bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature-sensitive mutants of Chandipura virus. II. Phenotypic characteristics of the six complementation groups
Authors: Gadkari D A , Pringle C R
Published: January 2020

Fifty temperature-sensitive (ts) mutants of the rhabdovirus Chandipura virus have been classified into six complementation groups designated ChI to ChVI. Group ChI contains 44 mutants, group ChII contains 2 mutants, and the remaining groups have 1 mutant each. Mutants in groups ChI, ChIII, ChIV, and ChVI had RNA-negative phenotypes in experiments measuring amplification of RNA synthesis at restrictive temperature. The two mutants in group ChII had RNA-positive phenotypes, and the virions were thermolabile. Mutant ts Ch851 of group ChV was also RNA positive, and the M polypeptide of this mutant appeared to be unstable in cells incubated at restrictive temperature. It is likely, therefore, that complementation groups ChII and ChV represent the genes coding for the two viral proteins of the virion envelope. No precise assignment can be made in the case of the four RNA-negative groups, since all the mutants examined showed some polymerase activity in vitro at restrictive temperature. An attempt to obtain polymerase mutants by screening for sensitivity to rifampin was not successful. Six temperature-dependent host range mutants (the tdCE phenotype) of Chandipura virus failed to multiply in chicken embryo cells at restrictive temperature, but otherwise they differed in their host range properties from similar mutants of vesicular stomatitis virus.
Frog Virus 3 Replication: Analysis of Structural and Nonstructural Polypeptides in Infected BHK Cells by Acidic and Basic Two-Dimensional Gel Electrophoresis
Authors: Elliott R. M. , Bravo R. , Kelly D. C.
Published: January 2020

Analysis of frog virus 3-infected BHK cells by two-dimensional, acidic and basic gel electrophoresis showed that at least 90 infected cell-specific polypeptides could be detected. These polypeptides represent between 70 and 85% of the coding capacity of the viral genome. The polypeptides were sequentially induced in at least three phases. The virus gradually suppressed host cell polypeptide synthesis during infection, although the synthesis of a few cell polypeptides may be “switched off” early in infection.
Recombinants between endogenous and exogenous avian tumor viruses: role of the C region and other portions of the genome in the control of replication and transformation
Authors: Tsichlis P N , Coffin J M
Published: January 2020

Endogenous retroviruses of chickens are closely related to exogenous viruses isolated from spontaneous tumors in the same species, yet differ in a number of important characteristics, including the ability to transform cells in culture, ability to cause sarcomas or leukemias, host range, and growth rate in cell culture. To correlate these differences with specific sequence differences between the two viral genomes, the genome RNA of transforming subgroup E recombinants between the Prague strain of Rous sarcoma virus, subgroup B (Pr-RSV-B), and the endogenous Rous-associated virus-0 (RAV-0), Subgroup E, and seven nontransforming subgroup E recombinants between the transformation-defective mutant of Pr-RSV-B and RAV-0 was examined by oligonucleotide fingerprinting. The pattern of inheritance among the recombinant viruses of regions of the genome in which Pr-RSV-B and RAV-0 differ allowed us to draw the following conclusions. (i) Nonselected parts of the genome were, with a few exceptions, inherited by the recombinant virus progeny randomly from either parent, with no obvious linkage between neighboring sequences. (ii) A small region in the Pr-RSV-B genome which maps in the 5' region was found in all transforming but only some of the nontransforming recombinants, suggesting that it plays a role in the control of the expression of transformation. (iii) A region of the Pr-RSV-B genome which maps between env and src was similarly linked to the src gene and may be either part of the structural gene for src or a control sequence regulating the expression of src. (iv) The C region at the extreme 3' end of the virus genome which is closely related in all the exogenous avian retroviruses but distinctly different in the endogenous viruses is the major determinant responsible for the differences in growth rate between RAV-0 and Pr-RSV-B. This latter observation allowed us to redefine the C region as a genetic locus, c, with two alleles cn (in RAV-0) and cx (in exogenous viruses).
Analysis of the nucleic acid components in reticuloendotheliosis virus
Authors: Simek S , Rice N R
Published: January 2020

Reticuloendotheliosis virus (REV) is known to be capable of transforming chicken bone marrow cells in vivo and embryo fibroblasts in vitro. As with spleen necrosis virus, we have found that sequences related to REV are found in DNA of several uninfected avian species. For example, about 15% of the [3H]cDNA synthesized in the endogenous reverse transcriptase reaction reassociated with DNA of uninfected chickens. Kinetic analysis revealed only a few (less than five) such sequences per haploid genome, and the thermal stability of the reassociated duplex indicated less than perfect complementarity. Comparison of REV propagated in an avian cell line with REV grown in a canine line has revealed clear differences between the two isolates. Viral RNA and [3H]cDNA of REV isolated from the transformed chicken bone marrow cell line appear to consist of at least three sequence classes. The most numerous of these classes is highly related to REV propagated in canine cells. Only slightly less abundant is a class unrelated to RNA isolated from the canine virus but highly related to sequences found in normal uninfected avian cellular DNA. A third component is present at about 1% the level of the most numerous class. Although REV appears to be unrelated to the other known avian retroviruses, distant relatedness between p30's of REV and various mammalian type C viruses has recently been reported. We have asked whether REV-related sequences can be detected in various mammalian DNAs and viral RNAs. Hybridization experiments performed at low stringency have revealed no such sequences.
Fluorescence changes of a membrane-bound dye during bacteriophage T5 infection of Escherichia coli
Authors: Glenn J , Duckworth D H
Published: January 2020

