bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide Sequence Analyses and Predicted Coding of Bunyavirus Genome RNA Species
Authors: Clerx-van Haaster Corrie M. , Akashi Hiroomi , Auperin David D. , Bishop David H. L.
Published: January 2020

We performed 3′ RNA sequence analyses of [
32
P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3′-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology
105:
564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3′-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3′-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3′-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.
Glycoprotein gE of herpes simplex virus type 1: effects of anti-gE on virion infectivity and on virus-induced fc-binding receptors
Authors: Para M F , Baucke R B , Spear P G
Published: January 2020

An Fc-binding glycoprotein, designated gE, was detected previously in cells infected with herpes simplex virus type 1 (HSV-1) and in virion preparations isolated from infected cells. For the studies reported here, we purified gE from HSV-1 strain HFEM(syn) by affinity chromatography and preparative electrophoresis and then immunized a rabbit to produce an antiserum to glycoprotein gE. We found that this antiserum selectively precipitated gE and its precursors from detergent-solubilized extracts of HSV-1 strain HFEM(syn)-infected HEp-2 cells, from extracts of other cell lines infected with the same virus, and from extracts of HEp-2 cells infected with several other HSV-1 strains. The antiserum did not precipitate any proteins from uninfected cells. The several forms of gE detected by immunoprecipitation accumulated in variable quantities in different cells infected with the different virus strains and also varied slightly with respect to electrophoretic mobility, suggesting some differences in the gE's from different HSV-1 strains and some effects of the host cell on the nature and extent of post-translational processing. One of the electrophoretic forms of gE previously detected in purified preparations of virions could be precipitated by anti-gE from extracts of purified HSV-1 strain HFEM(syn) virions. Moreover, anti-gE neutralized HSV-1 infectivity, but only in the presence of complement. Finally, F(ab')2 fragments of the anti-gE immunoglobulin partially inhibited the binding of 125I-labeled immunoglobulin G to the Fc receptors on HSV-1-infected cells.
Interference among defective interfering particles of vesicular stomatitis virus
Authors: Rao D D , Huang A S
Published: January 2020

Three defective interfering (DI) particles of vesicular stomatitis virus (VSV), all derived from the same parental standard San Juan strain (Indiana serotype), were used in various combinations to infect cells together with the parental virus. The replication of their RNA genomes in the presence of other competing genomes was described by the hierarchical sequence: DI 0.52 particles greater than DI 0.45 particles less than or equal to DI-T particles greater than standard VSV. The advantage of one DI particle over another was not due simply to multiplicity effects nor to the irreversible occupation of limited cellular sites. Interference, however, did correlate with a change in the ratio of plus and minus RNA templates that accumulated intracellularly and with the presence of new sequences at the 3' end of the DI genomes. DI 0.52 particles contained significantly more nucleotides at the 3' end that were complementary to those at the 5' end of its RNA than did DI-T or DI 0.45 particles. The first 45 nucleotides at the 3' ends of all of the DI RNAs were identical. VSV and its DI particles can be separated into three classes, depending on their terminal RNA sequences. These sequences suggest two mechanisms, one based on the affinity of polymerase binding and the other on the affinity of N-protein binding, that may account for interference by DI particles against standard VSV and among DI particles themselves.
Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus
Authors: Goff Stephen P. , Tabin Clifford J. , Wang Jean Yin-Jen , Weinberg Robert , Baltimore David
Published: January 2020

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10
4
transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3′ end of the genome was not present. The transforming gene was thus localized to the 5′ portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3′ ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.
Expression of Middle-Component RNA of Cowpea Mosaic Virus: In Vitro Generation of a Precursor to Both Capsid Proteins by a Bottom-Component RNA-Encoded Protease from Infected Cells
Authors: Franssen Henk , Goldbach Rob , Broekhuijsen Martien , Moerman Marja , van Kammen Albert
Published: January 2020

The expression of the middle-component (M) RNA of cowpea mosaic virus was studied by means of in vitro translation. In both the wheat germ extract and the rabbit reticulocyte lysate, M RNA was translated into two overlapping polypeptides of 95 and 105 kilodaltons. Incubation of these polypeptides with 30,000 ×
g
supernatant fractions from cowpea mesophyll protoplasts inoculated with complete virus or with separate bottom (B) components alone resulted in extensive processing, yielding polypeptides of 60, 58, 48, and 47 kilodaltons. Similar proteolytic activity was found associated with the in vitro translation products from the bottom-component RNA, demonstrating that the protease present in infected cells is encoded by B RNA. Using antisera raised against the separate capsid proteins VP23 and VP37, it was shown that the 60-kilodalton cleavage product is the precursor to both capsid proteins. Cleavage of nascent 95- and 105- kilodalton polypeptides by the in vivo protease demonstrated that this capsid protein precursor is located C terminally within both polypeptides and that the synthesis of these two overlapping polypeptides is the result of two initiation sites on middle-component RNA. In addition, a second virus-induced proteolytic activity, capable of releasing VP23 from the 95- and 105-kilodalton polypeptides, was detected in leaves of infected plants, but not in infected mesophyll protoplasts. A model for the expression of the middle-component RNA is presented.
Shope Fibroma Virus I. Biological and Molecular Properties of a Cytocidal and a Noncytocidal Strain
Authors: Pogo Beatriz G. T. , Freimuth Paul , Stein Adriane
Published: January 2020

