bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies
Authors: Yewdell J W , Caton A J , Gerhard W
Published: January 2020

The influenza virus hemagglutinin contains four major regions that are recognized by antibodies able to neutralize viral infectivity. To investigate the effect of an antibody response directed against each of these sites on viral evolution, influenza virus A/PR/8/34 (H1N1) was grown in allantois-on-shell cultures in the presence of a mixture of monoclonal antihemagglutinin antibodies. This selection mixture contained antibodies (two or three antibodies per antigenic site) whose concentrations were adjusted to achieve equal neutralization titers against each of the four antigenic sites. By varying the ratio of input virus to selection mixture concentration, we observed that variant viruses emerged under conditions of partial neutralization. Each of the four variants characterized in detail differed from the parental virus in its interaction with cellular receptors and exhibited minimal changes in antigenicity. Thus, these variants were virtually indistinguishable from wild-type viruses, as assessed by the binding of 103 monoclonal antihemagglutinin antibodies in an indirect radioimmunoassay. Despite this, many of the same antibodies demonstrated decreased titers to the variants in hemagglutination inhibition tests. The magnitude of the differences depended on the indicator erythrocytes used (much greater differences were detected with chicken erythrocytes than with human erythrocytes). Hemagglutination mediated by the variants was more resistant to neuraminidase treatment of erythrocytes than hemagglutination mediated by the parental virus. These findings are consistent with the idea that the variants were initially selected by virtue of their increased avidity for host cell receptors. Sequencing of viral RNA revealed that each of the variants differed from the parental virus by a single amino acid alteration in its HA1 subunit. Two of the changes were close to the proposed receptor binding site on hemagglutinin and could directly alter receptor binding, while a third was located near the trimer interface and may have increased receptor binding by altering monomer-monomer interactions.
Characterization of a gene coding for M proteins which is involved in host range restriction of an avian influenza A virus in monkeys
Authors: Buckler-White A J , Naeve C W , Murphy B R
Published: January 2020

The nucleotide sequence of the region of RNA segment 7 coding for the M1 and M2 proteins of avian influenza A/Mallard/New York/6750/78 was determined, and the deduced amino acid sequences were compared to other avian and human M protein sequences. The M2 proteins of the avian and human viruses have diverged much more than the M1 proteins, although amino acids specific for avian and human viruses were found in both M1 and M2 proteins.
Transforming Sloan-Kettering viruses generated from the cloned v-ski oncogene by in vitro and in vivo recombinations
Authors: Stavnezer E , Barkas A E , Brennan L A , Brodeur D , Li Y
Published: January 2020

The Sloan-Kettering viruses (SKVs) are replication-defective retroviruses that transform avian cells in vitro. Each of the three SKV isolates is a mixture of viruses with genomes ranging in size from 4.1 to 8.9 kilobases (kb) with a predominant genome of 5.7 kb. Using a cDNA representing a sequence, v-ski, that is SKV specific and held in common by the multiple SKV genomes, we generated a restriction map of the 5.7-kb SKV genome and molecularly cloned a ski-containing fragment from SKV proviral DNA. Southern hybridization and sequence analysis showed that the cloned DNA fragment consisted of the 1.3-kb ski sequence embedded in the p19gag sequence and followed by the remaining 5' half of the gag gene and small portions of both the pol and env genes. A large deletion encompassing the 3' half of gag and the 5' 80% of pol was mapped to a position about 1 kb downstream from the 3' ski-gag junction. To determine whether the cloned ski sequence had transforming activity, the ski-containing fragment and a cloned Rous-associated virus 1 (RAV-1) genome were used to construct an analog of the 5.7-kb SKV genome, RAV-SKV. Cotransfection of chicken embryo cells with RAV-SKV and RAV-1 yielded foci of transformed cells whose morphology was identical to that induced by the natural SKVs. The transformed transfected cells produced transforming virus with a 5.7-kb ski-containing genome and synthesized a gag-containing polyprotein of 110 kilodaltons (kDa). Several nonproducer clones of RAV-SKV-transformed cells were analyzed, and most were found to synthesize a 5.7-kb SKV RNA and a 110-kDa polyprotein. One clone was found to contain an 8.9-kb SKV RNA, and this clone synthesized a 125-kDa polyprotein. Since both the 5.7- and 8.9-kb genomes and the 110- and 125-kDa polyproteins had been identified in studies on the natural SKVs, the present results not only demonstrate the transforming activity of these individual SKVs but also suggest mechanisms for their generation.
Evidence for circular permutation of the prophage genome of Bacillus subtilis bacteriophage phi 105
Authors: Marrero R , Yasbin R E
Published: January 2020