The fluorescence intensity of membrane-bound N-phenyl-1-naphthylamine increases dramatically when T5 bacteriophage infect colicin Ib plasmid-containing hosts. This dramatic increase is not seen during normal infections or in infections wherein either the plasmid or the phage contain mutations which allow productive infection to occur. Two smaller increases in fluorescence intensity are seen, however, in all T5 infections in which the characteristic two-step injection of DNA can proceed.
Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants
Authors: D'Halluin J C , Milleville M , Martin G R , Boulanger P
Published: January 2020

The nature, polypeptide composition, and antigenic composition of the particles formed by six human adenovirus type 2 temperature-sensitive (ts) mutants were studied. ts115, ts116, and ts125 were phenotypically fiber-defective mutants, and ts103, ts104, and ts136 failed to synthesize detectable amounts of fiber plus penton base at 39.5 degrees C. The mutants belonged to five complementation groups, one group including ts116 and ts125. Except for ts103 and ts136, the other mutants were capable of producing particles at 39.5 degrees C. ts116 and ts125 accumulated light assembly intermediate particles (or top components) at nonpermissive temperatures, with few virus particles. The sodium dodecyl sulfate polypeptide pattern of ts116- or ts125-infected cells, intermediate particles, and virus particles showed that polypeptide IV (fiber) was smaller by a molecular weight of 2,000 than that in the wild-type virion and was glycosylated. In fiber plus penton base-defective ts104-infected cells, equivalent quantities of top components and viruses with a buoyant density (rho) of 1.345 g/ml (rho = 1.345 particles) were produced at 39.5 degrees C. These rho = 1.345 particles corresponded to young virions, as evidenced by the presence of uncleaved precursors to proteins VI, VIII, and VII. These young virions matured upon a shift down. Virus capsid vertex antigenic components underwent a phase of eclipse during their incorporation into mature virus particles. No antigenic penton base or IIa was detected in intermediate particles of all the ts mutants tested. Only hexon and traces of fiber antigens were found in ts104 young virions. Penton base and IIIa appeared as fully antigenically expressed capsid subunits in mature wild-type virions or ts104 virions after a shift down. The ts104 lesion is postulated to affect a regulatory function related in some way to penton base and fiber overproduction and the maturation processing of precursors PVI, PVII, and PVII.
Virus and host cell DNA syntheses during infection of Acholeplasma laidlawii by MVL3, a nonlytic cytocidal mycoplasmavirus
Authors: Haberer K , Maniloff J
Published: January 2020

The replication of mycoplasmavirus MVL3 in Acholeplasma laidlawii K2 host cells was studied by analysis of infected-cell lysates using sedimentation in sucrose gradients and DNA-DNA hybridization. Viral DNA replication was found to involve intermediates sedimenting faster than free viral DNA, which is a linear, double-stranded molecule of about 26 x 10(6) daltons. After the shutdown of cellular DNA synthesis, viral DNA synthesis continued for many hours. The fate of cellular and parental viral DNAs was examined.
DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro
Authors: Prives C , Beck Y , Shure H
Published: January 2020

Simian virus 40 large T- and small t-antigens have been shown previously to share immunological determinants and common sequences and to have roles in virus-induced cell transformation. However, only large T-antigen is a DNA binding protein. Under all conditions tested, small t-antigen did not interact with DNA. Large T-antigen synthesized in infected cells bound to both native calf thymus and simian virus 40 DNAs. As its binding efficiency was less than 100%, it is likely that there are different forms of T-antigen which vary in their affinity for DNA. Large T-antigen synthesized in cell-free protein-synthesizing systems primed by simian virus 40 mRNA also bound to DNA-cellulose, whereas small t-antigen similarly synthesized in vitro did not. An 82,000-molecular-weight T-antigen polypeptide synthesized in cell-free protein-synthesizing systems primed by simian virus 40 complementary RNA transcribed in vitro from simian virus 40 DNA by Escherichia coli RNA polymerase bound efficiently to simian virus 40 DNA. As this product did not share sequences with the small t-antigen, it can be concluded that the amino-terminal portion of the T-antigen is not required for some of its specific DNA binding properties.
Control of Early Gene Expression of Bacteriophage T4: Involvement of the Host
rho
Factor and the
mot
Gene of the Bacteriophage
Authors: Linder Claës H. , Sköld Ola
Published: January 2020

