycoprotein (gIII) inhibit virus adsorption and neutralize virus infectivity in the absence of complement. However, all react with surface components of the virion, indicating that the proteins with which they react are exposed on the surface of the virions. A nomenclature for the pseudorabies virus glycoproteins is proposed.
Characterization and myocarditic capabilities of coxsackievirus B3 variants in selected mouse strains
Authors: Gauntt C J , Gomez P T , Duffey P S , Grant J A , Trent D W , Witherspoon S M , Paque R E
Published: January 2020

Two variants of coxsackievirus B3 (CVB3) were compared with the original myocarditic parent variant (CVB3m) for myocarditic properties in several strains of mice. The ts1R variant produced little to no myocarditis in any of the nine mouse strains examined. The ts10R variant and CVB3m could be differentiated on the basis of the extent of myocarditis induced in mice of selected H-2b and H-2k haplotypes and in the female versus the male responses of two other inbred strains. Virus quantities recovered from the hearts of myocarditic mice did not correlate with the extent of disease. The three variants could not be differentiated on the basis of: (i) rate and extent of adsorption to heart tissue homogenates, (ii) kinetic neutralization rates with antiserum directed against CVB3m, (iii) 125I labeling of surface regions of polypeptides on purified particles, or (iv) rates of heat inactivation of infectivity at 50 degrees C. These data suggest that differences in pathogenicity cannot be attributed to major alterations in capsid polypeptides. Oligonucleotide fingerprint maps of T1 RNase digests of the genomes of purified particles of the three CVB3 variants showed distinct differences. Thus, the extent of myocarditis induced by CVB3 variants in a mouse model is affected by some subtle expression of the genome, presumably not involving capsid polypeptides, as well as by the haplotype and sex of a given mouse host species.
A tandemly reiterated DNA sequence in the long repeat region of herpes simplex virus type 1 found in close proximity to immediate-early mRNA 1
Authors: Rixon F J , Campbell M E , Clements J B
Published: January 2020

The 3' end of immediate-early mRNA 1 was mapped precisely within the IRL/TRL genome regions, and the DNA sequences around the 3' end were determined. An AATAAA polyadenylation signal was present 17 base pairs upstream of the 3' end, and eight tandemly repeated copies of a 16-base-pair sequence (GGGGGTGCGTGGGAGT) plus one further closely related copy were located 20 base pairs downstream. Other tandem reiterations present in the herpes simplex virus genome are described and their properties are considered.
Simian virus 40 replication pause sites map with the nucleosomes
Authors: Trifonov E N , Mengeritsky G
Published: January 2020

The locations of replication pause sites in the simian virus 40 minichromosome which were determined by sizing cloned fragments of nascent DNA (Zannis-Hadjopoulos et al., J. Mol. Biol. 165:599-607, 1983) were compared with the positions of simian virus 40 nucleosomes in the genome, as obtained by sequence-directed mapping (G. Mengeritsky and E. N. Trifonov, Nucleic Acids Res. 11:3833-3851, 1983; Mengeritsky and Trifonov, Cell Biophys. 6:1-8, 1984). Clear correlation between these two maps is demonstrated, suggesting that nucleosomes hinder propagation of the replication forks.
Genetic analysis of dar, uvsW, and uvsY in bacteriophage T4: dar and uvsW are alleles
Authors: Wu J R , Yeh Y C , Ebisuzaki K
Published: January 2020

A genetic study of the T4 dar (DNA arrested synthesis restoration) mutations was performed by two- and three-factor crosses. The dar mutations restore the viability of gene 59 mutants. Mapping studies of the dar mutations indicated that the dar gene extended over 16 map units. This high recombination frequency is attributed to an increased level of recombination in the dar region. Two other mutations, uvsY and uvsW, known to be located in the vicinity of dar, were studied. These studies indicated that the uvsY and dar mutations were located in separate genes but that dar and uvsW were allelic. The genes are ordered as follows: gene 24, dar(uvsW), uvsY, and gene 25 in clockwise order.
Structural characterization of virion proteins and genomic RNA of human parainfluenza virus 3
Authors: Storey D G , Dimock K , Kang C Y
Published: January 2020