The biological and molecular properties of two strains of Shope fibroma virus (SFV) were compared. SFV-I was highly cytocidal to most of the cell lines tested and produced pocks in the chorioallantoic membrane of chick embryos. By contrast, SFV-W did not produce cytopathic effects in any of the cell lines or in the chorioallantoic membrane, but it induced characteristic foci 3 to 4 days after infection. Both strains produced tumors when inoculated into the skin of susceptible rabbits. Maximal infectivity in BSC-1 cells was reached by both strains between 24 to 48 h after inoculation. Viral DNA synthesis also took place at the same time, although cells infected with SFV-I incorporated three times more [
3
H]thymidine than cells infected with SFV-W. Sedimentation analysis and hydroxylapatite chromatography of the two viral DNAs indicated that their molecular weights were similar and that both were naturally cross-linked. Digestion with three restriction endonucleases, however, revealed that they had different restriction sites. When SFV-I and vaccinia DNA were compared, the restriction patterns were more alike. Analysis of the virion structural proteins by gel electrophoresis indicated that SFV-I, SFV-W, and vaccinia virus had many polypeptides in common, although there were distinctive differences among the three viruses. Finally, the results of plaque neutralization tests with different antisera showed that SFV-I and SFV-W shared common antigens and that vaccinia antiserum inhibited SFV-I but not SFV-W. We conclude that the SFV-I genome contains information for both cytolysis and tumorigenesis. This unusual virus may be a recombinant between an orthopoxvirus and a leporipoxvirus.
Initiation and regulation of simian virus 40 early transcription in vitro
Authors: Lebowitz P , Ghosh P K
Published: January 2020

We have studied initiation and regulation of early transcription of simian virus (SV40) DNA in vitro by eucaryotic RNA polymerase II, using both a crude HeLa cell extract and a partially purified calf thymus polymerase supplemented with a HeLa cell S100 fraction. Analysis of initiation sites by primer-directed cDNA synthesis and sequencing of cDNA's has revealed that early transcription is initiated at a multiplicity of sites corresponding to the 5' termini of early viral mRNA's. The pattern of in vitro initiation closely resembles the pattern of 5' termini of early mRNA's late in the lytic cycle, with principal initiations between residues 5184 to 5194, upstream from the early Hogness-Goldberg (TATA) sequence, and at residue 5123, well downstream from this sequence. In vitro transcription is initiated to a lesser extent at sites between residues 5150 and 5155, the principal 5' termini of early mRNA's in transformed cells and early in lytic infection, located 21 to 26 nucleotides downstream from the TATA sequence. Initiation occurs at identical sites and with similar efficiencies on form I and linearized DNA templates. There are minor differences in the efficiency of initiation at specific sites by the two transcriptional systems. Studies using a DNA template cleaved just downstream from the TATA sequence and a second template cleaved through a pair of 72-base-pair tandem repeats starting 87 nucleotides upstream from the TATA sequence have revealed that neither the TATA sequence nor the repeats are essential for early transcription in vitro. However, removal of the TATA and upstream sequences shifts initiation of transcription principally to the residue 5123 site. Comparison of the relative efficiencies of transcription on intact wild-type DNA, the two cleaved DNAs, and DNA from a deletion mutant suggests that all or most of the sequences constituting an early promoter lie within the genomic region 60-70 to 140 nucleotides upstream from the principal 5' termini of the early mRNA's.
Adsorption of the tailed mycoplasma virus L3 to cell membranes
Authors: Haberer K , Maniloff J
Published: January 2020

The adsorption properties of the tailed bacteriophage L3 to Acholeplasma laidlawii cells were studied. Adsorption followed a biphasic curve. Reversibility and virus heterogeneity were not sufficient to explain the break in the adsorption curve. Binding studies showed that each colony-forming unit could bind about 350 virions. The electrostatic nature of L3 adsorption was indicated by the effect of cations, pH, and temperature on the adsorption rate constant. L3 adsorption appeared to have a requirement for Ca2+, which could not be replaced by the mono- and divalent cations examined. Ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid inhibition of adsorption was totally reversed by added Ca2+. The effects of EDTA, proteases, and lectins on absorption indicated that membrane proteins are the L3 receptors. The model for L3 adsorption is a multivalent one involving lateral diffusion of adsorbed virions and receptor proteins.
Altered pharmacological properties of liposome-associated human interferon-alpha
Authors: Eppstein D A , Stewart W E
Published: January 2020