Many early mRNA species of bacteriophage T4 are not synthesized after infection of
Escherichia coli
in the presence of chloramphenicol. This has been interpreted as a need for T4 protein(s) to be synthesized to allow expression of some early genes, e.g., those for deoxycytidinetriphosphatase, deoxynucleosidemonophosphate kinase and UDP-glucose-DNA β-glucosyltransferase. In the experiments described here, early mRNA of bacteriophage T4 was allowed to accumulate during chloramphenicol treatment. After the addition of rifampin to inhibit further RNA synthesis, and subsequent removal of chloramphenicol, the accumulated mRNA was permitted to express itself into measured enzyme activities. It was shown that the early mRNA species coding for deoxycytidinetriphosphatase and UDP-glucose-DNA β-glucosyltransferase could be formed in the presence of chloramphenicol if the
E. coli
host cell carried a mutation in the structural gene for the RNA chain termination factor
rho.
This was interpreted to mean that T4 protein(s) with anti-
rho
activity is normally required for the expression of these two early genes. An altered
rho
-factor could not, however, relieve the need of phage protein synthesis for the formation of another early mRNA, that coding for deoxynucleosidemonophosphate kinase. In this case the
mot
gene of T4 seemed to be involved, since the primary infection of
E. coli
cells with the
mot
gene mutant
ts
G1 did not allow subsequent deoxynucleoside monophosphate kinase mRNA synthesis after wild-type phage infection in the presence of chloramphenicol. In control experiments, deoxynucleoside monophosphate kinase mRNA synthesis induced by wild-type phage superinfecting in the presence of chloramphenicol was facilitated by the primary infection with T4 phage containing an unmutated
mot
gene.
Oligonucleotide Sequence Analyses Indicate that Vesicular Stomatitis Virus Large Defective Interfering Virus Particle RNA Is Made by Internal Deletion: Evidence for Similar Transcription Polyadenylation Signals for the Synthesis of All Vesicular Stomatitis Virus mRNA Species
Authors: Clerx-Van Haaster Corrie M. , Clewley Jon P. , Bishop David H. L.
Published: January 2020

RNase T
1
oligonucleotide fingerprint analyses of three vesicular stomatitis virus Indiana serotype small defective interfering (DI) particle RNA species indicate that they only have oligonucleotides derived from the 5′ region of the viral genome. These studies also indicate that these three DI RNAs have partial L gene sequences as well as two 5′ viral oligonucleotides (59 and 70) that are not transcribed into L (or other) mRNA species (J. P. Clewley and D. H. L. Bishop, J. Virol.
30
:116-123, 1979). Analyses of the large DI RNA (LT DI) reveal a different origin. The LT DI RNA has oligonucleotides derived from both the 3′ end of the genome (including all the large oligonucleotides identified for N, NS, M, and G genes), in addition to at least one of the 5′-proximal L gene oligonucleotides (47), as well as all seven oligonucleotides (3, 38, 42, 43, 44B, 59, and 70) that are not protected from nuclease digestion after the formation of mRNA-viral RNA duplexes (Clewley and Bishop). It appears therefore that the genesis of LT RNA involves a deletion of internal L gene sequences from the viral RNA. Oligonucleotide sequence analyses have been undertaken on several of the vesicular stomatitis viral RNA oligonucleotides, including all seven (3, 38, 42, 43, 44B, 59, and 70) that are not transcribed into mRNA. The analyses confirm that oligonucleotides 59 [3′...GAACACCAAAAAUAAAAAAUA(G)...5′] and 70 [3′...GACCAAAACACCA(G)...5′] are at the 5′-end region of the viral genome. Oligonucleotide 38 [3′...GAAAUUCAUACUUUUUU(U)(G)...5′] may represent the termination signal for L mRNA synthesis (R. A. Lazzarini, personal communication). Oligonucleotide 43 [3′...GUAUACUUUUUUU(G)...5′] corresponds to the sequence shown to be the N gene mRNA polyadenylation signal (D. J. McGeoch, Cell
17
:673-681, 1979). The other three oligonucleotides share a common feature with oligonucleotides 43 and 38, viz., a stretch of 6 or 7 U residues preceded by an AUAC sequence. Thus the sequence of oligonucleotide 3 is 3′...GAAUUAAUAUAAAAUUAAAAAUUAAAAAUACUUUUUU(U)(G)...5′, whereas that of oligonucleotide 42 is 3′...GAUACUUUUUUUCAU(U)(G)...5′, and that of oligonucleotide 44B is 3′...G(U)AUACUUUUUU(G)...5′. These sequence analyses suggest a common polyadenylation signal for the synthesis of all vesicular stomatitis virus mRNA species, i.e., the sequence (3′)...AUACUUUUUU(U)...(5′).
Head maturation pathway of bacteriophages T4 and T2. V. Maturable epsilon-particle accumulating an acridine-treated bacteriophage T4-infected cells
Authors: Schaerli C , Kellenberger
Published: January 2020