Analysis of DNA extracted from Bacillus subtilis lysogenic for bacteriophage phi 105 was performed by restriction endonuclease digestion and Southern hybridization using mature phi 105 DNA as a probe. The data revealed that the phi 105 prophage is circularly permuted. Digests using the enzymes EcoRI, SmaI, PstI, and HindIII localized the bacteriophage attachment site (att) to a region 63.4 to 65.7% from the left end of the mature bacteriophage genome. The phi 105 att site-containing SmaI C, PstI J, and HindIII L fragments were not present in digests of phi 105 prophage DNA. phi 105-homologous "junction" fragments were visualized by probing digests of prophage DNA with the purified PstI J fragment isolated from the mature bacteriophage genome. The excision of the phi 105 prophage was detected by observing the appearance of the mature PstI J fragment and the concomitant disappearance of a junction fragment during the course of prophage induction.
Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen
Authors: Gurney E G , Tamowski S , Deppert W
Published: January 2020

We isolated 16 new monoclonal antibodies that recognize large T antigen of simian virus 40 and mapped the epitopes to three distinct regions of the large T antigen. Also, 3 of the 16 recognized the large T antigen of the human papovavirus BKV.
Activation of enhancer sequences in type II human T-cell leukemia virus and bovine leukemia virus long terminal repeats by virus-associated trans-acting regulatory factors
Authors: Rosen C A , Sodroski J G , Kettman R , Haseltine W A
Published: January 2020

The ability of the sequences present in the long terminal repeats (LTRs) of human T-cell leukemia viruses type I and II (HTLV-I and HTLV-II) and of bovine leukemia virus to function as enhancer elements was investigated. Recombinant plasmids that contained the HTLV-I, HTLV-II, and bovine leukemia virus LTRs at a distance from a simian virus 40 promoter element located 5' to the bacterial gene encoding chloramphenicol acetyltransferase (EC 2.3.1.28) were constructed. We report that all three LTR sequences contain enhancer elements capable of increasing the level of gene expression directed from a distal heterologous promoter. The enhancer present in the HTLV-I LTR was active in uninfected cells of lymphoid and nonlymphoid origin. In contrast, the enhancer activity of the HTLV-II and bovine leukemia virus LTR sequences was evident only in virus-infected cells. This activity is likely due to virus-associated trans-acting transcriptional factors previously shown to be present in HTLV- and bovine leukemia virus-infected cells. The implication of these observations for virus replication and transforming activity are discussed.
Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods
Authors: Marshall R L , Rodriguez L L , Letchworth G J
Published: January 2020

Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus-neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope.
Conservative replication and transcription of Saccharomyces cerevisiae viral double-stranded RNA in vitro
Authors: Nemeroff M E , Bruenn J A
Published: January 2020

All double-stranded RNA viruses have capsid-associated RNA polymerase activities. In the reoviruses, the transcriptase synthesizes the viral plus strand in a conservative mode and the replicase synthesizes the viral minus strand, again conservatively. In bacteriophage phi 6 and in some fungal viruses, the transcriptase activity is semiconservative, acting by displacement synthesis. In this work we demonstrate Saccharomyces cerevisiae viral RNA replication in vitro for the first time and, using more sensitive techniques than those previously used, show that both the transcriptase and the replicase appear to act conservatively, like those of reovirus. There is therefore clearly no universal life cycle for the double-stranded RNA viruses.
Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence
Authors: Deiss L P , Frenkel N
Published: January 2020

Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence.
Mitogenic activity of the adenovirus type 12 E1A gene induced by hormones in rat cells
Authors: Oda K , Masuda-Murata M , Shiroki K , Handa H
Published: January 2020

