bluuebunny/crossref_metadata_2025 huggingface dataset:
Interstrain Variation of the Major Internal Structural Component (p30
gag
) of Two Murine Oncornaviruses: Comparative Immunochemical, Biochemical, and Biophysical Analysis
Authors: Burnette W. Neal , Mitchell William M.
Published: January 2020

The major internal structural protein (p30
gag
) of the Moloney leukemia virus and the endogenous Y-1 murine oncornavirus was examined for biochemical and biophysical manifestations of interstrain antigenic variation. Although the two viral proteins share murine group-specific antigenic determinants, the Y-1 virus p30 appeared to have both a lower relative number of such determinants and a decreased affinity at the cross-reactive sites for Moloney virus p30 monospecific antibodies. Further, immunological analysis indicated the presence of unique antigenic sites on the Moloney virus p30 not shared by the analogous Y-1 virus molecule. The two polypeptides copurified and had similar isoelectric points (pH 6.2 to 6.3) and sedimentation coefficients (2.47S). However, equilibrium sedimentation yielded a significant mass difference between the two proteins, 28,300 ± 600 and 31,000 ± 300 daltons for the Moloney and Y-1 virus molecules, respectively. Amino acid analysis indicated a concomitant increase in total residues for the Y-1 virus p30, although a number of residues appeared to have been conserved between the two viral proteins. Conformational studies and hydrodynamic calculations demonstrated marked secondary and tertiary structural differences; with the Y-1 virus p30 being an asymmetric prolate ellipsoid containing 27 to 28% α-helix and Moloney virus p30 being somewhat more spherical and possessing an α-helical content of 50 to 55%. Two-dimensional mapping of
125
I-labeled tryptic peptides of each p30 suggested that considerable sequence heterogeneity is responsible for many of the biophysical, biochemical, and immunochemical differences in these two analogous structural proteins.
Identification of a common antigen of herpes simplex virus bovine herpes mammillitis virus, and B virus
Authors: Norrild B , Ludwig H , Rott R
Published: January 2020

In immunoelectrophoretic analyses one common antigen was demonstrated in antigen preparations from herpes simplex virus types 1- and 2- (HSV-1 and HSV-2), bovine herpes mammillitis (BHM) virus-, and B virus-infected cells solubilized by Triton X-100. The antigen was also demonstrated in solubilized purified HSV-1 and BHM virus. The common antigen was identified as antigen 11 of HSV-1 or HSV-2. Differences were found in the polypeptide composition of the related antigens when isolated from the four different herpesviruses, but a glycopolypeptide with a molecular weight of 125,000 was present in each of the four different antigen preparations, indicating that this polypeptide carried the common antigenic determinants.
Isolation and preliminary characterization of temperature-sensitive mutants of encephalomyocarditis virus
Authors: Radloff R J
Published: January 2020

Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.
Purification of virus-specific RNA from chicken cells infected with avian sarcoma virus: identification of genome-length and subgenome-leghth viral RNAs
Authors: Parsons J T , Lewis P , Dierks P
Published: January 2020

Avian sarcoma virus (ASV)-specific RNA was purified from ASV-infected cells by using hybridization techniques which employ polydeoxycytidylic acid-elongated DNA complementary to ASV RNA as well as chromatography on polyinosinic acid-Sephadex columns. The purity and nucleotide sequence composition of purified, virus-specific RNA were established by rehybridization experiments and analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. Polyadenylic acid-containing RNA purified from ASV-infected cells contained approximately 1 to 4% virus-specific RNA, compared with 0.06 to 0.15% observed in uninfected cells. Sucrose gradient analysis of virus-specific RNA isolated from ASV-infected cells revealed two major classes of polyadenylated viral RNA with sedimentation values of 36S and 26-28S. Cells infected with transformation-defective ASV (virus containing a deletion of the sarcoma gene) contained 34S and 20-22S viral RNA species. Double-label experiments employing infected cells labeled initially for 48 h with [3H]uridine and then for either 30, 60, or 240 min with [32P]phosphate showed that the intracellular accumulation of genome-length RNA (36S) was significantly faster than that of the 26-28S viral RNA species.
New late gene, dar, involved in the replication of bacteriophage T4 DNA. II. Overproduction of DNA binding protein (gene 32 protein) and further characterization
Authors: Wu J R , Yeh Y C
Published: January 2020