The virion proteins and genomic RNA of human parainfluenza virus 3 have been characterized. The virion contains seven major and two minor proteins. Three proteins of 195 X 10(3) molecular weight (195K), 87K, and 67K are associated with the nucleocapsid of the virion and have been designated L, P, and NP, respectively. Three proteins can be labeled with [14C]glucosamine and have molecular weights of 69K, 60K, and 46K. We have designated these proteins as HN, F0, and F1, respectively. HN protein has interchain disulfide bonds, but does not participate in disulfide bonding to form homomultimeric forms. F1 appears to be derived from a complex, F1,2, that has an electrophoretic mobility similar to that of F0 under nonreducing conditions. A protein of 35K is associated with the envelope components of the virion and aggregates under low-salt conditions; this protein has been designated M. The genome of human parainfluenza virus 3 is a linear RNA molecule with a molecular weight of approximately 4.6 X 10(6).
Association of poliovirus proteins with the endoplasmic reticulum
Authors: Tershak D R
Published: January 2020

Poliovirus proteins, except P3-7c, are associated with the endoplasmic reticulum after extraction of the cytoplasm and centrifugation of membranes to equilibrium in sucrose gradients. Proteins P3-2, P2-X, and P3-9 are found preferentially among the rough endoplasmic reticulum, whereas P3-7c is located in smooth endoplasmic reticulum fractions. P3-7c is probably not membrane associated, since it can be separated from membranes after centrifugation in buffer. However, P3-4a, P2-5b, P2-X, and P3-9 are avidly bound to membranes and cannot be dislodged with high-ionic-strength buffer containing EDTA or 4 M urea. These proteins are digested by trypsin, indicating peripheral rather than internal localization.
Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules
Authors: Kikuchi G E , Coligan J E , Holland T C , Levine M , Glorioso J C , Nairn R
Published: January 2020

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.
Effects of lysosomotropic weak bases on infection of BHK-21 cells by Sindbis virus
Authors: Cassell S , Edwards J , Brown D T
Published: January 2020

The effects of the lysosomotropic weak bases chloroquine, ammonium chloride, and amantadine as well as dansylcadaverine (an inhibitor of receptor mediated endocytosis) on the replication of Sindbis virus in tissue-cultured cells was examined. Chloroquine had no effect on the expression of virus-induced homologous interference. None of these drugs significantly affected the ability of a complex of a cell and single virion to form an infectious center. Chloroquine and ammonium chloride were found to inhibit the synthesis of virus RNA in established infections when added early in infection. These drugs also inhibited the production of progeny virions when added any time after infection. These results suggest that the antiviral activity of these agents may not be due to an ability to prevent transport of the virus genome into the cell cytoplasm.
Middle tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with pp60c-src
Authors: Bolen J B , Israel M A
Published: January 2020

We have found that lysis of mouse embryo cells infected with the polyomavirus host range transformation-defective (hr-t) mutant NG59 under gentle conditions that avoid ionic detergents results in detectable NG59-encoded middle tumor antigen (MTAg) associated with pp60c-src. This MTAg-pp60c-src complex could be immunoprecipitated from NG59-infected cell lysates by either sera from animals bearing polyomavirus-induced tumors or by monoclonal antibodies directed against MTAg. Immune complex kinase assays revealed that, whereas the pp60c-src associated with NG59 MTAg possessed tyrosyl kinase activity, the NG59 MTAg in this complex was not phosphorylated in these in vitro reactions. These results demonstrate that the point insertion mutation found in this transformation-deficient strain of polyomavirus encodes MTAg molecules capable of associating with pp60c-src and defines a limited region within MTAg which appears to be critical for stable MTAg-pp60c-src interactions.
Assembly-controlled autogenous modulation of bacteriophage P22 scaffolding protein gene expression
Authors: Casjens S , Adams M B , Hall C , King J
Published: January 2020

In the assembly of bacteriophage P22, precursor particles containing two major proteins, the gene 5 coat protein and the gene 8 scaffolding protein, package the DNA molecule. During the encapsidation reaction all of the scaffolding protein molecules are released intact and subsequently participate in further rounds of DNA encapsidation. We have previously shown that even though it lies in the center of the late region of the genetic map, the scaffolding protein gene is not always expressed coordinately with the remainder of the late proteins and that some feature of the phage assembly process affects its expression. We present here in vivo experiments which show that there is an inverse correlation between the amount of unassembled scaffolding protein and the rate of scaffolding protein synthesis and that long amber fragments of the scaffolding protein can turn down the synthesis of intact scaffolding protein in trans. These results support a model for scaffolding protein regulation in which the feature of the assembly process which modulates the rate of scaffolding protein synthesis is the amount of unassembled scaffolding protein itself.
In vitro transcription of herpes simplex virus genes: identification of a new initiation site and second intervening sequence in the immediate-early RNA-5 gene
Authors: Fontichiaro K L , Beck T W , Millette R L
Published: January 2020