Several lines of rat 3Y1 cells in which expression of the adenovirus type 12 E1A gene can be regulated by dexamethasone were established by introduction of recombinant vector DNA containing the adenovirus type 12 E1A gene placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines (gMA cells) produced low basal levels of the E1A transcripts and proteins in normal medium and much higher levels upon addition of dexamethasone to the medium. When dexamethasone was added to density-arrested cells, DNA synthesis was induced in 10 to 40% of the cells, the percentage depending on the cell line. DNA synthesis was increased to up to 60% of the cell population by further addition of epidermal growth factor. Indirect immunofluorescence detection of E1A proteins in gMA cells treated with dexamethasone indicated that the intensity of fluorescence in cells varied and that the proportion of cells synthesizing DNA was correlated with the proportion that exhibited strong fluorescence. These results indicate that the E1A gene has a function to trigger the synthesis of cellular DNA.
Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion
Authors: Hoekstra D , Klappe K
Published: January 2020

A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost.
Studies on emerging radiation leukemia virus variants in C57BL/Ka mice
Authors: Rassart E , Shang M , Boie Y , Jolicoeur P
Published: January 2020

To analyze the emergence of radiation leukemia virus (RadLV) variants in primary X-ray-induced C57BL/Ka thymoma and to identify the virus responsible for the very high leukemogenic potential of passaged Kaplan strain BL/VL3 preparation, we cloned several primary and passaged ecotropic RadLV infectious genomes. By restriction analysis, we found that BL/VL3 cells harbor three related but different ecotropic RadLVs. Their restriction map differs significantly from those of primary RadLVs. Hybridization analysis also indicated that BL/VL3 and primary RadLVs differ in their p15E and long terminal repeat (LTR) regions. As compared with the LTR sequence of the putative parental endogenous ecotropic provirus, the LTR sequence of primary weakly leukemogenic RadLV has only one change, a C-rich sequence, generating a 6-base-pair direct repeat just in front of the promotor. The LTR of the primary nonleukemogenic RadLV only showed few base changes, mainly clustered in R and U5. The LTR from a moderately leukemogenic passaged BL/VL3 RadLV had conserved the C-rich sequence and acquired a 43-base-pair direct repeat in U3 and several other point mutations, small insertions, and deletions scattered in U3, R, and U5. All cloned primary RadLVs were fibrotropic, and some were weakly leukemogenic. All cloned BL/VL3 RadLVs were thymotropic and nonfibrotropic. The block of their replication was found to be after the synthesis of unintegrated linear and supercoiled viral DNA. Most of the BL/VL3 RadLVs were moderately leukemogenic, and one (V-13) was highly leukemogenic, being as virulent as the Moloney strain. We propose a model for the emergence of the RadLV variants and show that the virus responsible for the high leukemogenic potential of BL/VL3 preparation is a nondefective, ecotropic, lymphotropic, nonfibrotropic, unique retrovirus which most likely arose from a parental primary RadLV similar to those studied here.
Transfer, by selective breeding, of the pathogenic Mtv-2 endogenous provirus from the GR strain to a wild mouse line free of endogenous and exogenous mouse mammary tumor virus
Authors: Morris D W , Young L J , Gardner M B , Cardiff R D
Published: January 2020

The GR laboratory mouse strain has five endogenous proviral copies of the mouse mammary tumor virus (MMTV). One of these, Mtv-2, is unique because it causes mammary carcinomas in virtually 100% of breeding GR females prior to 1 year of age. To facilitate studies of this locus in particular, and mammary tumorigenesis in general, we genetically tailored a new mouse line, WXG-2, which bears Mtv-2 as its only endogenous MMTV provirus. The WXG-2 line was constructed by making hybrids between the GR strain and a wild mouse line free of both endogenous and exogenous MMTV, backcrossing to the MMTV-free line, and fixing the Mtv-2 locus in a population with the desired genotype. Mammary tumors were observed in 5 of the 20 hybrid females carrying the endogenous Mtv-2 provirus. The WXG-2 line represents a new model system for studying MMTV-induced mammary tumorigenesis in the absence of multiple endogenous proviruses.
Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus
Authors: Keegan K , Collett M S
Published: January 2020