We have previously shown that the arrested DNA synthesis of mutant defective in T4 phage gene 59 can be reversed by a mutation in dar. In this paper, we have examined the effect of the dar mutation on the kinetics of gene 32 protein (DNA binding protein) synthesis, DNA packaging, progeny formation, and several other porcesses. Several lines of evidence are presented showing that the regulation of synthesis of gene 32 protein is abnormal in dar 1-infected cells. In these cells, gene 32 protein, an early protein, is also expressed late in the infectious cycle. Our data also indicate that the packaging og DNA into T4 phage heads is delayed in dar mutant-infected cells, and this in turn results in a 6- to 8-min delay in intracellular progeny formation, although the synthesis of late proteins appears to be normal, as shown by gel electrophoresis. We have also studied the phenotypes of the double mutant dar-amC5 (gene 59). The increased sensitivity to hydroxyurea caused by a mutation in the dar gene can be alleviated by a second mutation in gene 59, but an increased sensitivity to UV irradiation caused by a mutation in gene 59 cannot be alleviated by a second mutation in the dar gene. Therefore, the double mutant still exhibits abnormalities in the repair of UV lesions.
Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores
Authors: Clinton G M , Burge B W , Huang A S
Published: January 2020

The proteins of vesicular stomatitis virus (VSV) were analyzed on the basis of charge as well as size in polyacrylamide gels containing urea and acetic acid. The phosphorprotein NS was resolved into two major species. The less phosphorylated NS1 species contained about 10% fewer phosphate residues than the second species, NS2. These two phosphorylated forms were compartmentalized both in the virus and in the infected cell cytoplasm. Cores from virions and the core-containing fraction of the infected cell cytoplasm contained only the NS1 form. All of the more highly phosphorylated NS2 form and some of the NS1 form were found to be free of cores, whether they were derived from virions or from the infected cell. Therefore, the degree of phosphorylation appeared to determine whether or not the NS protein became bound to VSV cores. Moreover, the amount of bound NS1 protein relative to nucleocapsids increased as the pH of the culture medium was raised from 6.6 to 7.4. Because an increased in pH increases VSV replication (Fiszman et al., J. Virol. 13:801-808, 1974; Palma and Huang, in W.S. Robinson and C.F. Fox, ed., Mechanisms of Virus Disease, ICN-UCLA Symposia, p. 87-100, 1974), the NS1 protein may either regulate overall VSV RNA synthesis or regulate the switch between transcription and replication.
Identification of a herpesvirus isolated from cytomegalovirus-transformed human cells
Authors: Geder L , Hyman R W , Figueroa M , Oakes J E , Iltis J P , Dawson M S , Rapp F
Published: January 2020

Human cells transformed by cytomegalovirus and transplanted to athymic nude mice yielded a cytopathic virus, Hershey Medical Center virus, following prolonged in vitro passage of the tumor cells. The virus is a double-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence tests, but indirect immunofluorescence tests revealed cytomegalovirus-related antigenicity. Further immunological tests revealed that Hershey Medical Center virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears that Hershey Medical Center virus is infectious bovine rhinotracheitis virus, which presumably appeared in the cell culture as a contaminant from fetal calf serum.
Evidence that the neck appendages are adsorption organelles in Bacillus subtilis bacteriophage phi29
Authors: Moreno F , Bluzat-Moreno F G
Published: January 2020

A mutant of Bacillus subtilis unable to adsorb phage phi29 efficiently has been isolated. This mutant can be infected by host range mutants of the phage. Since the host range mutations map in cistron 12, which codes for neck appendage protein, this would tend to confirm that these organelles are involved in viral adsorption.
Growth control in simian virus 40-transformed rat cells: temperature-independent expression of the transformed phenotype in tsA transformants derived by agar selection
Authors: Rassoulzadegan M , Perbal B , Cuzin F
Published: January 2020

Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.
Biochemical properties of p15-associated protease in an avian RNA tumor virus
Authors: Dittmar K J , Moelling K
Published: January 2020

It was observed that the viral structural protein p15 from avian myeloblastosis virus emerges from ion-exchange column chromatography along with a proteolytic activity. p15 is apparently pure, as judged by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and isoelectric focusing. Increase and decrease in proteolytic activity coincided exactly with increasing and decreasing amounts of p15 during ion-exchange chromatography and during size fractionation by gell filtration. The proteolytic activity cleaved various substrates such as bovine serum albumin, ovalbumin, concanavalin A, and casein after denaturation by sodium dodecyl sulfate and heat. Highest enzyme activity was observed around pH 5.7. As judged from its cleavage pattern and its response to proteolytic inhibitors, the proteolytic activity appears papain-like, and the protease responsible for it may be classified as a thiol protease. If added to immunoprecipitated viral polyprotein precursor Pr76, p15 resulted in cleavage of Pr76,which could be inhibited by antibodies against p15.
Molecular diversity among five different endogenous primate retroviruses
Authors: Bryant M L , Sherr C J , Sen A , Todaro G J
Published: January 2020

