bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment
Authors: Polvino-Bodnar M , Shedd D , Miller G
Published: January 2020

We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes.
Genetic analysis of the attenuation phenotype of poliovirus type 1
Authors: Omata T , Kohara M , Kuge S , Komatsu T , Abe S , Semler B L , Kameda A , Itoh H , Arita M , Wimmer E
Published: January 2020

Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation.
Localization of structural proteins in African swine fever virus particles by immunoelectron microscopy
Authors: Carrascosa J L , González P , Carrascosa A L , Garciá-Barreno B , Enjuanes L , Viñuela E
Published: January 2020

Seven African swine fever virus structural proteins were localized in the virion by immunoelectron microscopy. African swine fever virus-infected cells were incubated, before or after embedding and thin sectioning, with monoclonal antibodies specific for different structural proteins, and after labeling with protein A-gold complexes, the samples were examined in the electron microscope. Proteins p14 and p24 were found in the external region of the virion, proteins p12, p72, p17, and p37 were found in the intermediate layers, and protein p150 was found in the nucleoid and in one vertex. A monoclonal antibody that recognized protein p150 as well as p220, a virus-induced, nonstructural protein, could also bind to a component present in the nucleus of both uninfected and virus-infected cells.
Expression of the protein product of the mouse mammary tumor virus long terminal repeat gene in phorbol ester-treated mouse T-cell-leukemia cells
Authors: Racevskis J
Published: January 2020

Exposure of C57BL/6 mouse EL-4 T-cell leukemia cells to phorbol ester (12-O-tetradecanoylphorbol-13-acetate) (TPA) induced the synthesis of protein products encoded by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) region. Analysis of TPA-treated EL-4 cells with antiserum raised against a synthetic peptide predicted by the MMTV LTR open reading frame sequence detected a polypeptide migrating in gels with an apparent molecular weight of 37,000 Mr, as well as three less prominent proteins with apparent molecular weights of 31,000, 34,000, and 39,000. Tryptic peptide analysis established the identity of the immunoprecipitated cellular proteins with the LTR proteins obtained from in vitro translation of MMTV genomic RNA. All four proteins were glycosylated and were derived from one initial nonglycosylated translation product of 21,000 Mr. The 21,000-Mr apoprotein could be detected after digestion with endoglycosidase F or pretreatment of cells with tunicamycin. Untreated EL-4 cells synthesized three species of MMTV mRNA: 35S, 24S, and 20S. TPA treatment resulted in an increased level of transcription of the three mRNAs and the appearance of a new 1-kilobase mRNA. At least 10 acquired MMTV proviruses are present in the EL-4 genome, and examination of the degree of proviral methylation revealed extensive demethylation. However, no qualitative differences in the state of proviral methylation were apparent between TPA-treated and untreated cells.
Cloning and characterization of oriL2, a large palindromic DNA replication origin of herpes simplex virus type 2
Authors: Lockshon D , Galloway D A
Published: January 2020

An origin of replication within the long unique sequence of herpes simplex virus type 2 designated oriL2 has been identified in a position homologous to its type 1 counterpart, oriL1, between map coordinates 0.398 and 0.413. The difficulties encountered in previous attempts to clone both oriL2 and oriL1 in an undeleted form were surmounted by minimizing the growth of the host Escherichia coli, using a recBC sbcB E. coli host, and purifying the full-length plasmid from delected forms by using a novel method which exploits the ability of a palindrome-containing plasmid to adopt a cruciform conformation, thereby decreasing its supercoiling. In a previously developed assay for functional origin activity, oriL2 was localized to a 241-base-pair ApaI-SstII fragment. DNA sequence analysis revealed a 136-base pair, almost perfect palindrome. Comparison with oriL1 showed a very high degree of conservation: the two origins differ in only 16 of the 144-base-pair oriL1 palindromic region. Most significantly, the differences between oriL1 and oriL2 mainly occur in pairs so as to generally preserve the potential for intrastrand base pairing. The central region of oriL2 is homologous with the shorter palindromic structures detected in origins located within the repetitive sequences of the short component of herpes simplex virus type 1 or 2.
Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants
Authors: Rutila J E , Imperiale M J , Brockman W W
Published: January 2020