Four distinct antigenic determinants along the G2 glycoprotein encoded by the M segment RNA of the Phlebovirus Rift Valley fever virus were localized. These epitopes were defined by four monoclonal antibodies, three of which were capable of neutralizing virus infectivity; one was nonneutralizing. Immunoprecipitation by these monoclonal antibodies of either denatured or native antigen characterized the epitopes as having linear or higher order structure. Molecular cloning of G2 glycoprotein-coding sequences into a bacterial expression plasmid utilizing a beta-galactosidase fusion protein system was employed for epitope localization. A nuclease BAL 31 plasmid expression library, in which processive regions of the 3' end of the G2 glycoprotein coding sequences were deleted, allowed for approximation of the carboxy-terminal limit of the antigenic determinants. Further subcloning of limited G2 polypeptide sequences into the bacterial expression vector permitted more refined localization of the epitopes. The characteristics of the immunoreactivity of these small peptide regions (between 11 and 34 amino acids) produced in bacteria as G2-beta-galactosidase fusion proteins were similar to those of the authentic Rift Valley fever virus G2 glycoprotein. These defined antigenic determinants and their importance in virus infectivity are discussed.
Regulation of herpes simplex virus-specific cell-mediated immunity by a specific suppressor factor
Authors: Horohov D W , Wyckoff J H , Moore R N , Rouse B T
Published: January 2020

Our study was designed to investigate the nature of an antigen-specific suppressor factor generated by antigen-stimulated herpes simplex virus (HSV)-immune splenocytes. Factor SF-200, a 90,000- to 100,000-dalton fraction obtained after Sephacryl gel filtration, suppressed the generation of HSV-specific cytotoxic T-lymphocyte and lymphoproliferative responses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of SF-200 indicated that it contained an I-J+, anti-idiotypic protein. It was possible to adsorb the suppressor activity of SF-200 to an anti-I-J immunoaffinity column. The suppressor activity could be eluted from the immunoaffinity column with a low-pH buffer. The acid-eluted material was determined to be both I-J+ and reactive with anti-HSV antiserum by Western blot analysis. Both SF-200 and the I-J+ suppressor activity suppressed only HSV-specific cell-mediated immunity responses. However, it was possible to generate nonspecific suppressor activity by incubating the I-J+ suppressor factor with Lyt 1+ splenocytes from HSV-immune mice. The implication of these results with respect to the model for a suppressor cell circuit regulating HSV-specific cell-mediated immunity responses is discussed.
Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C
Authors: Robbins A K , Watson R J , Whealy M E , Hays W W , Enquist L W
Published: January 2020

A pseudorabies virus (Becker strain) glycoprotein gene was located in the UL region at map position 0.40. The gene was identified by using open reading frame Escherichia coli plasmid expression vectors and specific antibody reagents. A 1.55-kilobase unspliced transcript from the gene was detected in pseudorabies virus-infected tissue culture cells. The DNA sequence revealed a single open reading frame of 1,437 base pairs encoding 479 amino acids. The predicted primary translation product has a molecular weight of 50,860 and contains features of a typical herpesvirus glycoprotein. An E. coli expression plasmid was constructed that contained essentially all of the open reading frame for this gene. Antibodies raised in rabbits against the protein expressed in bacteria by this plasmid immunoprecipitated pseudorabies virus-specific glycoproteins of 92,000 and 74,000 daltons from infected cell extracts. It is likely that these two forms represent different glycosylation states of the protein.
Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infection
Authors: Klinkert M Q , Theilmann L , Pfaff E , Schaller H
Published: January 2020

The presence of the two "large" surface proteins of hepatitis B virus (HBV), P39 and GP42 of pre-S1-hepatitis B surface antigen, was assayed in the serum of an experimentally infected chimpanzee by using antibodies to a pre-S1-specific fusion protein synthesized in Escherichia coli. The immune response to pre-S1-hepatitis B surface antigen was monitored by using the pre-S1 fusion protein as an antigen. pre-S1 proteins were detected in the serum early in the course of infection and prevailed as long as hepatitis B surface antigen did, together with hepatitis B e antigen and viral DNA. Thus, the pre-S1 antigen can be considered a novel diagnostic marker for acute HBV infection. Antibodies to pre-S1, both immunoglobulin M and G classes, were also detected early in infection, shortly after the appearance of the pre-S1 antigen, suggesting its strong immunogenicity in vivo. The anti-pre-S1 antibodies therefore also represent an early serological marker for acute HBV infection and, owing to their early appearance and persistence, may play a role in the neutralization of the virus.
Protection against canine distemper virus in dogs after immunization with isolated fusion protein
Authors: Norrby E , Utter G , Orvell C , Appel M J
Published: January 2020