Human interferon-alpha was associated in different ways with positively (stearylamine) and negatively (phosphatidylserine) charged phosphatidylcholine multilamellar vesicles, depending on the presence or absence of a cholesterol component. Inclusion of cholesterol resulted in interferon that was significantly (P = 0.0001) more deeply internalized within the liposomes, such that detergent disruption was necessary before most of the interferon activity was expressed. Interferon was stably associated with stearylamine-containing liposomes, both with and without a cholesterol component. However, inclusion of cholesterol in the phosphatidylserine-containing liposomes was necessary for stable association of the interferon for more than 2 days at 4 degrees C or for more than 24 h at 37 degrees C. After intramuscular injection into mice, liposome-associated interferon in reverse-phase evaporation vesicles was retained at the local site of injection significantly longer than free interferon. Even 3 days after intramuscular injection, stearylamine-containing liposomes with or without cholesterol resulted in local interferon levels that were comparable to the peak levels obtained 2 to 4 h after free interferon was injected. In contrast, free interferon was not detectable in the local muscles 24 h after injection of 10(4.6) U. Liposomes containing phosphatidylserine and cholesterol resulted in intermediate levels of local interferon retention; without a cholesterol component, phosphatidylserine-containing liposomes resulted in no increased local interferon retention compared with the results when free interferon was injected.
Organization of type C viral DNA sequences endogenous to baboons: analysis with cloned viral DNA
Authors: Battula N , Hager G L , Todaro G J
Published: January 2020

Unintegrated linear and circular forms of baboon endogenous type C virus M7 DNA were prepared from M7-infected cells by chromatography on hydroxyapatite columns, and the circular DNAs were purified in cesium chloride-ethidium bromide equilibrium density gradients. The circular DNAs were linearized by digestion with EcoRI, which had a unique site on the viral DNA. The linearized DNA was then inserted into lambda gtWES. lambda B at the EcoRI site and cloned in an approved EK2 host. Molecularly cloned full-length M7 DNA was restricted with BamHI, and the resulting five subgenomic fragments were then subcloned individually in plasmid pBR322. The organization and sites of integration of the approximately 100 copies of M7 DNA sequences endogenous to baboons were investigated by digesting the DNA with restriction enzymes and identifying the virus-specific fragments by hybridization to labeled probes made by using the molecularly cloned full-length and subgenomic fragments of the viral DNA. We found that most of the endogenous sequences had sizes and organizations similar to those of the unintegrated viral DNA and therefore approximately similar to the RNA of the infectious virus. A few of the multiple sequences had deletions in the 3' end (envelope region), and some of the sequences either lacked or contained modified BamHI restriction sites on the 5' end of the viral DNA. The endogenous viral DNA sequences were nontandem, uninterrupted, and colinear with the DNA of the infectious virus, and they were integrated at different sites in the baboon DNA, like the M7 proviral DNA sequences acquired upon infection.
Characterization of genome structure of amphotropic and ecotropic wild mouse retroviruses
Authors: Lai M M , Shimizu C S , Rasheed S , Pal B K , Gardner M B
Published: January 2020

We studied the RNA genomes of several wild mouse type C retroviruses by using RNase T1-oligonucleotide fingerprinting. The amphotropic and ecotropic viruses of field strain 1504 produced very similar oligonucleotide fingerprints, but each also had several unique oligonucleotides. All of these unique oligonucleotides were located in the env gene region and were probably responsible for the host range differences between these viruses, as well as the lymphomagenic and paralytogenic properties of the viruses. We obtained similar results with the amphotropic and ecotropic viruses of another field strain (4070), which was isolated from a mouse from a different trapping area. The amphotropic viruses of several field strains (strains 1504, 4070, and 1313) were more closely related than the ecotropic viruses of different strains (strains 1504, 4070, and 4996). These findings suggested that the genetic sequences of the amphotropic viruses are more conserved than those of ecotropic viruses isolated from the same wild mice.
Inhibition of Sindbis virus maturation after treatment of infected cells with trypsin
Authors: Adams R H , Brown D T
Published: January 2020