We used sodium bisulfite mutagenesis to introduce point mutations within the early region of the simian virus 40 genome. Seventeen mutants which contained amino acid changes in the amino-terminal half of the large T antigen coding sequence were assayed for their ability to replicate viral DNA and to induce transformation in the established rodent cell line Rat-3. The mutants fell into four basic classes with respect to these two biological functions. Five mutants had wild-type replication and transformation activities, six were totally defective, three were replication deficient and transformation competent, and two were replication competent and transformation deficient. Within these classes were mutants which displayed intermediate phenotypes, such as four mutants which were not totally deficient in viral replication or cellular transformation but instead showed reduced large T antigen function relative to wild type. Three large T mutants displayed transforming activity that was greater than that of wild type and are called supertransforming mutants. Of the most interest are mutants differentially defective in replication and transformation activities. These results both support and extend previous findings that two important biological functions of large T antigen can be genetically separated.
RNA-binding proteins of bovine rotavirus
Authors: Boyle J F , Holmes K V
Published: January 2020

Two major bovine rotavirus proteins have RNA-binding activity as shown by an RNA overlay-protein blot assay. Of the six proteins in purified virions, only one showed RNA-binding activity. This 92,000-molecular-weight (92K) protein was present in both single- and double-shelled particles. Its RNA-binding activity was blocked by preincubation with monospecific antibody to VP2. Thus, the 92K RNA-binding protein in rotavirus virions is VP2, the second most abundant protein in single-shelled particles. In infected cell extracts, numerous cellular RNA-binding proteins and two virus-specific RNA-binding proteins were detected, VP2 and a 31K nonstructural (NS31) protein. VP2 bound single-stranded RNA in preference to double-stranded RNA, whereas NS31 bound both single- and double-stranded RNA equally well. Binding did not appear to be nucleotide sequence specific, because RNA from uninfected cells and an unrelated RNA virus bound to VP2 and to NS31 as did rotavirus RNA. This technique showed that both cellular and rotavirus RNA-binding proteins also bound DNA. VP2 interacted with rotavirus RNA over a broad pH range, with an optimum at pH 6.4 to 6.8, and at NaCl concentrations between 0 and 100 mM. The RNA-binding activity of NS31 exhibited similar pH and NaCl dependency. Sequence-specific nucleic acid binding could be detected by this method. When labeled synthetic oligodeoxyribonucleotides corresponding to the 3' and 5' plus-sense terminal sequences of rotavirus gene segments were used as probes, the 3' synthetic oligodeoxyribonucleotide bound to one 48K protein in control and infected cells. This suggests that there may be a specific functional interaction between the 48K cellular protein and this 3'-terminal noncoding region of the rotavirus genome or mRNA. These data show that the RNA overlay-protein blot assay is a useful test to identify some cellular and viral proteins with RNA-binding activity. For bovine rotavirus, the evidence suggests that, of all the virus-specific proteins, VP2 and NS31 are most likely to interact with RNA during transcription and replication or virus assembly or both.
Enhanced rate of conversion or recombination of markers within a region of unique sequence in the herpes simplex virus genome
Authors: Pogue-Geile K L , Spear P G
Published: January 2020

Insertion mutants of herpes simplex virus type 1, containing a second copy of the sequences of BamHI fragment L (map coordinates 0.706 to 0.744) inserted in inverted orientation into the thymidine kinase gene (at map coordinate 0.315), have been further characterized. We reported previously that, as a result of intramolecular or intermolecular recombination between copies of the BamHI-L sequence at the normal locus and inserted locus, a high proportion of progeny genomes exhibited either inversions of the unique sequence flanked by these inverted repeats or other rearrangements. Now we report that a genetic marker (syn-1 or syn-1+) originally present only in the inserted copy of BamHI fragment L appears in progeny at both the normal and inserted loci, and vice versa, at high frequency. Because these phenomena have not been observed with other insertion mutants containing duplications of other sequences from unique regions of the genome, we conclude that BamHI fragment L contains an element that enhances the rate of homologous recombination in adjacent sequences, resulting in genome rearrangements and gene conversion-like events.
Characterization of the restricted component of Epstein-Barr virus early antigens as a cytoplasmic filamentous protein
Authors: Luka J , Miller G , Jörnvall H , Pearson G R
Published: January 2020