Canine distemper virus attachment (hemagglutinin [H] equivalent) and fusion (F) antigens were purified by affinity chromatography with monoclonal antibodies. The purified antigens were used to immunize groups of three dogs. Radioimmune precipitation assays with sera from these animals showed that the F antigen preparation was pure and induced only an F polypeptide-specific antibody response but that the H antigen preparation had a slight contamination by the F antigen. Immunized animals were challenged with virulent canine distemper virus. Two animals in each group developed pronounced humoral and cellular immune responses after challenge. Among these infected animals, only the dogs immunized with H antigen developed symptoms, albeit mild. In contrast, three nonimmunized control animals developed severe disease, with a fatal outcome in two cases. The complete resistance against challenge in two dogs was interpreted to reflect in one case anti-F immunity and in the other case most likely a high level of anti-H immunity. It is suggested that the F antigen may be of particular interest for the development of morbillivirus and possibly other paramyxovirus subunit or synthetic vaccines, because it can induce immunity capable of blocking virus infection and in situations of virus replication prevent the emergence of symptoms.
Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis
Authors: Carrasco L , Bravo R
Published: January 2020

The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various times postinfection were also analyzed. At least 13 proteins labeled with [3H]glucosamine were detected in vaccinia-infected HeLa cells.
Recent clinical isolates of cytomegalovirus suppress human cytomegalovirus-specific human leukocyte antigen-restricted cytotoxic T-lymphocyte activity
Authors: Schrier R D , Oldstone M B
Published: January 2020

Peripheral blood mononuclear cells harvested from healthy adults seropositive for human cytomegalovirus (HCMV) and cultured with laboratory strain AD-169 demonstrated human leukocyte antigen-restricted and HCMV-specific killing on target cells infected with either HCMV laboratory strain AD-169 or recent low-passage HCMV isolates. These results indicated that the determinants recognized by cytotoxic T lymphocytes (CTLs) are shared among different strains of HCMV. However, when low-passage isolates, rather than high-passage AD-169 virions, were used to stimulate CTL activity, the lytic response was significantly lower against all targets. Mixing of AD-169 and low-passage HCMV isolates induced low CTL activity. Collectively, the findings suggest that low-passage HCMV isolates have dual effects--antigenic stimulation and immunosuppression--whereas laboratory strain AD-169 is primarily immunogenic. The study of several recent isolates indicated that they varied in their ratio of immunostimulation to suppression, that infectious virus was necessary to produce suppression, and that suppressive isolates did not have to be present at the initiation of culture to exert their suppressive effects.
Dispersed chromosomal localization of the proto-oncogenes transduced into the genome of Mill Hill 2 or E26 leukemia virus
Authors: Symonds G , Quintrell N , Stubblefield E , Bishop J M
Published: January 2020

Both Mill Hill 2 and E26 retroviruses have transduced two cellular genes--c-myc and c-mil/mht (Mill Hill 2) and c-myb and c-ets (E26). We localized the genes transduced by these viruses to different chromosomes: c-myc and c-myb to relatively large chromosomes and c-mil/mht and c-ets to microchromosomes. Thus, like avian erythroblastosis virus, each of these retroviruses has transduced two cellular genes unlinked in the chicken genome.
A mouse c-myc retrovirus transforms established fibroblast lines in vitro and induces monocyte-macrophage tumors in vivo
Authors: Baumbach W R , Keath E J , Cole M D
Published: January 2020