We have used partially purified preparations of RNA polymerase II from mock-infected and herpes simplex virus type 1-infected HEp-2 cells to transcribe the herpes simplex virus type 1 strain F BamHI Z DNA fragment containing the promoter for immediate-early RNA-5 (alpha 47), a 1.8-kilobase, spliced RNA. In agreement with the in vivo transcriptional regulation of herpes simplex virus type 1 immediate-early genes, electrophoretic analyses of runoff and truncated transcripts from this template showed that RNA polymerase II from mock-infected cells initiates transcription more selectively than does that from the herpes virus-infected cells at the immediate-early RNA-5 promoter. S1 nuclease mapping of the 5' ends of in vivo- and in vitro-synthesized mRNA placed the initiation site ca. 110 base pairs upstream from the previously published site and also demonstrated the presence of a second, smaller intervening sequence between this new cap site and the previously characterized intervening sequence. S1 analyses also suggested that some splicing of the larger but not the smaller intervening sequence occurred in vitro.
Analysis of herpesvirus saimiri structural proteins with monoclonal antibodies
Authors: Dahlberg J E , Zintz E , Ablashi D V
Published: January 2020

Herpesvirus saimiri is a lymphotrophic virus isolated from squirrel monkeys that causes highly malignant lymphomas in owl monkeys, marmosets, and rabbits, but not in its natural host. We have been interested in exploring immunological and biological aspects of this phenomenon and describe in this report the isolation of 27 monoclonal antibodies (MCAs) to herpesvirus saimiri which were grouped into 11 distinct sets based on the proteins they immunoprecipitated. In total, these 11 groups of MCAs identify the majority of the proteins present in the virion. Immunofluorescence was used to study how the viral antigens are compartmentalized in infected cells. One antibody produced intense nuclear staining and immunoprecipitated both the largest protein seen in gels (150,000 daltons) and one of the smallest (13,000 daltons). All of the other groups of MCAs principally stained the cytoplasm of infected cells. One of the unexpected results of this study was the observation that a majority of the MCAs precipitated more than one protein (15 of 27 antibodies, 7 of 11 groups). Whereas one group of MCAs, which normally precipitated four proteins, identified only single polypeptides after dithiothreitol pretreatment, the other sets of antibodies continued to recognize two or more viral antigens under reducing conditions. Immunoprecipitation of viral proteins with polyvalent sera obtained from virus-infected rabbits and monkeys was carried out to verify and extend previously published reports on the proteins of herpesvirus saimiri.
Integration of spleen focus-forming virus proviruses in Friend tumor cells
Authors: Moreau-Gachelin F , Robert-Lezenes J , Wendling F , Tavitian A , Tambourin P
Published: January 2020

Using the Southern blot procedure, we studied the presumed spleen focus-forming virus (SFFV) provirus integration sites in the genome of the premalignant and the malignant cells isolated during the course of Friend erythroleukemia. Two restriction endonucleases, PstI and BamHI, discriminated the presumed integrated SFFV proviruses from the endogenous xenotropic-mink cell focus-forming viral sequences. No SFFV integration sites were detectable in the premalignant cells, suggesting a random integration of SFFV proviruses in the genome of these cells. In contrast, SFFV proviruses were detected at a single or very few sites in the genome of all malignant cells we analyzed. These results indicate that the event leading to the malignant transformation in acute Friend leukemia is clonal. In two of the six animals examined, tumors cells isolated from the spleens and the livers of individual mice showed identical SFFV integration patterns. This last result suggests that in some cases different tumors in a same leukemic animal could be derived from a unique clonal event.
Mapping of a vaccinia host range sequence by insertion into the viral thymidine kinase gene
Authors: Gillard S , Spehner D , Drillien R
Published: January 2020