A maturable head-related particle of bacteriophage T4 has been identified and characterized. This epsilon-particle has the same size as the prehead, but its shell is made of the cleaved product of gene 23 (gp23*). It contains internal matter, most likely the processed core proteins, which is lost or modified by experimental manipulations. It accumulates, together with partially filled ("grizzled") heads, in T4 infected cells that are treated with 9-aminoacridine. On sections of "well-preserved" cells the epsilon-particles are not identifiable with certainty; a more or less empty breakdown product of them becomes visible when cytoplasmic leakage is induced. The number of particles per cell is then in agreement with the biochemically and with the number of particles counted in lysates. Morphologically and biochemically, the isolated epsilon-particles closely resemble the empty small particles of 17- -infected cells described in previous papers of this series. Both are composed of gp23* and are still unexpanded, so that they are not yet able to bind the minor head proteins soc and hoc. We discuss the possibility of the epsilon-particle being an intermediate on the normal T4 wild-type head maturation pathway.
Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses
Authors: Chang H W , Garon C F , Chang E H , Lowy D R , Hager G L , Scolnick E M , Repaske R , Martin M A
Published: January 2020

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.
Characterization of an Extremely Basic Protein Derived from Granulosis Virus Nucleocapsids
Authors: Tweeten Kathleen A. , Bulla Lee A. , Consigli Richard A.
Published: January 2020

Nucleocapsids were isolated from purified enveloped nucleocapsids of
Plodia interpunctella
granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of
Pieris rapae
and from the nuclear polyhedrosis viruses of
Spodoptera frugiperda
and
Autographa californica.
The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex.
Complementation analysis of measles virus mutants isolated from persistently infected lymphoblastoid cell lines
Authors: Ju G , Birrer M , Udem S , Bloom B R
Published: January 2020

Human lymphoblastoid cell lines persistently infected with measles virus release a heterogeneous population of virions. At least 80% of the infectious particles were temperature sensitive for plaque formation at 39 degrees C. Plaque-purified temperature-sensitive mutants from four persistently infected human lymphoblastoid cell lines were shown to be heterogeneous with respect to efficiency of plating at 31 and 39 degrees C, as well as to antigen and RNA production at 39 degrees C. The heterogeneity was confirmed by complementation analysis in which 21 temperature-sensitive isolates were found to represent at least four of the five previously described complementation groups of measles virus. Two isolates complemented four reference temperature-sensitive mutants. These isolates either represent new complementation groups or are members of the fifth complementation group, group E. The majority of isolates were found to have multiple mutations, and group B mutants (RNA-) predominated. Two temperature-sensitive isolates were able to interfere with production of parental measles virus at both permissive and nonpermissive temperatures.
Radioimmunological comparison of the DNA polymerases of avian retroviruses
Authors: Bauer G , Temin H M
Published: January 2020

125I-labeled DNA polymerases of avian myeloblastosis virus and spleen necrosis virus were used in a radioimmunological characterization of avian retrovirus DNA polymerases. It was shown that avian leukosis virus and reticuloendotheliosis virus DNA polymerases do not cross-react in radioimmunoassays. Within the avian leukosis virus species, species-specific and type-specific antigenic determinants of the DNA polymerase were defined. The previous finding of genus-specific antigenic determinants in avian myeloblastosis virus and Amherst pheasant virus DNA polymerases was confirmed and extended to members of all subgroups of avian leukosis virus. It was shown that there is little immunological variation between the DNA polymerases of the four members of the reticuloendotheliosis virus species. Particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against any of the sets of antigenic determinants defined in this study.
Integration Pattern of Human JC Virus Sequences in Two Clones of a Cell Line Established from a JC Virus-Induced Hamster Brain Tumor
Authors: Wold William S. M. , Green Maurice , Mackey Jesse K. , Martin Jonathan D. , Padgett Billie L. , Walker Duard L.
Published: January 2020

The physical state of the JC virus (JCV) genome was studied in two clonal cell lines (clones 2 and 7) derived from a tissue culture cell line (HJC-15) established from a hamster brain tumor induced by JCV. Saturation-hybridization and reassociation kinetic analyses, using in vitro
32
P-labeled JCV DNA, indicated that clone 7 and 2 cells contain 9 to 10 and 4 to 5 copies per cell, respectively, of all or most of the viral genome. Both cell DNAs were analyzed by using the Southern blotting procedure with three restriction endonucleases:
Xho
I, which does not cleave JCV DNA;
Eco
RI, which cleaves once; and
Hind
III, which cleaves three times. With each DNA, a variety of JCV-specific DNA fragments were detected. The following conclusions are possible: (i) JCV DNA is integrated into cell DNA in both clonal lines; (ii) both clonal lines contain multiple copies of the viral genome integrated in a tandem head-to-tail orientation; (iii) neither clonal line contains detectable free-form I, II, or III JCV DNA; (iv) each clonal line contains multiple independent sites of JCV DNA integration; and (v) most or all of the sites of integration on the cellular or the viral genome, or both, are different in clone 7 DNA than in clone 2 DNA. Thus, although both clone 7 and clone 2 cells were established from the HJC-15 tumor cell line, they differ in the copy number and integration pattern of JCV DNA.
Inhibition by lipiarmycin of bacteriophage growth in Bacillus subtilis
Authors: Osburne M S , Sonenshein A L
Published: January 2020