Brief treatment of Sindbis virus-infected BHK-21 or Vero cells with low concentrations of trypsin irreversibly blocked further production of progeny virions after removal of the enzyme. The inhibitory effects of the trypsin treatment could only be demonstrated in cells in which virus infection was established; optimal inhibition occurred at ca. 3 h postinfection. Production of virus structural proteins PE2, E1, and C occurred at normal levels in inhibited cells. PE2 and E1 were also transported to the cell plasma membrane during inhibition; however, PE2 was not cleaved to E2, and little capsid protein became membrane associated relative to control cells. Although trypsin treatment had no effect on Sindbis protein synthesis, the production of both 26S and 42S RNA was greatly reduced. Similar trypsin treatment of BHK cells infected with vesicular stomatitis virus had no detectable effect on the course of virus infection.
Uniform Nomenclature for Monoclonal Antibodies Directed Against Virus-Coded Proteins of Simian Virus 40 and Polyoma Virus
Authors: Crawford Lionel , Harlow Ed
Published: January 2020

A uniform nomenclature has been agreed upon for monoclonal antibodies directed against virus-coded proteins of simian virus 40 and polyoma virus. Blocks of numbers from PAb1 to PAb999 have been allocated to workers involved in the isolation of monoclonal antibodies of this type. The correspondence between PAb numbers and previously used names is given.
Herpesvirus sylvilagus I. Polypeptides of virions and nucleocapsids
Authors: Cohrs R , Rouhandeh H
Published: January 2020

Herpesvirus sylvilagus was propagated in juvenile cotton tail rabbit kidney cells and purified from the cytoplasmic fraction of the infected cells. The purification procedure included zonal centrifugation through a 5 to 30% dextran t-10 gradient, followed by equilibrium centrifugation in a 5 to 50% potassium tartrate gradient. H. sylvilagus formed one band after centrifugation through the tartrate gradient at a density of 1.22 g/cm3. Contamination of the purified virus preparation by cellular proteins was less than 0.2% as determined by the removal of radioactivity from an artificially mixed sample containing [35S]methionine-labeled control cells and nonlabeled infected cells. H. sylvilagus nucleocapsids were isolated from infected cell nuclei and purified by sedimentation through a 36% sucrose cushion, followed by equilibrium centrifugation in 5 to 50% tartrate gradient. Forty-four polypeptides ranging in molecular weight from 18,000 to 230,00 were resolved when [35S]methionine-labeled enveloped H. sylvilagus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Seventeen polypeptides found within the enveloped virus were also identified with the nucleocapsid. Six additional nucleocapsid polypeptides han no counterparts within the enveloped virus. The major polypeptide within both the virus and the nucleocapsid had a molecular weight of 150,000.
Restriction Endonuclease Mapping of the Proviral DNA of the Exogenous RIII Murine Mammary Tumor Virus
Authors: Etkind Polly R. , Szabo Paul , Sarkar Nurul H.
Published: January 2020

Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme
Hpa
I, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this
Hpa
I fragment with
Kpn
I,
Hin
dIII,
Eco
RI,
Bam
HI,
Bgl
II,
Pst
I,
Sst
I,
Sal
I, and
Xho
I enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three
Bam
HI and two
Eco
RI cleavage sites, a
Hpa
I cleavage site in the terminal 3′-5′ repeat unit of the provirus, and the absence of an
Xho
I cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with
Pst
I are different from the sizes of the fragments obtained by
Pst
I cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three
Sst
I (
Sac
I) cleavage sites, whereas the C3H provirus has only two
Sst
I sites.
Hpa
I digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells.
Pst
I digestion of the
Hpa
I 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a
Pst
I-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb
Pst
I fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.
Mobility of endogenous ecotropic murine leukemia viral genomes within mouse chromosomal DNA and integration of a mink cell focus-forming virus-type recombinant provirus in the germ line
Authors: Quint W , van der Putten H , Janssen F , Berns A
Published: January 2020

Characterization of endogenous ecotropic Akv proviruses in DNA of low and high leukemic mouse strains revealed the presence of one to six copies of the Akv genome per haploid genome equivalent integrated in the germ line. Low leukemic strains analyzed so far contained only one complete copy of the Akv proviral DNA. The site of integration varied among strains, although genetically related strains often carried the Akv proviral gene in the same chromosomal site. The different substrains of the AKR mouse displayed the presence of variable numbers (two to six) of Akv genomes. In all substrains one Akv genome was present in an identical chromosomal site; this locus probably comprised the progenitor genome. Closely related substrains had several Akv proviral DNAs integrated in common sites. The accumulation of Akv genomes in the germ line of the AKR/FuRdA strain is likely the result of independent integration events, since backcross studies with the Akv-negative 129 strain showed random segregation of all six proviral loci. The AKR/Cnb strain carried a recombinant provirus in the germ line. This provirus resembled in structure the AKR mink cell focus-forming viruses, which are generated by somatic recombination during leukemogenesis. Therefore, the germ-line amplification of Akv proviral DNAs occurs most likely through infection of embryonic cells by circulating virus.
Adsorption of Rous sarcoma virus to genetically susceptible and resistant chicken cells studied by laser flow cytometry
Authors: Notter M F , Leary J F , Balduzzi P C
Published: January 2020