Activation of the c-myc proto-oncogene, in the form of DNA rearrangements that lead to constitutive expression, has been implicated in the genesis of a wide range of tumors. Therefore, it is of great interest to determine the influence of c-myc oncogene activation on cellular growth control, especially in primary cells. To facilitate the efficient transfer of an activated c-myc oncogene, we developed a mouse retrovirus that contains the c-myc protein-coding sequences and which can be transmitted in the presence of a Moloney murine leukemia virus helper or established as a helper-free stock with a retrovirus-packaging cell line. The virus can transform established lines of mouse fibroblasts to anchorage-independent growth; the transformed cells are tumorigenic in nude mice. However, the virus was not capable of inducing foci of transformed cells on confluent monolayers. In addition to studies on established cell lines, the effect of the c-myc retrovirus on primary cells was examined. Infection of bone marrow cells gave rise to partially transformed mononuclear phagocytes which were entirely dependent upon an exogenous supply of the monocyte-specific colony-stimulating factor CSF-1 for proliferation. Infection in vivo induced monocyte-macrophage tumors with a latency period of 8 to 10 weeks.
Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus
Authors: Wu C A , Harper L , Ben-Porat T
Published: January 2020

Serial passage of pseudorabies virus (PrV) at high multiplicity yields defective interfering particles (DIPs), but the sharp cyclical increases and decreases in titer of infectious virus that are observed upon continued passage at high multiplicity of most DIPs of other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471-479). We have studied the dynamics of the interactions of the virions present in a population of DIPs to assess the cis functions for which the genomes of the DIPs are enriched. The defective genomes present in one population of DIPs, [PrV(1)42], replicate preferentially over the nondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIP DNA is transient and disappears at later stages of infection. The defective DNA does not appear to be encapsidated preferentially over the nondefective DNA present in this virion population, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion population has become modified and has acquired reiterations of sequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIP population compete for encapsidation more effectively than do the genomes of standard PrV. These results indicate that the defective genomes in the population of virions studied are enriched not only for an origin of replication but probably also for sequences necessary for efficient cleavage-encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired the ability to compete with the defective genomes for cleavage-encapsidation.
cis Functions involved in replication and cleavage-encapsidation of pseudorabies virus
Authors: Wu C A , Harper L , Ben-Porat T
Published: January 2020

Serial passage at high multiplicity of pseudorabies virus generates defective interfering particles (DIPs) whose genomes consist at least in part of reiterations of segments of DNA in which sequences originating from different regions of the genome have become covalently linked (F. J. Rixon and T. Ben-Porat, Virology 97:151-163). To determine whether some cis functions present in these reiterated DNA sequences may be responsible for the amplification of DIP DNA, BamHI restriction fragments of this DNA were cloned. These fragments were analyzed and tested for their ability to promote the amplification of covalently linked pBR325 DNA when cotransfected into cells with helper pseudorabies virus DNA. The cloned DIP BamHI DNA fragments consisted of various combinations of sequences originating from either one or both ends as well as sequences from the middle of the unique long (UL) segment of the genome. Only plasmids with inserts consisting of segments of defective DNA originating from the middle of the UL, as well as from both ends of the genome, were able to replicate and be encapsidated autonomously. This finding indicated that signals present at both ends of the genome may be necessary for efficient cleavage-encapsidation. To confirm this observation, we constructed plasmids in which DNA segments containing an origin of replication and sequences from either one or both ends of the virus genome were linked. These experiments showed that efficient cleavage-encapsidation requires the presence of sequences derived from both ends of the genome. Two origins of replication, one at the end of the UL segment and one in the middle of the UL segment, were also identified.
Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli
Authors: Tanese N , Roth M J , Goff S P
Published: January 2020

Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion proteins in Escherichia coli, and the purified proteins were used to elicit antibodies in Escherichia coli, and the purified proteins were used to elicit antibodies in rabbits. The sera were used to examine the mature pol gene products contained in virion particles and identified the reverse transcriptase and a second protein, P46pol, encoded by the 3' portion of the gene. The P46 protein was not phosphorylated and was present at the same molar abundance as the reverse transcriptase. The sera were also used to detect the Pr200gag-pol intracellular precursor protein and to analyze its processing to the mature forms. The proteins formed by several Moloney MuLV mutants were analyzed. Further tests revealed cross-reactivity with Friend MuLV and feline leukemia virus proteins, but not with avian retrovirus proteins.
Genetic reassortants of lymphocytic choriomeningitis virus: unexpected disease and mechanism of pathogenesis
Authors: Riviere Y , Oldstone M B
Published: January 2020