We have used lipiarmycin, a specific inhibitor of initiation of transcription, to study the role of host RNA polymerase in the transcription programs of various phages of Bacillus subtilis. Unlike rifampin, lipiarmycin preferentially inhibits transcription dependent on the sigma subunit of RNA polymerase because it inactivates holoenzyme at a much greater rate than it does core enzyme. With phage SP01, addition of lipiarmycin at a middle-to-late time of infection did not inhibit phage production even though phage production was sensitive to addition of rifampin at that time. This result is consistent with the notion that unmodified host RNA polymerase holoenzyme becomes dispensable after transcription of early classes of SP01 genes, even though host core enzyme is required for synthesis of all classes of phage RNA. SP01-modified forms of RNA polymerase, which lack sigma subunit but contain phage-coded polypeptides and are able to transcribe middle and late genes, were resistant to lipiarmycin in vitro. For phage phi 105, phage development was sensitive to both lipiarmycin and rifampin in wild-type cells and resistant to both drugs in resistant mutant cells, leading to the conclusion that the activity of host holoenzyme was required for phage RNA synthesis. Growth of phage PBS2, which was resistant to rifampin, was sensitive to the addition of lipiarmycin at early times of infection of a wild-type host strain. In a lipiarmycin-resistant mutant host, PBS2 growth was resistant to lipiarmycin. This result suggests that host holoenzyme plays a previously unanticipated role in transcription of PBS2 genes.
Extra RNAs of von Magnus particles of influenza virus cause reduction of particular polymerase genes
Authors: Ueda M , Nakajima K , Sugiura A
Published: January 2020

Extra RNAs, or RNA species other than eight gene segments, in von Magnus particles of the influenza virus WSN strain were studied by polyacrylamide gel electrophoresis and oligonucleotide mapping. From the original virus stock, various cloned stocks were obtained, each giving rise to a characteristic set of extra RNAs. One cloned virus stock contained a large number of von Magnus particles. The RNA pattern was characterized by two prominent extra RNAs (X1 and X2) and a decrease in the content of two polymerase genes, P1 and P2. Segregation of the two extra RNAs was carried out by coinfection of cells with a von Magnus particle and infectious virions. The results showed that the presence of one of the extra RNAs (X2) was associated with a reduction in the amount of the P1 gene and that the presence of the other extra RNA (X1) was associated with a reduction in the amount of the P2 gene. Oligonucleotide mapping showed that both extra RNAs, X1 and X2, were derived from the P1 gene. The results suggested that an extra RNA did not necessarily cause the reduction of the progenitor polymerase gene, but might cause the reduction of another polymerase gene.
Viral DNA in bursal lymphomas induced by avian leukosis viruses
Authors: Neiman P , Payne L N , Weiss R A
Published: January 2020

Avian leukosis viruses (ALV) induce malignant lymphoma of the bursa of Fabricius. Viral DNA in tumors and normal tissues from infected birds were analyzed by using restriction endonucleases. Viral DNA fragments diagnostic of the exogenous ALV were easily detected in tumors, uninvolved bursal tissue, kidney, and erythrocyte nuclei. Exogenous viral DNA was more difficult to detect in liver. Using a restriction endonuclease (SacI) which cleaves linear unintegrated ALV DNA in a single site to define integration sites in DNA from the various tissues, we were able to detect ALV DNA only in tumor tissue. We concluded that the proviral DNA detected in the various nontumor tissue must be integrated in multiple sites. The appearance of ALV integration sites uniquely in tumors suggests that they are clonal growths. Furthermore, the data suggested the presence of a single exogenous integration site for the ALV provirus in each of six early neoplastic bursal nodules. This provirus appeared to retain the organization of EcoRI and BamHI recognition sequences present in the genome of virus used to infect the birds. The ALV integration site appeared different in each of the tumors studied. In a widespread metastatic lymphoma, multiple ALV integration sites were found as well as structural alterations in at least some copies of the ALV provirus.
Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus
Authors: Sherr C J , Fedele L A , Oskarsson M , Maizel J , Woude G V
Published: January 2020

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.
Specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication
Authors: Ramabhadran T V , Thach R E
Published: January 2020

Effect of protein synthesis inhibitors on encephalomyocarditis virus production in L-cells was studied. Inhibition of initiation by hypertonicity, harringtonine, or pactamycin decreased viral protein synthesis to a lesser extent than that of host. Virus yield was unaffected or actually enhanced by low concentrations of these inhibitors. On the contrary, the elongation inhibitors cycloheximide, anisomycin, and emetine, shown previously to inhibit viral protein synthesis preferentially, had a greater effect on virus yield than on overall protein synthesis. These results support our earlier proposal that the antiviral activity of cycloheximide derives from its specific effect on the rate of elongation of protein synthesis, and that elongation inhibitors in general may show varying degrees of specific antiviral activity.
Comparative biochemical studies of type 3 poliovirus
Authors: Minor P D
Published: January 2020