Quantitative binding of Rous sarcoma virus (RSV) of different antigenic subgroups to chicken cells was examined by using a laser flow cytometer/cell sorter. RSV of subgroups A, C, and E, labeled with the fluorescent membrane probe rhodamine-18, bound 2 to 10 times more to genetically susceptible chicken embryo fibroblasts than to resistant cells, as measured by flow cytometry on a single-cell basis. This suggested that susceptible cells possess both specific and nonspecific receptors for virus adsorption, whereas resistant cells bind virus only by means of nonspecific sites. Polybrene at low concentration increased eightfold the binding of virus. Higher levels of Polybrene inhibited adsorption. Cell binding sites were saturable, and attachment of labeled virus could be partially blocked by preexposure of cells to unlabeled RSV. Virus surface glycoproteins played an important role in adsorption, since their removal with bromelain decreased binding of virus to susceptible cells. Maximal binding of RSV to both susceptible and resistant cells occurred within 10 min, although the level of binding was up to 10-fold higher for susceptible cells. Binding to all cell types showed a broad distribution. This implies that there are considerable differences in the number of virions bound per cell.
DNA sequence of the 5' terminus containing the replication origin of parvovirus replicative form DNA
Authors: Rhode S L , Klaassen B
Published: January 2020

The nucleotide sequence of the 5' terminus of the parvovirus H-1 was determined. There are two orientations of the 242-base-pair terminal palindrome in native replicative form DNA, one inverted with respect to the other. Adjacent to the terminal palindrome is an AT-rich region that is noncoding and contains a 55-base-pair tandem repeat. The addition mutant of H-1, DI-1, was also sequenced in this region and shown to have three copies of the tandem repeat sequence. Similarly, the related parvovirus H-3 contains only one copy of this repeat sequence. This region contains the replication origin for parvovirus replicative form DNA replication. Some of the implications of these results are discussed.
Correlation of the Genetic Map and the Endonuclease Site Map of
Bacillus subtilis
Bacteriophage SP02
Authors: Graham S. , Sutton S. , Yoneda Y. , Young F. E.
Published: January 2020

By marker rescue of bacteriophage SP02
sus
mutants with purified bacteriophage SP02 DNA fragments, 11 of the 17 known bacteriophage SP02
sus
loci were assigned to discrete DNA fragments. The left-most genetic locus,
susA
, was found to reside near one bacteriophage SP02 terminus (
Eco
RI-C1 fragment), whereas the right-most genetic locus,
susP
, was found to reside near the other bacteriophage SP02 terminus (
Eco
RI-C2 fragment). The physical locations of the intervening genetic loci were found to be consistent with the previously determined genetic order. Evidence was also obtained which suggested that at least one end of a transforming DNA fragment is degraded during DNA uptake by the competent bacterium.
Surface antigenic determinants of mammalian "hepadnaviruses" defined by group- and class-specific monoclonal antibodies
Authors: Cote P J , Dapolito G M , Shih J W , Gerin J L
Published: January 2020

The hepatitis B-like viruses (human hepatitis B virus, woodchuck hepatitis virus, ground squirrel hepatitis virus, and duck hepatitis B virus) are hepatotropic DNA viruses which have been referred to collectively as "hepadnaviruses." Using a murine monoclonal antibody (101-2) to the surface antigen of woodchuck hepatitis virus, we have shown that the surface antigens of mammalian hepadnaviruses (HBsAg, WHsAg, and GSHsAg) are antigenically related via a common determinant (HV/101). Furthermore, analysis with other monoclonal antibodies to WHsAg revealed that WHsAg and GHsAg are antigenically distinct, although the antigens had more determinants in common with each other than with HBsAg. The hepadnavirus group-specific antibody (101-2) reacted with HBsAg subtypic variants in a group-specific rather than subtype-specific manner. In conjunction with observations with an HBsAg-specific, group-reactive monoclonal antibody (BX259), the present data suggest that there are at least two group-reactive epitopes of HBsAg: one which is virus specific (HBV/259) and one which is common to two other mammalian hepadnaviruses (HV/101).
Avian sarcoma virus UR2 encodes a transforming protein which is associated with a unique protein kinase activity
Authors: Feldman R A , Wang L H , Hanafusa H , Balduzzi P C
Published: January 2020

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.
Genome of Reticuloendotheliosis Virus: Characterization by Use of Cloned Proviral DNA
Authors: Rice Nancy R. , Hiebsch Ronald R. , Gonda Matthew A. , Bose Henry R. , Gilden Raymond V.
Published: January 2020