Four monoclonal antibodies produced against the restricted component of the Epstein-Barr virus (EBV) early antigen (EA-R) precipitated a polypeptide with an approximate molecular weight of 85,000. Three of these antibodies prepared against the native 85,000-molecular-weight protein (85K protein) reacted by immunofluorescence with acetone-fixed smears but not methanol-fixed smears of EBV-producing cells activated with tumor-promoting agent and sodium butyrate. The fourth monoclonal antibody which was produced against the denatured 85K protein reacted with both acetone-fixed cells and methanol-fixed cells. Blocking of direct immunofluorescence by the different monoclonal antibodies established that these monoclonal antibodies were directed against three different epitopes expressed on the 85K protein. The cytoplasmic staining pattern produced by each antibody was granular during the first 24 to 28 h after induction, developed into filamentous structures about 36 h after induction, and then began to aggregate after 48 h. Similar structures were observed in human placental cells transfected by EBV DNA and stained with three of the monoclonal antibodies. These results suggest that the EA-R polypeptide is assembled into filaments during the EBV lytic cycle. The significance of this in regards to replication has yet to be determined. Biochemical characterization of this major EA-R component did not reveal any major differences in this protein isolated from different cell lines.
Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type
Authors: VandePol S B , Holland J J
Published: January 2020

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.
Identification of a protein encoded by the trans activator gene tatIII of human T-cell lymphotropic retrovirus type III
Authors: Goh W C , Rosen C , Sodroski J , Ho D D , Haseltine W A
Published: January 2020

The human T-cell lymphotropic virus type III (HTLV-III/LAV) is a retrovirus associated with acquired immune deficiency syndrome. The region on the viral genome that is necessary for trans-activation of the HTLV-III/LAV long terminal repeat called tatIII has previously been determined to lie between nucleotides 5365 and 5607. Here we report that a bacterial fusion protein containing amino acid sequences specified by the first coding exon of the tatIII gene is recognized by some patient antisera. We also demonstrate that lymphoid and epithelial cells that express the trans activator function express a 14-kilodalton (kDa) protein recognized by a patient antiserum that reacts with the bacterial tatIII fusion protein. Cells transiently transfected with a deletion mutant of the trans activator protein produce a 12-kDa protein rather than the 14-kDa protein. These observations indicate that the tatIII region contains a functional gene and is capable of expressing a protein that migrates with an apparent molecular size of 14 kDa in some lymphoid and epithelial cells transfected with plasmids containing the tatIII region. We propose that the product of the trans activator gene be designated p14tat-III.
Further characterization of the vesicular stomatitis virus temperature-sensitive O45 mutant: intracellular conversion of the glycoprotein to a soluble form
Authors: Chen S S , Huang A S
Published: January 2020

Reexamination of the viral products of tsO45, a glycoprotein mutant of vesicular stomatitis virus, showed that at 39 degrees C there was a conversion of the glycoprotein (G) to a truncated, soluble form, Gs, which subsequently appeared in the extracellular medium. The half-life for this intracellular conversion and extracellular appearance was about 2 h at 39 degrees C. Gs was precipitated by a monoclonal antibody to the ektodomain but not by an antipeptide serum made against the first 15 amino acids at the carboxy terminus of G. Gs was also resistant to endoglycosidase H digestion. On the basis of pulse-chase experiments, the generation of Gs most probably occurred in the rough endoplasmic reticulum. This additional phenotype of the tsO45 mutant provides another approach for studying the generation and subsequent transport of a secreted protein in fibroblast cells.
The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences
Authors: Wheeler E F , Roussel M F , Hampe A , Walker M H , Fried V A , Look A T , Rettenmier C W , Sherr C J
Published: January 2020

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180gag-fms encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180gag-fms) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120v-fms, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. Both constructs were biologically active when transfected into NIH 3T3 cells and produced morphologically transformed foci at equivalent efficiencies. When transfected into a cell line (psi 2) expressing complementary viral gene functions, G418-resistant (Neor) cells containing either of these vector DNAs produced high titers of transforming viruses. Analysis of proteins produced in cells containing the vector lacking gag gene sequences showed that gP180gag-fms was not synthesized, whereas normal levels of both immature gp120v-fms and mature gp140v-fms were detected. The glycoprotein was efficiently transported to the cell surface, and it retained wild-type tyrosine kinase activity. We conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180gag-fms is mediated by signal peptidase and that the amino termini of gp140v-fms and the c-fms gene product are identical.
Monoclonal antibody analysis of p53 expression in normal and transformed cells
Authors: Yewdell J W , Gannon J V , Lane D P
Published: January 2020