A study of the biochemistry of type 3 poliovirus strains which involves the examination of the virus-coded polypeptides in infected cells and the preparation of oligonucleotide maps is reported. The polypeptide patterns were shown to be a relatively stable property of virus strains and distinguished Sabin vaccine strains from wild strains of poliovirus type 3. This approach may be of value in deciding the origin (vaccine or nonvaccine) of field isolates of poliovirus. Oligonucleotide maps were found to be sensitive indicators of differences among strains and appear to form a basis for determining genetic relationships among strains. The nucleotide maps of two viruses isolated from human cases of paralytic poliomyelitis temporally associated with the administration of attenuated vaccine suggested a vaccine origin for the strain. In one case the nucleotide map was indistinguishable from that of the vaccine strain.
Characterization of Herpes Simplex Virus Type 1 RNA Present in the Absence of De Novo Protein Synthesis
Authors: Anderson Kevin P. , Costa Robert H. , Holland Louis E. , Wagner Edward K.
Published: January 2020

We used herpes simplex virus type 1 (HSV-1) DNA and restriction fragments of HSV-1 DNA covalently coupled to cellulose as a reagent to isolate for further characterization the major and minor HSV-1 immediate-early mRNA species in HeLa cells infected and maintained in the absence of de novo protein synthesis. Five major and several minor immediate-early mRNA species were characterized. One major species was a 4.2-kilobase mRNA mapping in the TR
S
/IR
S
region with its 3′ end distal to the U
S
region; this mRNA encoded a 170,000-dalton polypeptide in vitro. A 2.8-kilobase mRNA, encoding a 120,000-dalton polypeptide, was mapped in the TR
L
/IR
L
region with its 3′ end directed toward the U
L
region. Three 1.8-kilobase mRNA species were mapped. One, mapping in the IR
S
region with its 3′ end in the U
S
, encoded a 68,000-dalton polypeptide. One mapped in the TR
S
region and had its 3′ end in the U
S
region; the third one encoded a 64,000-dalton polypeptide and mapped in the U
L
region near the IR
L
region. One minor species 5.2 kilobases in size was clearly detectable mapping in the U
L
region. Furthermore, there were indications that one or more immediate-early mRNA species approximately 3 kilobases in size hybridized to regions near the TR
L
and in or near the TR
S
/IR
S
regions. Nuclear immediate-early RNA mapped only in those regions where polyribosomal immediate-early mRNA mapped, although minor differences were seen. Finally, we demonstrated that at least three major immediate-early mRNA's—4.2 kilobases, 2.8 kilobases, and the 1.8-kilobase one mapping in the IR
S
/U
S
region—continued to appear on polyribosomes as functional mRNA late after infection.
Regulation of Brome Mosaic Virus Gene Expression by Restriction of Initiation of Protein Synthesis
Authors: Chroboczek Jadwiga , Puchkova Ludmiła , Zagórski Włodzimierz
Published: January 2020

The translation of total and individual brome mosaic virus (BMV) RNAs was examined in a wheat germ cell-free system in the presence of various inhibitors. Inhibitors of the initiation of polypeptide synthesis, e.g., potassium ions, 7-methylguanosine 5′ -monophosphate, and aurintricarboxylic acid, were shown not only to inhibit overall BMV protein synthesis but also to change the ratio of BMV polypeptides synthesized. Under conditions restrictive for initiation, the translation of nonstructural BMV genes was suppressed, but coat protein synthesis proceeded at a high rate. A similar discrimination among BMV messengers was exerted by a regulatory protein kinase isolated from wheat germ. These results suggest that the regulation of the expression of BMV genes is based on a difference in the mechanism of formation of initiation complexes for individual BMV messages.
Bacteriophage phi W-14-infected Pseudomonas acidovorans synthesizes hydroxymethyldeoxyuridine triphosphate
Authors: Neuhard J , Maltman K L , Warren R A
Published: January 2020

The infection of Pseudomonas acidovorans with bacteriophage phi W-14 leads to the gradual disappearance of dTTP from the cells and to the appearance of hydroxymethy dUTP (hmdUTP). Infected-cell contain dUMP hydroxymethylase and activities converting hmdUMP to humdUDP and hmdUTP. Hydroxymethylase appears immediately after infection, reaching a maximum 20 min later. Thymidylate synthase activity decreases to less than 10% of the preinfection level during the initial 40 min after infection. Newly replicated DNA contains 2 to 3% hydroxymethyluracil. Although uracil is released from newly replicated DNA by acid hydrolysis, uracil is not incorporated as such into phi W-14 DNA, and dUTP is not present in the acid-soluble pool of infected cells. It is concluded that the thymine and alpha-putrescinylthymine in phi W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level and that an intermediate in one or both of these conversions is degraded to uracil by acid hydrolysis. The modification of hydroxymethyluracil is coupled tightly to replication.
Site on the vesicular stomatitis virus genome specifying polyadenylation and the end of the L gene mRNA
Authors: Schubert M , Keene J D , Herman R C , Lazzarini R A
Published: January 2020