Reticuloendotheliosis virus is an avian type C retrovirus that is capable of transforming fibroblasts and hematopoietic cells both in vivo and in vitro. This virus is highly related to the three other members of the reticuloendotheliosis virus group, including spleen necrosis virus, but it is apparently unrelated to the avian leukosis-sarcoma virus family. Previous studies have shown that it consists of a replication-competent helper virus (designated REV-A) and a defective component (designated REV) that is responsible for transformation. In this study we used restriction endonuclease mapping and heteroduplex analysis to characterize the proviral DNAs of REV-A and REV. Both producer and nonproducer transformed chicken spleen cells were used as sources of REV proviral DNA; this genome was mapped in detail, and fragments of it were cloned in λgtWES·λB. The infected canine thymus line Cf2Th(REV-A) was used as a source of REV-A proviral DNA. The restriction maps and heteroduplexes of the REV and REV-A genomes showed that (proceeding from 5′ to 3′) (i) REV contains a large fraction of the REV-A
gag
gene (assuming a gene order of
gag-pol-env
and gene sizes similar to those of other type C viruses), for the two genomes are very similar over a distance of 2.1 kilobases beginning at their 5′ termini; (ii) most or all of REV-A
pol
is deleted in REV; (iii) REV contains a 1.1 kilobase segment derived from the 3′ end of REV-A
pol
or the 5′ end of
env
or both; (iv) this
env
region in REV is followed by a 1.9-kilobase segment which is unrelated to REV-A; and (v) the helper-unrelated segment of REV extends essentially all of the way to the beginning of the 3′ long terminal repeat. Therefore, like avian myeloblastosis virus but unlike the other avian acute leukemia viruses and most mammalian and avian sarcoma viruses, REV appears to be an
env
gene recombinant. We also found that the REV-specific segment is derived from avian DNA, for a cloned REV fragment was able to hybridize with the DNA from an uninfected chicken. Therefore, like the other acute transforming viruses, REV appears to be the product of recombination between a replication-competent virus and host DNA. Two other defective genomes in virus-producing chicken cells were also cloned and characterized. One was very similar to REV in its presumptive
gag
and
env
segments, but instead of a host-derived insertion it contained additional
env
sequences. The second was similar (but not identical) to the first in its
gag
and
env
regions and appeared to contain an additional 1-kilobase inversion of REV-A sequences.
Detection of herpes simplex virus type 2 glycoproteins expressed in virus-transformed rat cells
Authors: Lewis J G , Kucera L S , Eberle R , Courtney R J
Published: January 2020

Rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2) were assayed for the expression of certain virus-specific glycoproteins on the surface membranes. Monospecific antisera to HSV-2-specific glycoproteins, designated gAgB, gC, and gX, were used in membrane immunofluorescence studies with HSV-2-transformed cell lines tREF-G-1, tREF-G-2, and a tumor-derived rat fibrosarcoma cells line produced in syngeneic rats inoculated with tREF-G-1 cells. Analysis of the three HSV-2-transformed cell lines showed that antisera to the gAgB and gX glycoproteins were reactive with these cells. In contrast, no significant reactivity was observed when anti-gC serum was reacted with the HSV-2-transformed cell lines. All three antiglycoprotein sera reacted positively with rat cells productively infected with HSV-2. Additionally, the HSV-2-transformed and tumor-derived cell lines showed positive internal immunofluorescence after reaction with antiserum to an early, nonstructural viral protein designated VP143 (molecular weight, 143,000). Infectivity of HSV-2 in standard plaque assays was neutralized by hyperimmune rat antisera to tREF-G-2 or rat fibrosarcoma cells and to HSV-2 virions and by sera from rats bearing the fibrosarcoma. Adsorption of rat-anti-HSV-2 serum with tREF-G-2 or rat fibrosarcoma cells reduced neutralizing activity to 10 and 12%, respectively, compared with 90% neutralization by antiserum adsorbed with nontransformed rat embryo fibroblast cells and 100% neutralization with unadsorbed antiserum. In summary, HSV-2-transformed rat cells retained and expressed genetic information necessary for the production of HSV-2 glycoproteins and a nonstructural protein after high passage in tissue culture or in the syngeneic host.
Ψ, a Temperate Phage of
Agrobacterium tumefaciens
, Is Mutagenic
Authors: Expert Dominique , Tourneur Jacques
Published: January 2020

A new temperate bacteriophage (phage Ψ) of
Agrobacterium tumefaciens
strain B91 is described. Many octopine-utilizing strains of
A. tumefaciens
harbor prophage Ψ or a phage that is similar if not identical to it. This phage has a very narrow host range, and we found that its growth is strongly reduced in strains which carry an octopine pTi plasmid. When sensitive bacteria are infected with Ψ, 1 to 3% of the survivors carry mutations on the chromosome, as well as on the pTi plasmid. This phenomenon appears to be a direct consequence of lysogenization. The possible mechanisms whereby such Ψ-induced mutations occur are discussed.
5-methyl-dCTP deaminase induced by bacteriophage XP-12
Authors: Wang R Y , Ehrlich M
Published: January 2020