A vaccinia virus mutant deleted of ca. 18 kilobase pairs at the left-hand end of the genome is unable to multiply on many human cell lines. To determine whether all or some of the deleted sequences were responsible for the host range property, the corresponding region from wild-type DNA was cloned in three pieces into a vaccinia transplacement vector containing the thymidine kinase gene on the HindIII J fragment. The next step was to transfer these pieces to the genome of the host range deletion mutant by in vivo homologous recombination around the thymidine kinase locus. Transfer of one 5.2-kilobase-pair EcoRI fragment was found to restore a wild-type phenotype on the host range mutant, thus demonstrating that only a small portion of the 18-kilobase-pair deletion contains the host range function(s). This result also illustrates that the method initially devised for inserting foreign genes into vaccinia virus DNA is useful for studies of the vaccinia genome.
Assignment of the temperature-sensitive lesion in the replication mutant A1 of vesicular stomatitis virus to the N gene
Authors: Marks M D , Kennedy-Morrow J , Lesnaw J A
Published: January 2020

The replication defect in the temperature-sensitive mutant A1 of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus was confirmed by the absence of intracellular nucleocapsids in infected cells incubated at the restrictive temperature. After preamplification, the relative yield of the A1 N protein accumulated intracellularly after 1 h of incubation at the restrictive temperature was decreased by 50% that of the wild-type or revertant A1 N protein. This difference was not as apparent in pulse-chase experiments. The functional lesion in A1 was correlated with a structural alteration in the N protein on the basis of the thermolability of the template activity of the A1 N protein-RNA complex in in vitro transcription reactions and the covariance of this phenotype with the temperature-sensitive phenotype in a spontaneous A1 revertant. This correlation was consistent with a direct role of the N protein in replication and allowed the assignment of the N gene to complementation group A.
Molecular cloning of the six mRNA species of infectious hematopoietic necrosis virus, a fish rhabdovirus, and gene order determination by R-loop mapping
Authors: Kurath G , Ahern K G , Pearson G D , Leong J C
Published: January 2020

Plasmids carrying cDNA sequences to the mRNA species of infectious hematopoietic necrosis virus were constructed and cloned into Escherichia coli. Characterization of 21 cloned plasmids by hybridization to mRNA blots identified sets of plasmids with homology to each of the six viral mRNA species. R-loop mapping with these cDNA plasmids determined that the gene order on the infectious hematopoietic necrosis virus genome is (3')N-M1-M2-G-NV-L(5').
Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants
Authors: Fujimura R K , Tavtigian S V , Choy T L , Roop B C
Published: January 2020

Segments of DNA that contained the DNA polymerase gene of bacteriophage T5 were isolated. The physical locus of the gene was identified by transforming Escherichia coli with purified DNA fragments generated by restriction enzyme digestions, and the transformed cells were used to rescue amber mutants of T5 with mutations in the gene for DNA polymerase. The method is applicable to any other gene that has mutations with low reversion frequencies. We studied the following mutations of the T5 DNA polymerase gene, reading from left to right by the standard convention (D. J. McCorquodale, Crit. Rev. Microbiol. 4:101-159, 1975): D7, D8, aml, ts5E-ts53, am6, and D9. These loci were found to reside within three pieces of DNA with a total length of 3,600 base pairs. Because the structural gene for T5 DNA polymerase is estimated to be 2,600 base pairs long, the whole structural gene may reside in these segments. These are located 58.3 to 61.3% of the distance from the left end of the DNA. The left-end piece of the DNA (1,100 base pairs) containing the polymerase gene has loci D7 and D8, and the right-end piece (1,600 base pairs) has locus D9, according to the results of the transformation assay. These results are consistent with the genetic map.
Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses
Authors: Ahlquist P , Strauss E G , Rice C M , Strauss J H , Haseloff J , Zimmern D
Published: January 2020

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex.
Sequence comparisons of the anemia- and polycythemia-inducing strains of Friend spleen focus-forming virus
Authors: Wolff L , Kaminchik J , Hankins W D , Ruscetti S K
Published: January 2020

A nucleotide sequence analysis carried out on the envelope gene of the anemia-inducing strain of the Friend spleen focus-forming virus (F-SFFVA) reveals that its product has some unique features in common with previously described polycythemia-inducing strains of F-SFFV (F-SFFVP). (i) It contains an amino terminus that is highly related to the gp70 of mink cell focus-inducing viruses, (ii) it is a fusion protein containing the amino terminus of gp70 and the carboxy terminus of p15E, and (iii) it lacks the R-peptide normally found at the carboxy end of the p15E region. Although the envelope genes of F-SFFVA and F-SFFVP are quite similar overall, they do show sequence variation, particularly at the 3' end in the p15E-related region. These variations may contribute to previously observed differences in the response of F-SFFVP- and F-SFFVA-infected erythroid cells to regulatory hormone or to differences in the way the envelope glycoproteins are processed. The long terminal repeat regions of F-SFFVA and the Lilly-Steeves strain of F-SFFVP were also sequenced and compared with each other and with a previously published sequence of another F-SFFVP long terminal repeat. The sequences were found to be reasonably similar to each other but different from their ecotropic parent, Friend murine leukemia virus, as a result of a deletion of one copy of the direct tandem repeat in the enhancer regions. The observation that all SFFVS have this common change in the long terminal repeat enhancer region raises the possibility that it is required for pathogenicity.
Isolation of different bacteriophages using the LamB protein for adsorption on Escherichia coli K-12
Authors: Charbit A , Hofnung M
Published: January 2020