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.
Molecular and biological properties of BK virus-IR, a BK virus variant isolated from a human tumor
Authors: Pagnani M , Negrini M , Reschiglian P , Corallini A , Balboni P G , Scherneck S , Macino G , Milanesi G , Barbanti-Brodano G
Published: January 2020

We describe the molecular and biological properties of BK virus (BKV)-IR, a new BKV variant isolated from a human tumor of pancreatic islets. BKV-IR bears a 253-base-pair (bp) deletion and an 80-bp insertion in the early region of the genome. The deletion abolishes the expression of small-t antigen. The inserted sequences, grouped in four clusters, produce rearrangements in the first and second enhancer elements. They are bound by 12-bp direct repeats and could form a 217-base stem-loop structure suggestive of an insertion sequence. As compared with wild-type BKV, BKV-IR transformed hamster cells with a reduced efficiency and induced ependymomas in hamsters at a lower frequency and with a longer latency period. Tumors induced by BKV-IR, however, showed features of higher malignancy. The possible role of the insertion sequence-like element in transformation by BKV-IR is discussed.
Physical and genetic characterization of the genome of Lactobacillus lactis bacteriophage LL-H
Authors: Trautwetter A , Ritzenthaler P , Alatossava T , Mata-Gilsinger M
Published: January 2020

Bacteriophage LL-H is a virulent phage of Lactobacillus lactis LL23. A restriction map of the phage genome was constructed with various restriction endonucleases. This chromosome has a 34-kilobase size and seems to be circularly permuted. We used a bank of LL-H restriction fragments to study the expression of five of the seven main phage particle proteins. Immunoblotting experiments permitted the mapping on the chromosome of several genes coding for phage particle proteins. We also show that the gene of the main capsid protein is expressed from its own promoter in an Escherichia coli strain.
Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture
Authors: Robbins A K , Whealy M E , Watson R J , Enquist L W
Published: January 2020

We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein gIII gene is not essential for growth in cell culture. This was accomplished by construction and analysis of viral mutants containing two defined deletion mutations affecting the gIII gene. These mutations were first constructed in vitro and introduced into Escherichia coli expression plasmids to verify structure and protein production. Each mutation was then crossed onto PRV by cotransfection of plasmid DNA and parental viral DNA by using gIII-specific monoclonal antibodies as selective and screening reagents. One resultant virus strain, PRV-2, contained an in-frame deletion of a 402-base-pair (bp) SacI fragment contained within the gIII gene. Another virus strain, PRV-10, contained a deletion of a 1,480-bp XhoI fragment removing 230 bp of the upstream, putative transcriptional control sequences and 87% of the gIII coding sequence. The deletion mutants were compared with parental virus by analysis of virion DNA, gIII specific RNA, and proteins reacting with gIII specific antibodies. Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two gIII specific monoclonal antibodies. However, two species of truncated glycosylated proteins were observed in PRV-2 infected cells that reacted with antiserum raised against bacterially produced gIII protein. PRV-10 produced no detectable gIII-specific RNA or protein. PRV-10 could be propagated without difficulty in tissue culture. Virus particles lacking gIII were indistinguishable from parental PRV virus particles by analysis of infected-cell thin sections in the electron microscope. We therefore conclude that expression of the gIII gene was not absolutely essential for PRV growth in tissue culture.
Expression of reverse transcriptase activity of human T-lymphotropic virus type III (HTLV-III/LAV) in Escherichia coli
Authors: Tanese N , Sodroski J , Haseltine W A , Goff S P
Published: January 2020

The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III provirus was inserted into a bacterial expression vector. The resulting gene fusion induced the formation of active reverse transcriptase that could be readily detected in extracts of bacterial cells. The activity exhibited the template and divalent cation requirements of the authentic enzyme. These constructs will be useful for safe and rapid analysis of potential inhibitors of this important enzyme.
Vesicular stomatitis virus N and NS proteins form multiple complexes
Authors: Davis N L , Arnheiter H , Wertz G W
Published: January 2020

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.
Localization of conserved and nonconserved epitopes within the Rous sarcoma virus-encoded src protein
Authors: Parsons S J , McCarley D J , Raymond V W , Parsons J T
Published: January 2020

We investigated the location of binding sites of pp60src-specific monoclonal antibodies by immunoprecipitating a panel of structurally altered src proteins. Two families of antibodies which recognized epitopes mapping to either amino acid residues 28 to 38 or 92 to 128 were identified. The highly conserved nature of the epitope defined by residues 92 to 128 suggests that it may represent an important functional region of the cellular src protein.
Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system
Authors: Muggeridge M I , Fraser N W
Published: January 2020