The 5'-terminal nucleotide sequence from positions 50 to 130 of vesicular stomatitis virus RNA was determined indirectly by using a defective interfering particle RNA which contains covalently linked genomic minus and antigenomic plus sense RNAs. The last 18 nucleotides of the L gene coding for in the viral polymerase were identified and isolated by specific duplex formation between 5' terminally labeled oligonucleotides from a small single-stranded defective interfering particle RNA and L gene mRNA. The L gene ends at position 60 from the 5' terminus of the vesicular stomatitis genome. The data demonstrated that the first seven adenine residues in the polyadenylic acid tail of L gene mRNA may be coded for in the genome and suggested that the viral transcriptase itself may carry out polyadenylation, possibly by chattering at the uridine-rich sequence at the end of the L gene. Analysis of the 5'-terminal sequence of vesicular stomatitis virus genomic RNA revealed that it might fold into a complex secondary structure with possibly 62% of the bases paired.
Effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus DNA replication
Authors: Colby B M , Shaw J E , Elion G B , Pagano J S
Published: January 2020

The effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus (EBV) DNA replication in the lymphoblastoid cell lines P3HR-1 and Raji is reported. Acyclovir at a concentration of 100 microM completely inhibited EBV DNA synthesis in superinfected Raji cells, but did not inhibit DNA synthesis in mock-infected cells. The number of EBV genome equivalents per cell in the virus-producing cell line P3HR-1 was significantly reduced by acyclovir, whereas the number of latent EBV genomes in Raji cells was not affected by the drug. In situ cytohybridization performed on untreated P3HR-1 cultures revealed the presence of relatively large amounts of EBV DNA in 15 to 20% of the cells. After a 100 microM drug treatment, no P3HR-1 cells contained levels of EBV DNA detectable by in situ cytohybridization. Indirect immunofluorescence studies demonstrated that during treatment with 100 microM acyclovir for 7 days, the percentage of P3HR-1 cells expressing viral capsid antigen was reduced. The EBV DNA remaining in P3HR-1 cells after treatment with 100 microM acyclovir (approximately 14 genomes per cell) had the properties of covalently closed circular DNA with an average molecular weight of 108 X 10(6), as determined by contour length measurements.
Monoclonal Antibodies Identify Individual Determinants on Mouse Mammary Tumor Virus Glycoprotein gp52 with Group, Class, or Type Specificity
Authors: Massey Richard J. , Arthur Larry O. , Nowinski Robert C. , Schochetman Gerald
Published: January 2020

Hybrid cell lines producing monoclonal antibodies against the C3H strain of mouse mammary tumor virus (C3H MMTV) were prepared by the fusion of mouse myeloma cells with the lymphocytes of BALB/c mice that were immunized with C3H MMTV. Approximately 10% of the hybrid cells initially plated after cell fusion produced immunoglobulins that reacted in antibody-binding assays with C3H MMTV; 40 of these cells were cloned, and 6 eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from serum and ascites fluid of syngeneic mice inoculated with the hybrid cells. All of the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the immunoglobulin M subclass and three produced immunoglobulin G2a. The monoclonal antibodies showed limited charge heterogeneity in light and heavy chains when analyzed by high-resolution, two-dimensional gel electrophoresis. Three serologically distinct specificities were observed when these ascites fluids were tested against different strains of MMTV. The antigenic determinants detected were the following: (i) a type-specific determinant unique to the C3H strain of MMTV; (ii) class-specific determinants shared between C3H and GR MMTVs; and (iii) a group-specific determinant found on C3H, GR, RIII, and the endogenous C3H (C3Hf) MMTVs. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the three patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule and also clearly show that one strain of MMTV can be distinguished from other strains.
Conserved sequence related to the 3'-terminal region of retrovirus RNA'S In normal cellular DNAs
Authors: Kominami R , Tomita Y , Connors E C , Hatanaka M
Published: January 2020

The nucleotide sequences related to the 3'-terminal protion of retrovirus genomic RNA have been detected in the DNA of animals, including humans. The DNA complementary to the 400 to 700 nucleotides from the 3'-terminal end of retrovirus RNA (cDNA3'), which contains the enriched conserved region, was hybridized with DNA from a variety of animal cells. Under the conditions of annealing in 0.72 M NaCl at 67 degrees C and hydroxyapatite chromatography at 55 degrees C, 20 to 50% of the radioactivity of the cDNA3' prepared from two retroviruses, a murine Rauscher virus (RLV) and a baboon virus (M7), annealed with normal cellular DNA of animals, including human tissue. The thermal denaturation profile revealed considerable mismatching between the duplex of the cDNA3' and human DNA, cDNA3' of retroviruses is most homologous to cellular DNA of the host species of origin and is less homologous to cellular DNA of species that are distant in the phylogeny of the host species. The conservation and evolution of nucleotide sequences related to the 3' end of retrovirus genomes in animal DNAs, including humans, suggest that the sequences may have important functions.
Recombination and linkage between structural and regulatory genes of herpes simplex virus type 1: study of the functional organization of the genome
Authors: Honess R W , Buchan A , Halliburton I W , Watson D H
Published: January 2020