Genetically transmitted retroviruses of Old and New World monkeys include type C viruses isolated from baboons (M7), macaque (MAC-1), and owl monkeys (OMC-1) and type D viruses from langurs (PO-1-Lu) and squirrel monkeys (SMRV, M534). Each of these isolates is unrelated to the others by nucleic acid hybridization criteria and contains a unique array of virion-associated proteins which can be resolved by agarose gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. The major structural protein of each virus has a distinct primary structure, as determined by two-dimensional tryptic peptide analysis, and is antigenically different from the others. The major virion phosphoproteins of endogenous primate type C viruses (pp15) are also different from those of type D viruses (pp13-pp14). Immunological and structural analyses show that the endogenous langur virus and the horizontally transmitted Mason-Pfizer virus of rhesus monkeys are closely related to one another, consistent with the sequence homology detected in their RNA genomes. Although certain radioimmunoassays detect interspecies antigenic determinants common to either the p30 or gp70 proteins of some of these viruses, no one assay has yet been designed which can detect all groups of endogenous primate retroviridae. The data lead to the conclusion that primates contain a minimum of three different sets of genetically transmitted type C and type D retroviral genes.
Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin
Authors: Morrison T G , McQuain C O , Simpson D
Published: January 2020

The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.
Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein
Authors: Gentsch J R , Bishop D H
Published: January 2020

Tryptic peptide analyses have been undertaken on the nucleocapsid (N) protein of snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses. Similar analyses have been performed on the N proteins of two recombinant viruses which have the large/medium/small RNA genome configurations: SSH/LAC/LAC and SSH/LAC/SSH. The results provide conclusive evidence that the S RNA of bunyaviruses codes for the the viral N protein.
Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes
Authors: Su Robert T. , DePamphilis Melvin L.
Published: January 2020

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for “normal” DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of “Okazaki pieces,” and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated α-
32
P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.
Replication-defective ecotropic murine leukemia viruses: Detection and quantitation of infectivity using helper-dependent XC plaque formation
Authors: Rein A , Bassin R H
Published: January 2020

Clones 8A and NP-N, which appear to be infected with replication-defective variants of murine leukemia virus, produce particles which do not form plques in the XC test. These particles formed XC plaques when amphotropic murine leukemia virus, which is XC negative, was added to the assay plates. This phenomenon can be used as a quantitiative infectivity assay for these replication-defective murine leukemia viruses.
Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies
Authors: Dudley Jaquelin P. , Butel Janet S. , Socher Susan H. , Rosen Jeffrey M.
Published: January 2020

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a

T
m

of 88.5°C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (<three molecules per cell) by RNA excess hybridization. Two of the three “virus-negative” cell lines were derived from tumors induced by the chemical carcinogen 7,12-dimethylbenz(α)anthracene, whereas the third tumor occurred spontaneously. Two lines from tumors induced by either viral (mammary tumor virus) or hormonal (17-β-estradiol) stimulus contained between three and nine molecules of MMTV RNA per cell by both RNA excess and cDNA excess hybridization. Clonal derivatives of these tumor lines had levels of viral RNA comparable to those of their parental lines. Therefore, it appears that the presence of detectable MMTV RNA sequences is not a necessary requirement for the maintenance of all murine mammary gland neoplasias.
Lysogenic conversion in Klebsiella pneumoniae: system which requires active immunity regulation for expression of the conversion phenomenon
Authors: Satta G , Pruzzo C , Debbia E , Calegari L
Published: January 2020

We have previously described Klebsiella pneumoniae MirM7b, which, although stably lysogenic for the inducible and nondefective phages FR2 and AP3, is not immune to superinfection by these same viruses. MirA12b, a strain which is lysogenic for FR2 and AP3 and immune to superinfection, has been derived from MirM7b. The sensitivity of this strain and that of the nonimmune parent to several bacteriophages have been compared in this work. It has been found that, whereas MirM7b is sensitive to coliphages P1, T3, T7, and phiI, MirA12b is fully resistant to all of them. It is shown that phages FR2 and AP3 convert Klebsiella strains to resistance to coliphage P1 and coliphages T3, T7, and phiI, respectively, and cause loss of surface antigens in lysogenic cells. To determine such a conversion, both FR2 and AP3 require expression of immunity to superinfection. This explains the differences that exist between MirM7b and MirA12b in both phage sensitivity and surface antigens. Hypotheses are presented to explain the peculiar need for an active superinfection repressor to express lysogenic conversion.
Low-multiplicity infection of Moloney murine leukemia virus in mouse cells: effect on number of viral DNA copies and virus production in producer cells
Authors: Fan H , Jaenisch R , MacIsaac P
Published: January 2020