After corneal inoculation, herpes simplex virus type 1 replicates in the mouse eye, trigeminal ganglia, and brainstem, producing first an acute and then a latent infection. Previous work from this laboratory focused on the structure of the viral DNA in this system. We have now examined the structure of the viral genome at the chromosome level by using micrococcal nuclease digestion. Studies with disaggregated cell preparations made from the brainstems of acutely infected mice show that the majority of the viral DNA is in a nonnucleosomal form; however, a nucleosomelike fraction was also consistently detected. A similar result was obtained for viral DNA in herpes simplex virus type 1-infected C1300 (clone NA) neuroblastoma cells (a neuronal cell line).
Morphology and Physiology of the Intracellular Development of
Bacillus subtilis
Bacteriophage φ25
Authors: Liljemark W. F. , Anderson D. L.
Published: January 2020

The morphology of the intracellular development of bacteriophage φ25 in
Bacillus subtilis
168M has been correlated with nucleic acid synthesis in infected cells. Host deoxyribonucleic acid (DNA) synthesis was shut off by a phage-induced enzyme within 5 min after infection, and another phage-mediated function extensively degraded host DNA at the time of cell lysis. Synthesis of phage DNA in infected cells began within 5 min and continued until late in the rise period. After phage DNA synthesis and coinciding with lysis, much of the unpackaged, newly synthesized phage DNA was degraded. Studies of thin sections of φ25 infected cells suggested that unfilled capsids may be precursors to filled capsids in the packaging process. To assess dependence of capsid formation on phage DNA replication, cells were either treated with mitomycin C and infected with normal phage or infected with ultraviolet-irradiated (99% killed) φ25. Only empty capsids were found in these cells, indicating that capsid production may be independent of the presence of newly synthesized viral DNA.
Interaction of Bacteriophage N1 with Cell Walls of
Micrococcus lysodeikticus
Authors: Lovett Paul S. , Shockman Gerald D.
Published: January 2020

Bacteriophage N1 does not irreversibly adsorb to cell walls isolated from its host
Micrococcus lysodeikticus
strain 1 (ML-1). ML-1 walls do bind the virus in a specific but completely reversibly union. Electron microscopic examination of OsO
4
-treated mixtures of phage and walls revealed phage bound to wall fragments by their tail tips, suggesting that reversible phage attachment to walls involves a “tail-first” adsorption of the virus. Treatment of ML-1 walls with fluorodinitrobenzene confers upon the walls the ability to inactivate N1 phage. The relationship between reversible phage attachment to walls and the mechanism of infection by N1 phage is discussed.
Proteins of Vesicular Stomatitis Virus
Authors: Kang C. Y. , Prevec L.
Published: January 2020

The antigens of the nucleoprotein core and the coat of vesicular stomatitis virus (VSV) particles of the Indiana serotype were prepared and purified by sucrose gradient fractionation. Antibody was prepared separately to each of the two antigen fractions. By immunological procedures, it was shown that soluble antigens of VSV preparations sedimenting at 20
S
and in the leading edge of the 6
S
region are antigenically related to VP3, the protein of the virus core, whereas the 6
S
soluble antigen cross-reacts only with viral coat antibodies. These results confirm previous results obtained by polyacrylamide gel analysis of the antigens. It has further been demonstrated that the 6
S
antigen is a glycoprotein. Comparing antigens of the New Jersey and Indiana serotype showed that the coat antigens of virus particles and the 6
S
antigen are immunologically distinct in the two serotypes. In complement-fixation tests, the core antigens and the soluble 20
S
antigens from one serotype showed a cross-reaction with antiserum prepared against core proteins of the other serotype.
Degradation of
Escherichia coli
B Deoxyribonucleic Acid After Infection with Deoxyribonucleic Acid-Defective Amber Mutants of Bacteriophage T7
Authors: Sadowski Paul D. , Kerr Carol
Published: January 2020