Reassortant viruses of different strains of lymphocytic choriomeningitis viruses cause lethal disease after inoculation into neonatal BALB/c WEHI mice, but, in contrast, parental strains or reciprocal reassortants do not cause lethal disease. The disease is characterized by inhibition of growth and death. The pathogenic mechanism is the induction of interferon combined with higher virus titers and subsequent liver necrosis. The generation of lethal reassortants from nonlethal parent viruses likely has implications for understanding the outbreaks of unanticipated virulent disease within a viral family.
Site-directed mutagenesis of polyomavirus middle-T antigen sequences encoding tyrosine 315 and tyrosine 250
Authors: Markland W , Oostra B A , Harvey R , Markham A F , Colledge W H , Smith A E
Published: January 2020

Tyrosine residues of middle-T and tyrosine phosphorylation are thought to be important in the transformation of cultured rodent cells by polyomavirus. Of the potential tyrosine sites in the carboxyl-terminal half of middle-T, tyrosines 297, 315, and 322 have been studied previously, whereas tyrosine 250 has not. Two mutant plasmids, XD121 and pT250, encode polyomavirus middle-T species in which the tyrosine 250 residue is affected. XD121 is a deletion mutant in which the region encoding tyrosine 250, together with three adjacent amino acids, is deleted, whereas pT250 is a point mutant in which the tyrosine 250 codon has been converted to a phenylalanine codon. The plasmids were handicapped in transforming ability, as judged by focus formation on a monolayer of Rat-1 cells. Both demonstrated a reduction in the number of foci produced and a lag in the time of appearance of foci when compared with wild-type plasmid. The importance of residue 250 in this phenotype was indicated by the observation that plasmids containing multiple mutations proximal to the tyrosine 250 codon were wild type in their transforming ability. Furthermore, a revertant of pT250 (pT250-w.t.), which utilized the alternative tyrosine codon of TAC, was shown to regain full transforming activity. A combined-mutant plasmid, pTH, encodes a middle-T species in which both tyrosines 250 and 315 are converted to phenylalanine. This plasmid was totally defective in the transformation of rodent cells in a focus formation assay; however, it did impart a small measure of anchorage-independent growth when the encoded protein was expressed in NIH 3T3 cells. The in vitro kinase activity and pp60c-src association of the mutant middle-T antigens were examined. These assays demonstrated a reduction in phosphate acceptor activity for the middle-T species encoded by pT250 and pTH. Quantitative kinase assays showed that all of the tyrosine-mutant middle-T species, encoded by pAS131 (containing the tyrosine 315 codon-to-phenylalanine codon mutation), pT250, and pTH, were able to enhance pp60c-src kinase activity but only at levels which were intermediate and which reflected their transforming abilities relative to wild type.
Intracellular maturation of mumps virus hemagglutinin-neuraminidase glycoprotein: conformational changes detected with monoclonal antibodies
Authors: Waxham M N , Merz D C , Wolinsky J S
Published: January 2020

Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.
Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction
Authors: Thomsen A R , Pisa P , Bro-Jørgensen K , Kiessling R
Published: January 2020

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.
Expression of the Epstein-Barr virus nuclear protein 2 in rodent cells
Authors: Dambaugh T , Wang F , Hennessy K , Woodland E , Rickinson A , Kieff E
Published: January 2020

A 3.0-kilobase-pair Epstein-Barr virus (EBV) DNA segment necessary for lymphocyte immortalization encodes at least part of a nuclear protein (EBNA2) which is characteristically expressed in latently infected, immortalized cells. A 1.5-kilobase open reading frame within this DNA segment has now been inserted into a murine leukemia virus (MuLV)-derived expression vector (pZIP-NEO-SV(X)1) which provides for transcription of heterologous DNA but not for translational start. Transfection of the recombinant DNA into NIH 3T3 cells resulted in expression of a full-sized EBNA2 which localized to the cell nucleus. Significant new evidence is thereby provided that this 1.5 kilobase open reading frame includes a translational start site and encodes the entire EBNA2 protein. Transfection of the recombinant DNA into a helper cell line (psi am22b) providing amphotropic MuLV-packaging functions resulted in the release of a recombinant MuLV carrying the EBNA2 gene. This recombinant virus can infect rodent cells and convert them to stable EBNA2 expression. Rat-1 cells infected with the MuLV EBNA2 recombinant expressed EBNA2 and grew more rapidly in medium supplemented with 1 or 0.5% fetal calf serum than did Rat-1 cells infected with MuLV vector lacking EBNA2. The Rat-1 cells expressing EBNA2 remained contact inhibited, anchorage dependent, and nontumorigenic in nude mice. Different EBV isolates have one of at least two EBNA2 alleles. Despite divergence between the two alleles, a human serum recognized the prototype EBNA2 allele (EBNA2A) as well as the variant EBNA2B allele characteristic of some Burkitt tumor EBV isolates. The EBNA2B allele was also expressed from the MuLV-derived vector. The reproducible expression of EBNA2A or EBNA2B from these recombinant vectors will facilitate analysis of the EBNA2A and EBNA2B phenotypes.
DNA-binding properties of the major structural protein of simian virus 40
Authors: Soussi T
Published: January 2020