Mouse cells infected with Moloney murine leukemia virus (M-MuLV) were prepared by two methods, and the number of M-MuLV-specific DNA copies in the infected cells was measured. The number of M-MuLV-specific DNA copies detected varied from one to eight per infected cell in different cell lines. Cells in which multiple rounds of viral infection occurred during establishment had on the average more viral DNA copies than cells in which infection at low multiplicity was performed, followed by cloning of the cells. However, even in cells derived by the low multiplicity of infection method, most cell lines carried more than one copy of M-MuLV-specific DNA. Virus production per cell was also measured, and no strict correlation was observed between the number of M-MuLV DNA copies present and the amount of virus produced.
Extent of Transcription of the E Strand of Polyoma Virus DNA During the Early Phase of Productive Infection
Authors: Acheson Nicholas H. , Miéville Françoise
Published: January 2020

Early polyoma virus-specific RNA, in nuclei and cytoplasm of cells labeled with [
3
H]uridine, was analyzed by hybridization with filter-bound
Hpa
II fragments of polyoma DNA. About 40% of labeled cytoplasmic virus-specific RNA hybridized with
Hpa
II fragment 2, which represents about 40% of the region coding for E-strand mRNA's; less than 5% hybridized with fragments 1 or 3, which lie outside this region. A somewhat lower proportion (about 30%) of labeled nuclear virus-specific RNA hybridized with fragment 2, and a small but significant fraction (7 to 14%) hybridized with fragments 1 and 3. About two-thirds of the nuclear RNA which hybridized to fragment 1 was complementary to the E strand, and one-third was complementary to the L strand. Results did not vary greatly in samples labeled for periods of from 15 min to 3 h. The major species of pulse-labeled nuclear polyoma-specific RNA sedimented at 22S and thus is slightly larger than the 19S cytoplasmic mRNA. These results show that most early nuclear RNA (∼75%) is transcribed from the region of the E strand, which codes for early mRNA's, and that there is probably a site at which transcription is terminated at the end of this region. However, a small amount of early nuclear RNA (∼15%) is transcribed from the remainder of the E strand, perhaps by readthrough of this termination signal. In addition, there is a small amount of transcription from the L strand, whose significance is unclear. Neither the L-strand transcripts nor the nonmessenger E-strand transcripts are transported to the cytoplasm.
Tryptic peptide analyses of polypeptides generated by premature termination of cell-free protein synthesis allow a determination of the Rauscher leukemia virus gag gene order
Authors: Murphy E C , Arlinghaus R B
Published: January 2020

Translation of Rauscher murine leukemia virus (R-MuLV) 35S RNA in an mRNA-dependent cell-free protein-synthesizing system yields polypeptides identical to authentic Pr65gag, the R-MuLV gag precursor, and Pr200gag-pol, the precursor to the R-MuLV reverse transcriptase. In addition to these polypeptides, the cell-free product contains a family of polypeptides of less than 65,000 molecular weight which appear to be generated by premature termination of protein synthesis within the viral gag gene. We compared the tryptic maps of several of these less than 65,000-molecular-weight premature termination polypeptides with that of full-size Pr65gag and found a progressive loss of tryptic peptides which could be assigned to known R-MuLV gag proteins. A 40,000-molecular-weight fragment, P40gag, lacked p10 and part of p30, placing p10 at the C terminus pf Pr65gag and p30 ajacent to it. Fragments of 33,000 (P33gag) and 27,000 to 28,000 (P27/28gag) molecular weight showed a successive loss of additional p30 tryptic peptides, but no loss of either p15 or p12. An 18,000-molecular-weight fragment lost p12 but retained p15. These data suggest an R-MuLV gag gene order of NH2-p15-p12-p30-p10-COOH.
Characterization of the amino-terminal tryptic peptide of simian virus 40 small-t and large-T antigens
Authors: Mellor A , Smith A E
Published: January 2020

Simian virus 40 small-t and large-T antigen were synthesized in vitro and labeled with methionine donated by initiator tRNA. Tryptic peptide fingerprinting was used to identify the amino-terminal peptide of the two proteins. Similar fingerprint analysis of small-t and large-T made in vitro in the absence of acetyl coenzyme A showed that the mobility of the amino-terminal peptide was changed under these conditions and suggested that it is acetylated. These data establish that the amino-terminal methionine residue of simian virus 40 small-t and large-T results from an initiation event, not post-translational cleavage, and provides additional evidence that the amino terminus of both proteins is acetylated. The identification of the amino-terminal peptide provides a useful marker for further studies on different forms of T-antigen from cells infected with and transformed by simian virus 40 and related viruses.
Structural proteins of equine infectious anemia virus
Authors: Cheevers W P , Ackley C M , Crawford T B
Published: January 2020