The degradation of bacterial deoxyribonucleic acid (DNA) was studied after infection of
Escherichia coli
B with DNA-negative amber mutants of bacteriophage T7. Degradation occurred in three stages. (i) Release of the DNA from a rapidly sedimenting cellular structure occurred between 5 and 6 min after infection. (ii) The DNA was cleaved endonucleolytically to fragments having a molecular weight of about 2 × 10
6
between 6 and 10 min after infection. (iii) These fragments of DNA were reduced to acid-soluble products between 7.5 and 15 min after infection. Stage 1 did not occur in the absence of the gene 1 product (ribonucleic acid polymerase sigma factor), stage 2 did not occur in the absence of the gene 3 product (phage T7-induced endonuclease), and stage 3 did not occur in the absence of the gene 6 product.
Comparison of Membrane Protein Glycopeptides of Sindbis Virus and Vesicular Stomatitis Virus
Authors: Burge Boyce W. , Huang Alice S.
Published: January 2020

A comparison has been made of the membrane glycoproteins and glycopeptides from two enveloped viruses, Sindbis virus and vesicular stomatitis virus (VSV). Glycopeptides isolated from Sindbis virus and VSV grown in the same host appear to differ principally in the number of sialic acid residues per glycopeptide; when sialic acid is removed by mild acid treatment, the glycopeptides of the two viral proteins are indistinguishable by exclusion chromatography. Preliminary evidence argues that the carbohydrate moiety covalently bound to different virus-specified membrane proteins may be specified principally by the host.
Characterization of Some Poliovirus Temperature-Sensitive Mutants and Poliovirus-Related Particle Formation Under Nonpermissive Conditions
Authors: Mikhejeva Angelika , Yakobson E. , Soloviev G. Y.
Published: January 2020

Investigation of 15 poliovirus temperature-sensitive (ts) mutants by using physiological tests [formation of virus-specific antigen and ribonucleic acid (RNA) under nonpermissive conditions] permitted us to divide them into three groups. From each group, one mutant was selected (ts 2, 5, 11), and a comparative study of poliovirus-related particle (5, 10, 73, and 150
S
) formation under permissive (36 C) and nonpermissive (40 C) conditions was carried out. The ts 2 and ts 11 are mutants with greatly reduced RNA synthesis which at 40 C produce particles with a sedimentation constant of 5
S
, and the ts 5 (RNA
+
) mutant produces both 5 and 10
S
particles. The relationship between different temperature-sensitive defects in the mutants is discussed. The results obtained indicate a possible role of 5
S
protein structures in morphogenesis of poliovirus.
Virus-Specific Deoxyribonucleic Acid in Simian Virus 40-Exposed Hamster Cells: Correlation with S and T Antigens
Authors: Levine Arthur S. , Oxman Michael N. , Henry Patrick H. , Levin Myron J. , Diamandopoulos George T. , Enders John F.
Published: January 2020

Several homologous hamster embryonic cell lines, transformed in association with simian virus (SV) 40 infection, were examined for the presence of deoxyribonucleic acid (DNA) complementary to SV40 ribonucleic acid (RNA) made in vitro. The methods employed permitted the detection of 10
−5
μg of viral DNA in 100 μg of cellular DNA, corresponding to one-fifth of an SV40 DNA molecule per cell. Those lines which contained both the SV40 surface (S) and tumor (T) antigens also contained DNA complementary to SV40 RNA synthesized in vitro. In contrast, neither of two lines which contained S, but not T, antigen contained detectable DNA complementary to SV40 RNA. These findings suggest that the production of S antigen does not depend upon the persistence of SV40 DNA in transformed cells.
λ Mutants Which Persist as Plasmids
Authors: Lieb M.
Published: January 2020

Lambda phages mutated in gene N do not kill sensitive host bacteria, but persist as plasmids. Plasmids are formed by genomes containing cI
+
, and also by
sus, ts
, or
c
mutants of cI. Bacteria infected with two or more phage particles give rise to clones in which most of the bacteria are carriers. The introduced λ genomes replicate more than once per bacterial division until there are 10 to 20 λ plasmids per host genome. In bacteria containing both F and λ plasmids, both replicate independently, and elimination by growth in acridine orange is also independent. Carriers of λ N
sus
plasmids are not immune, and there is complementation between the plasmids and superinfecting λ mutants.
In Vitro Product of a Ribonucleic Acid Polymerase Induced by Influenza Virus
Authors: Mahy B. W. J. , Bromley P. A.
Published: January 2020