We investigated whether the VP1 protein of simian virus 40 binds to DNA. In vitro DNA-binding experiments clearly indicate that VP1 bound strongly to double-stranded and single-stranded DNA, with a higher affinity for the latter; additional experiments show that VP1 did not bind to a specific sequence of simian virus 40 DNA.
Nucleotide sequence and protein overproduction of bacteriophage T4 thioredoxin
Authors: LeMaster D M
Published: January 2020

The bacteriophage T4 thioredoxin gene was cloned and physically mapped to 47.6 kilobases from the reference BamHI site. The DNA sequence is consistent with that reported from earlier protein sequence studies. The gene was subcloned into a lambda pL overexpression vector which allowed for the isolation of approximately 5 mg/liter.
Structure of
Bacillus subtilis
Bacteriophage φ25 and φ25 Deoxyribonucleic Acid
Authors: Liljemark W. F. , Anderson D. L.
Published: January 2020

The structure of
Bacillus subtilis
bacteriophage φ25 and φ25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of φ25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of φ25 is 130 nm in length and includes a large, complex tail plate. Phage φ25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of φ25 DNA contain numerous random interruptions. Chemical analysis of φ25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.
Process of Infection with Bacteriophage φX174
Authors: Funk Fred D. , Sinsheimer Robert L.
Published: January 2020

Twenty-two new amber and ochre mutants of φX174 were isolated and classified into complementation groups. Three ochre mutants gave positive complementation tests with reference mutants in the seven previously defined groups and thus represent an eighth cistron. Studies of the physiology of infection in the nonpermissive condition for mutants in cistron VIII yielded the following information. (i) Replicative-form synthesis proceeds at a normal rate, and is turned off at the usual time. (ii) Synthesis of single-stranded deoxyribonucleic acid (DNA) is delayed until nearly 40 min after infection (in the absence of lysis), at which time a slow synthesis of infectious phage particles commences. The synthesis of infectious particles at late times is interpreted as a consequence of “leakage,” and indicates that the cistron VIII product is required in very small quantities. (iii) During the normal period of single-strand synthesis, most of the replicative-form DNA is found in a form with properties similar to those of the transient intermediates of single-strand DNA synthesized during normal infection.
Heat-Sensitive Early Function in Induced λ N
sus
Lysogens
Authors: Cross Ronald A. , Lieb Margaret
Published: January 2020

Mutations in gene N of λ prevent killing of the host bacterium after infection. However, derepression of N
sus
prophages in nonpermissive (pm
−
) bacteria results in death of the lysogens. When prophages in pm
−
(λCI
ts
A-N
sus
) lysogens are derepressed by raising the temperature to 45 C, the cells remain viable as long as they are at 45 C. However, they cannot form colonies at 33 C unless they have been superinfected, at the high temperature, by λCI
+
-N
sus
phage which produces repressor at 45 C. A large fraction of these “rescued,” heat-inducible lysogens are lysogenized by the superinfecting phage, but lysogenization is not required for rescue. In pm
−
(λCI
ts
A-N
sus
) lysogens growing at 45 C, the rate of deoxyribonucleic acid (DNA) synthesis shows a characteristic increase after the temperature is lowered. This increased DNA synthesis, which is correlated with loss of rescue potential, does not occur as long as the cultures are maintained at 45 C.