Equine infectious anemia virus was found to be comprised of fourteen polypeptides of molecular weight ranging from 10,000 to 79,000. Eighty percent of the virion protein was accounted for by five polypeptides, including two non-glycosylated components (p29 and p13) comprising one-half of the virion protein and three glycoproteins (gp77/79, gp64, and gp40).
Adenovirus type 12 gene 401 function and maintenance of transformation
Authors: Ledinko N , Schaeufele J , Soorma O
Published: January 2020

Rat (3Y1) and hamster embryo brain cells were transformed by wild-type adenovirus type 12 or the DNA-minus temperature-sensitive mutant ts401. The ts401-transformed 3Y1 cells, but not the wild-type transformants, displayed a temperature-sensitive response with respect to the following characteristics of the transformed phenotype: morphology, saturation density, growth rate, cloning in soft agar, colony formation on plastic at low cell densities in 1% serum medium, and the T antigen(s). Temperature shift-down experiments showed that the density-dependent inhibition of growth of the ts401-transformed cells was reversible, as was, to some extent, the low efficiency of colony formation at low cell densities in 1% serum. Examination of hamster transformants for their ability to clone in soft agar at permissive and nonpermissive temperatures showed that this property was temperature dependent, again only in the ts401 transformants and not in the wild-type transformants. Alteration in uptake of 2-deoxyglucose or in intracellular cyclic AMP content was not a characteristic of the adenovirus-transformed phenotype in the 3Y1 cells. The findings suggest that an active 401 function is required for maintenance of the adenovirus-transformed cell pheno-type.
Bovine coronavirus genome
Authors: Guy J S , Brian D A
Published: January 2020

The tissue culture-adapted strain (Mebus) of the bovine coronavirus was grown to titers of greater than 10(7) 50% tissue culture infective doses per ml in secondary bovine embryo kidney cells, and the RNA was isotopically labeled with [3H]uridine. The RNA was extracted from purified virus and was found to have the following properties. (i) It consisted primarily of a homogeneous large-molecular-weight species which comigrated electrophoretically with vesicular stomatitis viral RNA and therefore had an apparent molecular weight of 3.8 X 10(6). (ii) It remained as a 3.8 x 10(6)-molecular-weight molecule after heat denaturation when rapidly harvested virus was examined. (iii) It was 80% susceptible to pancreatic RNase A digestion in high (0.3 M) NaCl, and the 20% resistant fraction was 4S to 7S in size. (iv) It was polyadenylated to the extent that 40 and 60% of the native RNA bound to polyuridylic acid-Sepharose and oligodeoxythymidylic acid-cellulose, respectively, under conditions of high (0.5 M) NaCl.
Influenza B virus genome: assignment of viral polypeptides to RNA segments
Authors: Racaniello V R , Palese P
Published: January 2020

It was shown that all eight RNA segments of influenza B viruses are most likely monocistronic and code for eight virus-specific polypeptides. A genetic map of the influenza B virus genome was established, and six polypeptides (P1 protein, nucleoprotein, hemagglutinin, neuraminidase, M protein, and nonstructural protein) were unambiguously assigned to specific RNA segments. Molecular weight estimates of the eight individual genes are obtained by using the glyoxal method. These results suggest that each influenza B virus RNA segment has a greater molecular weight than the influenza A virus RNA segment which codes for the analogous gene product.
Physical characterization of the superhelical DNA genome of an enveloped mycoplasmavirus
Authors: Nowak J A , Maniloff J
Published: January 2020

Mycoplasmavirus MVL2 is a nonlytic enveloped virion containing DNA. This DNA has been shown to be a double-stranded circular superhelical molecule of 11.8 kilobase pairs (7.8 X 10(6) daltons). The superhelix density is greater than that of phi X174 RFI but less than that of PM2 phage DNA. A physical map of the MVL2 genome has been obtained using restriction endonucleases.
Subcellular distribution of newly synthesized virus-specific polypeptides in Moloney murine leukemia virus infected cells
Authors: Shanmugam G
Published: January 2020

Immune precipitation analysis of pulse-labeled proteins present in subcellular fractions of mouse embryo cells infected with Moloney murine leukemia virus showed the presence of anti-gp70 serum-precipitable viral envelope gene products mainly in the microsomal fractions of these cells. In contrast, anti-p30 serum-specific gag (group specific antigen) gene products were found to be distributed in similar amounts in both the microsomal and postmicrosomal supernatant fractions of pulse-labeled cells.
Inhibition of entry of the lipid-containing bacteriophage PR4 by fatty acid derivatives
Authors: Sands J A , Reinhardt A , Auperin D , Landin P
Published: January 2020