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8
S
region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18
S
. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11
S
, but molecules which may be replicative intermediate, sedimenting at 14 to 20
S
, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18
S
region. An 11
S
double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus.
Properties of Light Particles Produced During Growth of Type 4 Adeno-Associated Satellite Virus
Authors: Torikai Katsutaka , Ito Michio , Jordan Liane E. , Mayor Heather D.
Published: January 2020

Adeno-associated satellite virus type 4, obtained by repeated undiluted passage, failed to produce distinct bands at the expected density of 1.43 g/cm
3
after density gradient centrifugation in CsCl. This phenomenon occurred regardless of the hemagglutinating activity of the starting material. Sharp bands were found at a density of 1.34 to 1.35 g/cm
3
. These bands contained adenovirions and numerous satellite particles. These latter particles could be distinguished by electron microscopy from standard dense satellite particles by their flattened profiles and deep penetration of negative stains. Dense bands of satellite virus at 1.43 g/cm
3
were constantly observed when the inoculum was comprised of highly diluted seed virus. Light satellite particles had a particle to HA ratio comparable with dense particles, but possessed low infectivity. Measurements of contour lengths of extracted deoxyribonucleic acid (DNA) indicate that light particles contain only a small amount of DNA, possibly less than 0.5 × 10
6
daltons, compared to 1.4 × 10
6
for the complete satellite DNA molecule.
Density Gradient Centrifugation Studies on Lymphocytic Choriomeningitis Virus and on Viral Ribonucleic Acid
Authors: Pedersen Ib R.
Published: January 2020

Lymphocytic choriomeningitis (LCM) virus, Traub strain, was purified from BHK-21 tissue culture medium. The virus was then analyzed by equilibrium centrifugation and rate zonal centrifugation in sucrose gradients. A buoyant density in sucrose of 1.18 g/ml was found and the
S
20, w
value was estimated to be about 470 to 500
S
. Furthermore, the
3
H-uridine-labeled ribonucleic acid (RNA) from virus was extracted from LCM virus and analyzed by rate zonal centrifugation. Two major and one minor single-stranded RNA components were found with sedimentation coefficients of 28, 22, and 18
S
.
Sedimentation Properties of Simian Virus 40-Specific Ribonucleic Acid Present in Green Monkey Cells During Productive Infection and in Mouse Cells Undergoing Abortive Infection
Authors: Martin Malcolm A. , Byrne Janet C.
Published: January 2020

The size distribution of polyribosome-associated simian virus 40 (SV40) ribonucleic acid (RNA) was examined at various times after productive infection. Eight hours after infection, virus-specific RNA was detected in the 14 to 17
S
region of a sucrose gradient by deoxyribonucleic acid (DNA)-RNA hybridization; RNA present in fractions sedimenting more rapidly did not react with SV40 DNA. At successively later times, SV40 RNA was detected in more rapidly sedimenting regions. By 24 hr, a portion of the SV40 RNA was detected in the 28
S
region, sedimenting slightly more rapidly than a MS2 RNA marker. Nuclear SV40 RNA, prepared from cells 48 hr after infection, was distributed in more rapidly sedimenting regions of the gradient, peaking at about 32 to 34
S
. Some nuclear virus-specific RNA could be detected in the 45 to 50
S
region. During the abortive infection of mouse cells, the sedimentation profile of SV40 RNA was very similar to that observed during the early phases of the lytic cycle.
Isolation and Properties of Poliovirus Minus Strand Ribonucleic Acid
Authors: Roy Polly , Bishop D. H. L.
Published: January 2020

Poliovirus minus strands were purified from double-stranded polio ribonucleic acid. The minus strands have a base ratio complementary to that of the viral ribonucleic acid and are not infectious.
Comparison of the Virion Polymerase of Reovirus with the Enzyme Purified from Reovirus-Infected Cells
Authors: Gomatos Peter J.
Published: January 2020

Reovirus has in its protein coat an enzyme which catalyzes the net synthesis of the three size classes of virus-specific, single-stranded ribonucleic acid (RNA). For synthesis of 24, 19, and 14
S
single-stranded RNA, Mn
++
was the preferred divalent cation, and ammonium sulfate at an optimal concentration of 4.2% of saturation was an absolute requirement. During synthesis, the parental double-stranded RNA was conserved in the viral core and the newly synthesized completed RNA chains were released as free RNA. The viral cores synthesizing RNA had properties consistent with the presence of nascent RNA on their outer surface. The enzyme-template complex from the infected cells described in an earlier paper was comprised of viral cores already active in the in vivo synthesis of single-stranded RNA. This pool of viral cores was newly made during infection, and exponential increase in the number of particles in this pool, as detected by the increase in enzymatic activity, occurred 2 hr earlier than that in mature virus.
Electron Microscopic Observations on Virus-Like Particles Associated with SH Antigen
Authors: Jokelainen P. T. , Krohn Kai , Prince A. M. , Finlayson N. D. C.
Published: January 2020