Bacteriophage XP-12, whose DNA contains 34 mol% 5-methylcytosine, induces the synthesis of a unique enzyme, 5-methyl-dCTP deaminase. The substrate for this enzyme, 5-methyl-dCTP, is produced by reactions catalyzed in part by other phage-induced enzymes, and the product of the reaction is dTTP. The deaminase therefore provides a novel pathway for biosynthesis of thymine residues for phage XP-12 DNA. Evidence is presented that this pathway is used for dTTP synthesis in XP-12-infected Xanthomonas oryzae.
Gene responsible for superinfection exclusion of heteroimmune corynebacteriophage
Authors: Groman N , Rabin M
Published: January 2020

Wild-type beta and gamma corynebacteriophages are heteroimmune and infect lysogens of each other productively. Unlike their wild-type counterparts, the bin mutants of each phage are excluded in lysogens carrying the heteroimmune phage. The wild-type phages overcome exclusion by means of the bin gene product which appears to act as an antirepressor. When repression is lifted, exclusion of bin mutants is abolished (N. Groman and M. Rabin, J. Virol. 28:28-33, 1978; J. Virol. 36:526-532, 1980). It has not been clear whether the excluding compound is the immune repressor itself or one whose synthesis is positively regulated by repressor. We have isolated beta exclusion mutants (xcl) that as prophage exhibited normal immune repression but no longer excluded gamma-bin mutants. Furthermore, we have shown that an xcl phage with an active immune repressor acted in trans to continue the positive regulation of exclusion by a second xcl+ prophage whose immune repressor was inactivated. From these results it was concluded that there is a gene distinct from the imm gene which is directly or indirectly responsible for exclusion. The xcl gene, mapped in prophage crosses, was located between imm and bin, that is, in the regulatory region of the phage genome. The simplest hypothesis compatible with the established observations is that beta immune repressor regulates the expression of the xcl and bin genes, the former positively and the latter negatively. It is likely that an analogous regulatory model applies to gamma phage since it has already been shown that both beta and gamma have bin alleles.
Complete nucleotide sequence of the polymerase 3 gene of human influenza virus A/WSN/33
Authors: Kaptein J S , Nayak D P
Published: January 2020

The complete nucleotide sequence of polymerase 3 (P3) gene of a human influenza virus (A/WSN/33) has been determined using cDNA clones except for the last 11 nucleotides which were obtained by direct RNA sequencing. The WSN P3 gene contains 2,341 nucleotides and codes for a protein of 759 amino acids (molecular weight 85,800). The WSN P3 protein, as deduced from the plus-strand DNA sequence, is basic and enriched in positively charged amino acids. In addition, it contains clusters of basic amino acids which may provide sites for the interaction of P3 protein with the capped primer, template, and/or other polymerase proteins during the transcriptive and replicative processes of influenza viral RNA.
Subgenomic mRNA in OK10 Defective Leukemia Virus-Transformed Cells
Authors: Saule S. , Sergeant A. , Torpier G. , Raes M. B. , Pfeifer S. , Stehelin D.
Published: January 2020

OK10, a defective leukemia virus, is produced as a defective particle by so-called nonproducer transformed quail fibroblasts. OK10 defective viral particles contain an 8-kilobases (kb)-long genomic RNA, lack any detectable reverse transcriptase activity, and are not infectious. We studied the genetic content of OK10 RNA extracted from both virions and infected cells. As shown by RNA-cDNA hybridizations in stringent conditions, about 77% (6.4 kb) of the OK10 8.0kb RNA was related to avian leukosis viruses in the three structural genes
gag, pol
, and
env
, as well as in the
c
region. The remainder of the OK10 genome-encoding capacity (≤1.6 kb) was homologous to the MC29-specific transforming sequence
myc
(m) and therefore has been named
myc
(o).
Eco
RI restriction analysis of the OK10 integrated proviral DNA with different probes indicated the presence of only one provirus in the OK10 QB5 clone, which agreed with the gene order: 5′-
gag
-Δ
pol
-
myc
(o)-Δ
env
-
c
- 3′. Heteroduplex molecules formed between the viral OK10 8.0-kb RNA and the 6.8-kb
Sac
I DNA fragment of the Prague A strain of Rous sarcoma virus confirmed that structure and indicated that the
myc
(o) sequence formed a continuous RNA stretch of 1.4 to 1.6 kb long between Δ
pol
and Δ
env
. We also examined the
myc
(o)-containing mRNA's transcribed in OK10-transformed cells. OK10-transformed quail fibroblasts (OK10 QB5) transcribed two mRNA species of 8.0 and 3.6 kb containing the
myc
(o) sequence. The genetic content of the 3.6-kb species made it a possible maturation product of the genome size 8-kb species by splicing out the
gag
and
pol
sequences. In OK10-transformed bone marrow cells (OK10 BM), a stable bone marrow-derived cell line producing OK10, the
myc
(o) sequence was found in four RNA species of 11.0, 8.0, 7.0, and 3.6 kb. Again, the genetic content of these mRNA's indicated that (i) the 3.6-kb species could be spliced out of the 8.0-kb-genome size mRNA and (ii) the 11.0-kb-long mRNA could represent a read-through of the OK10 provirus, the corresponding maturation product being, then, a 7.0-kb mRNA. The 7.0- and 3.6- kb mRNA's both contained the
myc
(o) sequence, but no sequences related to the
gag
or
pol
gene. In conclusion, whereas the
myc
sequences have been generally thought to be expressed through a
gag-onc
fusion protein, as for MC29 and CMII viruses, our experiments indicate that they could also be expressed as a non-
gag
-related product made from a subgenomic mRNA in the OK10-transformed cells.
Genome of
Bacillus subtilis
Bacteriophage SPP1: Structure and Nucleotide Sequence of
pac
, the Origin of DNA Packaging
Authors: Deichelbohrer Inge , Messer Walter , Trautner Thomas A.
Published: January 2020