Amphiphilic fatty acid derivatives having a 14- to 20-carbon cis-unsaturated alkyl chain were found to be potent inhibitors of the entry process of the lipid-containing bacteriophage PR4.
Genes controlling receptors for ecotropic and xenotropic type C virus in Mus cervicolor and Mus musculus
Authors: Marshall T H , Rapp U R
Published: January 2020

Gene loci controlling cell surface receptors for murine leukemia virus were studied by using murine X Chinese hamster hybrid cells. Hybrids which exclusively segregate murine chromosomes were made by fusing Mus cervicolor and Mus musculus lymphocytes to hamster fibroblasts. Sensitivity to Moloney murine leukemia virus infecotion and specific binding of the envelope glycoprotein of Rauscher murine leukemia virus (gp70) cosegregate and isozyme analysis show an association with chromosome 5 in both species. With the possible exception of one clone, no evidence was found for a proviral integration site independent of chromosome 5. Evidence is presented for additional unlinked ectropic and xenotropic receptors independent of chromosome 5.
Molecular genetics of herpes simplex virus. III. Fine mapping of a genetic locus determining resistance to phosphonoacetate by two methods of marker transfer
Authors: Knipe D M , Ruyechan W T , Roizman B
Published: January 2020

We have transferred a genetic locus determining resistance to phosphonoacetic acid (PAAr) from one herpes simplex viral genome to another by two methods of marker transfer. One method requires recombination between an intact DNA molecule and a restriction endonuclease DNA fragment, whereas the other requires repair of a partial heteroduplex formed between the two DNA molecules. These two methods mapped the PAAr locus between positions 0.45 and 0.53 map units on the physical map of the viral DNA. Fine mapping of the PAAr locus showed that it maps at or near an EcoRI restriction endonuclease site at either 0.46 or 0.49 map units. We also describe and compare the two methods of marker transfer.
Structure of the murine leukemia virus envelope glycoprotein precursor
Authors: Witte O N , Wirth D F
Published: January 2020

The glycosylated env gene precurosr (Pr80env) of Moloney murine leukemia virus has been isolated by selective immunoprecipitation. Use of the drug tunicamycin to inhibit nascent glycosylation or specific cleavage with endoglycosidase H demonstrated that the precursor contained an apoprotein with a molecular weight of 60,000. The finished virion glycoprotein (gp70) was largely resistant to the action of endoglycosidase H. Chromatography of the glycopeptides of Pr80env in conjunction with endoglycosidase H digestion studies suggested that the precursor contained two distinct major glycosylation sites. Analysis of partial proteolytic cleavage fragments of Pr80env before and after endoglycosidase H treatment placed the two glycosylation sites within a 30,000-dalton region of the apoprotein sequence. Kinetic experiments showed that carbohydrate processing as well as proteolytic cleavage are late steps in the maturation of Pr80env.
Novel bacteriophage lambda mutation affecting lambda head assembly
Authors: Georgopoulos C P , Bisig R , Magazin M , Eisen H , Court D
Published: January 2020

A novel phage lambda mutation, called dc10, which interferes with proper lambda head assembly has been isolated and characterized. Phage lambda carrying this mutation is (i) unable to form plaques at 30 or 37 degrees C but does so at 42 degrees C and (ii) unable to form plaques at 42 degrees C on pN-constitutive hosts. Both properties are due to dc10 since all phage revertants for one phenotype simultaneously lose the other phenotype and vice versa. The dc10 mutation has been mapped in the B gene and has been shown to be dominant over the corresponding wild-type product. At 30 degrees C the dc10 mutation results in the formation of abnormal petit lambda heads made up of pE, pB, pC, and pNu3. Under pN-constitutive conditions, the dc10 mutation results in the formation of abnormal petit lambda heads made of pE, X1, and X2 only. A model to explain the data is presented.
Reticuloendotheliosis virus: detection of immunological relationship to mammalian type C retroviruses
Authors: Charman H P , Gilden R V , Oroszlan S
Published: January 2020

Reticuloendotheliosis virus (REV) p30 shares cross-reactive determinants and a common NH2-terminal tripeptide with mammalian type C viral p30's. An interspecies competition radioimmunoassay was developed, using iodinated REV p30 and a broadly reactive antiserum to mammalian virus p30's. The avian leukosis-sarcoma viruses and mammalian non-type C retroviruses did not compete in this assay. Previous data indicating that the REV group is not represented completely in normal avian cell DNA lead us to speculate that this may be the first example of interclass transmission, albeit in the remote past, among the Retroviridae.
Development of Escherichia coli virus T1: escape from host restriction
Authors: Wagner E F , Auer B , Schweiger M
Published: January 2020