Phenotypic and genetic properties of 12 markers in structural and regulatory functions of herpes simplex virus type 1 were characterized, and their recombination and segregation behavior was investigated and interpreted with reference to available information on their physical locations. The markers were: (i) ts markers in a structural glycoprotein (tsB5) and in alpha (immediate early; tsLB2, tsc75) or beta (early, delayed early; tsB1) functions with regulatory effects; together with (ii) plaque morphology (syn), phosphonoacetate resistance (Pr), and thymidine kinase (TK) phenotypes; and (iii) electrophoretically distinct variants of glycosylated (glycoprotein C, gpC; ICP10) and non-glycosylated [VP(13-14), VP23] structural and nonstructural [ICP(47-48)] polypeptides. Mean two-factor recombination frequencies ranged from 2% (for noncomplementing mutants tsLB2 and tsc75) to 35 to 40% (for unlinked markers) and were influenced by the relative contributions of parental viruses to the mixed infection. Even with control of this variable, standard deviations of mean measures of recombination frequency ranged from a minimum of 14% (with n greater than or equal to 10) to 65% (with n = 3) of mean values; no recombination frequencies higher than 55% were observed. Differences in mean two-factor recombination frequencies between a small number of loosely linked markers were, therefore, not reliable measures of real differences in linkage. Measurements of the segregation of unselected markers among recombinant progeny were, therefore, used as measures of linkage. These experiments (i) established a linkage group for markers in the long unique region of the genome additional to, but consistent with, existing physical data, i.e., TK-syn-tsB5-(tsB1.Pr)-[gpC.VP(13-14)]; (II) identified markers, e.g., ICP(47-48), linked to regulatory mutations (tsLB2, tsc75) in redundant DNA sequences; and (iii) used the segregation of these regulatory mutations and linked markers among unselected progeny to demonstrate the linkage groups: Pr-syn-TK-tsc75-ICP(47-48), [VP(13-14).gpC]-Pr-syn-TK, and TK-tsc75-[VP(13-14).gpC]. These results were most simply explained if bi- or intermolecular recombination occurred between circular molecules or molecules catenated "head-to-tail" and were incompatible with intermolecular recombination as the mechanism of isomerization of herpes simplex virus DNA.
Electron Microscopy of Vesicular Stomatitis Virus Replicative Ribonucleoproteins
Authors: Naeve Clayton W. , Summers Donald F.
Published: January 2020

The objective of this investigation was to examine by electron microscopy the replicative ribonucleoprotein (RNP) structures synthesized in vesicular stomatitis virus-infected HeLa cells. Pulse-labeled in vivo products of vesicular stomatitis virus replication and transcription can be separated by centrifugation in Renografin gradients. Transcription complexes are dissociated, allowing nascent messenger RNPs to remain at the top of the gradient, whereas RNPs biochemically consistent with replication complexes sediment to the middle of the gradient. Examination of these structures by electron microscopy revealed that all exist as coiled or helical RNPs having dimensions of ∼20 by 700 nm. These structures can be further subdivided into three major morphological classes: (i) linear forms (20 by 769 ± 158 nm), which have both ends free; (ii) circular forms (20 by 679 ± 95 nm), which appear to have both ends joined; and (iii) complex forms, which include those structures which are branched replicative complexes as well as those which are random. To distinguish random complexes and possible transcriptive complex contaminants from replicative complexes, it was necessary to uncoil the RNP structures with EDTA so that length measurements could be made relating the nascent strand length to its position on the template. After EDTA treatment, the linear RNPs uncoiled (10 by 4,035 ± 3,802 nm), and the circular morphology virtually disappeared. However, a new form appeared which was one-half the length and double the width (20 by 2,103 ± 306 nm) of full-length RNPs and contained a loop at one end and two free ends at the other (α-form RNP). The distribution and length analysis of these structures, plus and minus EDTA, suggest that the α-form RNPs arise by EDTA-induced uncoiling of circular forms held together at the ends. Close scrutiny of uncoiled complex RNPs revealed no single-strand RNP templates with single-strand nascents. However, several complexes were observed which appeared to contain α-form templates with single-strand nascent RNPs. Length measurements suggest these complexes are neither random nor transcriptive, but are replicative. These experiments suggest that replication may, in part, occur on circular coiled RNP templates.
Expression of superinfection immunity to bacteriophage phi 105 by Bacillus subtilis cells carrying a plasmic chimera of pUB110 and EcoRI fragment F of phi 105 DNA
Authors: Cully D F , Garro A J
Published: January 2020

A 2.1-megadalton, EcoRI-generated fragment of Bacillus subtilis phage phi 105 DNA was cloned into plasmid pUB110. The hybrid plasmid produces a biologically active product which renders B. subtilis immune to infection by phi 105.