The structural aspects of SH antigen-containing particles were investigated. These studies confirmed the existence of a large spherical particle (ca. 43 nm) and smaller (ca. 20 nm) rod- and sphere-shaped particles. The large particle consists of an outer and inner membrane and a core of “nucleic acid” as seen by positive staining techniques. The outer membrane of the large particle appears to be similar to that of the 20-nm diameter spheres and rods known to possess the SH antigen.
Epstein-Barr Viral Antigen in Single Cell Clones of Two Human Leukocytic Lines
Authors: Miller Mary H. , Stitt Daphne , Miller George
Published: January 2020

A method was devised for obtaining single cell clones of human leukocytic cell lines in the presence of a human placental cell feeder layer. Clones of two lines, LS-B and EB
3
, which contain Epstein-Barr viral (EBV) antigen in approximately 1% of cells were tested for EBV antigen. Since all EB
3
clones and LS-B clones contained EBV antigen, it is concluded that in vitro EBV genome is associated with all of the cells.
LPP-1 Infection of the Blue-Green Alga
Plectonema boryanum
Authors: Sherman Louis A. , Haselkorn Robert
Published: January 2020

Host and viral deoxyribonucleic acid (DNA) metabolism in LPP-1-infected
Plectonema boryanum
was studied by equilibrium centrifugation in CsCl gradients. Approximately 50% of the host DNA is degraded to acid-soluble material between 3 and 7 hr after infection. Most of the acid-soluble product is reincorporated into viral DNA. Incorporation of exogenous
3
H-adenine into viral DNA can be detected very early after infection (within the first 2 hr), but the bulk of viral DNA synthesis occurs between 6 and 8 hr. Both the breakdown of host DNA and the synthesis of viral DNA require protein synthesis during the first few hours of infection.
Equal Incorporation of Both Parental Bacteriophage T7 Deoxyribonucleic Acid Strands into Intracellular Concatemeric Deoxyribonucleic Acid
Authors: Ihler Garret M. , Thomas Charles A.
Published: January 2020

After infection of
Escherichia coli
B with radiolabeled T7 bacteriophage, the parental deoxyribonucleic acid label was found in both polynucleotide chains of the intracellular T7 concatemer.
Mammalian cell transformation by a murine retrovirus vector containing the avian erythroblastosis virus erbB gene
Authors: Gazit A , Pierce J H , Kraus M H , Di Fiore P P , Pennington C Y , Aaronson S A
Published: January 2020

A recombinant murine retrovirus vector containing the v-erbB gene of avian erythroblastosis virus was constructed to investigate v-erbB as a transforming gene for mammalian cells. A restriction fragment containing the v-erbB sequences from a molecular clone of avian erythroblastosis virus was inserted into a Moloney murine leukemia virus vector. The construct, designated MuLV/erbB, transformed NIH 3T3 cells at a high efficiency in the DNA transfection assay. Individual MuLV/erbB transfectants grew in soft agar and were tumorigenic. The transfectants contained v-erbB DNA sequences, expressed v-erbB-specific transcripts, and synthesized v-erbB-related glycoproteins. The majority of transfectants produced two major v-erbB gene products of 58 and 66 kilodaltons. However, some transfectants produced much smaller v-erbB-specific proteins. Tunicamycin experiments revealed that the size heterogeneity observed between different transfectants was not due to variations in glycoprotein processing, implying that, in some cases, alterations in the MuLV/erbB genome occurred during the transfection process. These findings indicate that expression of the complete v-erbB gene product is not required for transformation of NIH 3T3 cells. A transmissible murine v-erbB (M-erbB) virus was generated by infection of nonproducer transfectants with amphotrophic murine leukemia virus. Transmission of the rescued M-erbB virus was confirmed by DNA, RNA, and protein analyses. The introduction of a transforming v-erbB gene into mammalian cells by virus infection provides a means of analyzing the mechanism by which this epidermal growth factor receptor-related gene alters the growth and differentiation of cells from various lineages.