The bacterial virus T1 grows interchangeably on different Escherichia coli strains (C, B, and K). This implies that T1 has an efficient mechanism to overcome the host restriction barrier. The DNA of T1 was found to be methylated independently of the hosts. The percentage of N6-methyladenine varied from 1.6 to 1.8, and the 5-methylcytosine content varied from 0.1 to 0.4%. In contrast, the range in percentage of N6-methyladenine and 5-methylcytosine found in the hosts was 0.7 to 2.4 and 0.0 to 1.1, respectively.
Fractionation of two protein kinases from avian myeloblastosis virus and characterization of the protein kinase activity preferring basic phosphoacceptor proteins
Authors: Rosok M J , Watson K F
Published: January 2020

Two protein kinase activities were fractionated from purified virions of avian myeloblastosis virus. Distinguishing characteristics of these two protein kinases included: (i) their binding properties during purification by ion-exchange chromatography; (ii) their estimated molecular weights; and (iii) their phosphoacceptor protein specificities. The protein kinase that bound to the anion exchanger DEAE-cellulose (pH 7.2) had an estimated molecular weight of 60,000 to 64,000 and preferred basic phosphoacceptor proteins. The protein kinase that bound to the cation exchanger phosphocellulose (pH 7.2) had an estimated molecular weight of 42,000 to 46,000 and preferred acidic phosphoacceptor proteins. The protein kinase preferring basic phosphoacceptor proteins was further purified and characterized. Optimal transfer of phosphate catalyzed by this enzyme required a divalent metal ion, a sulfhydryl-reducing agent, and ATP as phosphate donor. GTP was not an effective phosphate donor at concentrations comparable to ATP; and the cyclic nucleotides cyclic AMP and cyclic GMP neither stimulated nor inhibited protein phosphorylation by the protein kinase. The specificity of the protein kinase for basic phosphoacceptor proteins extended to proteins from avian myeloblastosis virus, in that the neutral to basic virion proteins p12, p19, and p27 served as phosphate acceptors. In addition, the protein kinase also appeared to phosphorylate itself. The role(s) of this virion-associated protein kinase is discussed.
Vesicular stomatitis virus glycoprotein is anchored to intracellular membranes near its carboxyl end and is proteolytically cleaved at its amino terminus
Authors: Chatis P A , Morrison T G
Published: January 2020

The intracellular vesicular stomatitis virus glycoprotein (G) is inserted into membranes such that a small portion of one end of the molecule is exposed on the cytoplasmic surface of the endoplasmic reticulum and is susceptible to proteolytic digestion (T.G. Morrison, C.O. McQuain, and D. Simpson, J. Virol. 28:368-374). We have determined that this region of the G protein contains two methionyl tryptic peptides. The methionyl tryptic peptides of the G protein have been ordered by the use of the antibiotic pactamycin, and the two methionyl tryptic peptides removed by proteolytic digestion of intracellular G protein have been shown to be derived from the carboxyl terminal end of the protein. In addition, we have found that the unglycosylated G protein synthesized in a reticulocyte cell-free reaction migrates on polyacrylamide gels slightly slower than the unglycosylated G protein synthesized in tunicamycin-treated infected cells. We have also compared these G proteins derived from different sources by partial proteolysis (D.W. Cleveland, S.G. Fischer, M.W. Kirschner, and V.K. Laemmli, J. Biol. Chem. 252:1102-1106) and by chymotryptic peptide analysis. We have found minor differences between the two proteins consistent with the removal of 10 to 15 amino acids from the amino terminus of the intracellular G protein.
Carbohydrate Components of Influenza C Virions
Authors: Nakamura Kiyoto , Herrler George , Petri Thomas , Meier-Ewert Herbert , Compans Richard W.
Published: January 2020

The carbohydrate components of influenza C virions grown in chicken kidney (CK) cells were analyzed by gel filtration following exhaustive digestion with Pronase. The [
3
H]glucosamine-labeled glycopeptides were larger and more heterogeneous than those of influenza A/WSN virions; three major size classes (G
1
, G
2
, and G
3
) were resolved. Treatment with
Vibrio cholerae
neuraminidase caused a decrease in size of G
1
and G
2
, along with release of about 16% of the
3
H label. The released sugar components were identified as
N
-acetylneuraminic acid by thin-layer chromatography. Peak G
3
was highly labeled with [
3
H]mannose, whereas G
1
and G
2
contained lower levels of mannose. The three major viral glycoproteins gp88, gp65, and gp30 were isolated from sodium dodecyl sulfate-polyacrylamide gels, and their glycopeptide components were analyzed after Pronase digestion. The three size classes of glycopeptides were obtained from any of the three glycoproteins; however, the relative amounts of the three components varied among the glycoproteins. Host cell-derived components, which appear to be mucopolysaccharides and glycoproteins, were found associated with influenza C virions grown in CK cells. These components contained glycopeptides that were mainly of sizes similar to peak G
2
from influenza C virions. Previous studies have shown that influenza A/WSN virus grown in several cell types contained only two size classes of glycopeptides. Two size classes comparable to peaks G
2
and G
3
from influenza C virions were also observed in influenza A/WSN grown in CK cells. Thus the large G
1
glycopeptides appear to be characteristic of influenza C virions.
Potassium Requirement for Synthesis of Macromolecules in
Bacillus subtilis
Infected with Bacteriophage 2C
Authors: Willis Dawn B. , Ennis Herbert L.
Published: January 2020