Ten phages which use the LamB protein for adsorption have been isolated from sewage waters. Nine have a shape similar to lambda and require only the LamB protein for adsorption. One has a shape similar to T phages and can use either the LamB or the OmpC protein. Preliminary characterization by a number of criteria showed that at least nine of these phages were different and also differed from other known phages which use the LamB protein, such as lambda, 21, and K10.
Virions and intracellular nucleocapsids produced during mixed heterotypic influenza infection of MDCK cells
Authors: Sklyanskaya E I , Varich N L , Amvrosieva T V , Kaverin N V
Published: January 2020

Phenotypically mixed virus yields, obtained by coinfection of MDCK cells with influenza A/WSN/33 and B/Lee/40 viruses, contained both A/WSN/33 and B/Lee/40 NP proteins, as revealed by polyacrylamide gel electrophoresis of the purified 14C-amino acids-labeled virus. Virions were lysed with 0.6 M KCl-Triton X-100 buffer, and nucleocapsids were immunoprecipitated with antibodies against NP protein of influenza A virus. Polyacrylamide gel electrophoresis of the immunoprecipitate revealed NP protein of A/WSN/33 but not of B/Lee/40 virus. However, in similar experiments with the lysates of doubly infected cells, the band of B/Lee/40 NP protein was revealed in the polyacrylamide gel electrophoresis patterns of the immunoprecipitates. In an attempt to analyze the RNA content of the immune complexes, we absorbed the lysates of doubly infected [3H]uridine-labeled cells with protein A-containing Staphylococcus aureus covered with antibodies against the NP protein of influenza A virus. RNA extracted from the immune complexes contained genomic RNA segments of both A/WSN/33 and B/Lee/40 viruses. In control samples containing an artificial mixture of cell lysates separately infected with each virus, the analysis revealed homologous components (i.e., A/WSN/33 NP protein or RNA segments) in the immune complexes. The results suggest the presence of phenotypically mixed nucleocapsids in the cells doubly infected with influenza A and B viruses; in the course of the virion formation, the nucleocapsids lacking the heterologous NP protein are selected.
Gene K of bacteriophage phi X174 codes for a protein which affects the burst size of phage production
Authors: Gillam S , Atkinson T , Markham A , Smith M
Published: January 2020

Site-directed mutagenesis has been used to produce a T----A change at nucleotide 70 of phi X174 genome. This generates an am codon, TAG, in the gene K reading frame without affecting the amino acid, leucine, encoded by the overlapping gene A. The gene K mutant produces small plaques on su- hosts. It has an identical latent period, but a more reduced burst size than that of the wild-type phi X174. The reduced burst size in the gene K mutant suggests that the gene K protein, although not essential, has a role in increasing infectivity by increasing the burst size three- to sixfold.
Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody
Authors: Lin J C , Choi E I , Pagano J S
Published: January 2020

We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme-linked immunosorbent assay) and 0.8 to 1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.
Expression of the cloned bacteriophage phi X174 A* gene in Escherichia coli inhibits DNA replication and cell division
Authors: Colasanti J , Denhardt D T
Published: January 2020

The A* gene of bacteriophage phi X174 has been cloned into the inducible expression vector pCQV2 under conditions allowing its lethal action to be controlled by the lambda cI857 repressor. Upon induction of expression, DNA synthesis in Escherichia coli carrying the recombinant plasmid is severely inhibited; however, these same cells permit beta-galactosidase induction at a rate similar to that observed in control cells at the inducing (for A*) temperature. Cells in which A* is expressed form filaments and produce more RecA protein, indicating at least a partial induction of the SOS response; however, there is no evidence of damage to the bacterial chromosome. It appears that the A* protein has as one function the inhibition of cell division and DNA replication but not transcription or protein synthesis during phage infection.