The DNA of
Bacillus subtilis
bacteriophage SPP1 is terminally redundant and partially circularly permuted. To explain these parameters, we followed the Streisinger-Botstein models of phage maturation and assumed that packaging of SPP1 DNA begins at a unique genomic site (“
pac
”) and proceeds sequentially from there. We describe the sequence of about 1,000 nucleotides surrounding
pac
. This together with size determinations of small,
pac
-terminated restriction fragments has revealed heterogeneity of the natural
pac
ends of SPP1 DNA. Such ends fell in each DNA strand into a region of five to seven nucleotides. However, within this range more than 50% of all molecules terminated with defined cytosines on both strands, generating a 3′ protruding terminus. The nucleotide sequence of the DNA segment surrounding
pac
did not reveal any features which would distinguish this region.
Two defective DNAs of human polyomavirus JC adapted to growth in human embryonic kidney cells
Authors: Yoshiike K , Miyamura T , Chan H W , Takemoto K K
Published: January 2020

Human polyomavirus JC (JCV) adapted to growth in human embryonic kidney (HEK) cells contains two or more species of shorter-length viral DNA even after two cycles of plaque purification in HEK cells. We have molecularly cloned JCV DNA from one plaque isolate and determined the physical map of its DNA. Using gel electrophoresis and electron microscopy, we found that the cloned DNA consisted of two classes of JCV DNA. One class of DNA had a deletion of 30% (between 0.7 and 1.0 map units from the EcoRI site) and an insertion of 1% at the same site in the late region. The other had a deletion of 35% (0.18 to 0.53 map units) and an insertion of 12% at the same site in the early region of the JCV genome. Both DNAs had added sequences near the origin of DNA replication. From their structure, the two DNAs appear to complement each other. These results indicate that the HEK-adapted JCV may be replicating by complementation between two defective mutants.
Differing populations of intracisternal A-particle genes in myeloma tumors and mouse subspecies
Authors: Shen-Ong G L , Cole M D
Published: January 2020

Intracisternal A-particle genes form a family of endogenous retrovirus-like genetic elements that are transcribed in mouse plasmacytomas (myeloma tumors). Two types of A-particle genes that can be differentiated by a sequence of 0.5 kilobase found in one type but not the other have been identified. Quantitative Southern blot analysis was used to measure the populations of different A-particle genes in DNAs from BALB/c mice, the Japanese subspecies Mus musculus subsp. molossinus, and myeloma tumors. The majority of the genes (715 copies per haploid genome or 76%) were found to be nearly identical except for small changes in conserved restriction enzyme sites. The second type of A-particle gene was much less abundant with 90 copies representing approximately 10%. The A-particle RNA in MOPC104E and MOPC315 was found to be colinear with a small portion of this latter type, comprising only 2% of the endogenous intracisternal A-particle sequences. Myeloma tumor DNA was found to have a two- to fourfold increase in the number of these genes, suggesting that the intracellular viruses have been activated to produce a double-stranded complementary DNA which subsequently integrated into the tumor genome. Analysis of M. musculus subsp. molossinus DNA revealed similar but shifted populations of A-particle genes, when compared with BALB/c DNA, except for the absence of a prominent EcoRI-HindIII band at 3.9 kilobases. This latter band, representing approximately 15% of the A-particle genes in BALB/c DNA, was shown to be a deletion variant of the most abundant gene family.