A mutant of
Bacillus subtilis
168 (strain 168 KW), defective in its ability to concentrate K
+
from low levels in the growth medium, was used to study the role of K
+
in the development of phage 2C. Both the final burst size and the duration of the rise period depended on the K
+
concentration in the medium. During normal infection (in the presence of K
+
), host deoxyribonucleic acid (DNA) synthesis stopped. The synthesis of host messenger ribonucleic acid (RNA) continued throughout infection, albeit at a steadily decreasing rate. The synthesis of ribosomal RNA and its subsequent incorporation into mature ribosomes also proceeded. In contrast to these findings, host DNA and messenger RNA synthesis were not inhibited in cells infected in the absence of K
+
. Only “early” phage messenger RNA was synthesized under these conditions of infection. Phage DNA synthesis was dependent on K
+
irrespective of the requirement for this cation in protein synthesis.
Ultrastructure and Sequential Development of Infectious Pancreatic Necrosis Virus
Authors: Moss L. Howard , Gravell Maneth
Published: January 2020

The morphology and sequential development of infectious pancreatic necrosis (IPN) virus, a pathogen of trouts, were studied by electron microscopy. Mature virions were seen in the cytoplasm of infected cells incubated at 24 C as early as 6 hr after infection. These virions were hexagonal in profile and approximately 55 nm in diameter. Generally between 8 to 10 hr after infection, virus crystals of various sizes were occasionally observed. Although virus replication did not appear to be confined to a particular cytoplasmic locus, mature virions were sometimes seen in association with unidentified tubular structures approximately 45 nm in outside diameter. Negative stains of virus revealed unenveloped icosahedra approximately 65 nm in diameter with probably 92 capsomeres. Contrary to a previous communication which reported IPN virus to have picornavirus-like morphology, we found it to morphologically resemble members of the reovirus group.
Nucleotide Distribution and Functional Orientation in the Deoxyribonucleic Acid of Phage φ80
Authors: Skalka A.
Published: January 2020

The distribution of nucleotides in the deoxyribonucleic acid (DNA) of φ80 was determined by density analysis of molecular fragments of known length and origin. One half of the molecule includes a long, fairly homogeneous segment that contains 55% guanine plus cytosine (GC). DNA in the other half contains about 50% GC, except for a short stretch near the molecular end where the GC content is higher. Transcription of φ80 DNA was studied by ribonucleic acid-DNA hybridization tests with isolated molecular halves. At times early in the growth cycle, messenger is synthesized at a constant, relatively low rate and originates almost exclusively from DNA in the lower-GC half. At later times, messenger represents both halves and is synthesized at a greatly increased rate. The DNA of φ80 is closely analogous, physically and functionally, to λ DNA. The similarity is most striking in the high-GC half, which is defined here as “left” since its molecular end contains a cohesive site homologous to that at λ's left end. The left halves of the DNA of the two phages contain primarily late-functioning genes, and possess similar nucleotide distributions and some similar base sequences. The right halves of the two DNA molecules are less similar. Both contain the early genes, but they differ considerably in GC content (45% in λ, 51% in φ80) and do not strongly cross-react in hybridization tests. The DNA of φ80 lacks the central 37%-GC segment found in λ.
Markers of Rubella Virus Strains in RK
13
Cell Culture
Authors: Fogel Alice , Plotkin Stanley A.
Published: January 2020

When tested on RK
13
cell cultures, strains of rubella virus could be differentiated by their ability to form small or large plaques. Large plaques were produced by the HPV-77 and Cendehill strains, and also by a laboratory stock strain (West Point), after only 14 passages in RK
13
culture. Five wild-type rubella viruses, isolated and passaged only a few times in African green monkey kidney tissue culture, grew well in RK
13
cell culture, but they were sensitive to agar inhibitors and, therefore, formed small plaques. On the other hand, RA27/3, an attenuated strain grown in WI-38 human fibroblast cells, developed low titers in RK
13
cells and also produced small plaques. We concluded that the morphological differences between small-plaque and large-plaque viruses depended on their sensitivity to agar inhibitors and on the
p
H of the medium during plaque formation.