bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Identification of Lymphocytic and Myelogenous Leukemia-Specific Sequences in Genomes of Gibbon Oncornaviruses
Authors: Sun Lily , Kawakami Thomas G.
Published: January 2020

Five gibbon ape leukemia virus substrains (two from gibbons with lymphocytic leukemia and three from gibbons with myelogenous leukemia) were examined for unique genomic sequences specific for each form of leukemia. By using sequential adsorption procedures, the genome from each gibbon ape leukemia virus was fractionated into four sets of distinct nucleotide sequences. Based on their hybridization specificities toward DNAs of leukemic tissues, these sequences were designated as follows: (i) “COM,” (ii) “LYM” or “MYE,” (iii) “UNI,” and (iv) “UND.” The COM fraction represented sequences common to all of the viral genomes. The LYM fraction, which was isolated only from gibbon ape leukemia viruses associated with lymphocytic leukemia, represented genomic sequences associated with lymphocytic leukemia since the RNA hybridized at a 4- to 15-fold-higher rate to infected tissue DNA from lymphocytic leukemic gibbons than to infected tissue DNA from myelogenous leukemic gibbons. The MYE fraction, which was isolated only from gibbon ape leukemia viruses associated with myelogenous leukemia, represented genomic sequences associated with myelogenous leukemia since the RNA hybridized at a 5- to 15-fold-higher rate to infected tissue DNA from myelogenous leukemic gibbons than to infected tissue DNA from lymphocytic leukemic gibbons. The UNI fraction contained sequences unique to one virus substrain. The UND fraction contained sequences which varied depending upon the substrains involved in the adsorption procedures. These findings suggest that each gibbon ape leukemia virus examined in this study contains subgenomic sequences that are specifically identifiable only with the form of leukemia from which the virus was isolated.
Comparative analysis of the genomes of feline leukemia viruses
Authors: Rosenberg Z F , Pedersen F S , Haseltine W A
Published: January 2020

The genomes of several strains of feline leukemia virus (FeLV) were compared by two-dimensional polyacrylamide gel electrophoresis of the large RNase T1-resistant oligonucleotides of the 70S RNA. Differences between each strain of FeLV tested were detected by this method. We estimate that the degree of sequence identity between the viruses is: FeLV A (Glasgow-1) to FeLV B (Snyder-Theilen), 52%; FeLV A (Glasgow-1) to FeLV C(Sarma), 66%; FeLV B(Snyder-Theilen) to FeLV C (Sarma), 37%. The fingerprints of two independent isolates of FeLV strains of subgroup A (Glasgow-1 and Rickard) were detectably different. We conclude that the RNase T1 oligonucleotide fingerprint pattern provides a useful tool for identification of FeLV strains.
Use of vesicular stomatitis virus pseudotypes to map viral receptor genes: Assignment of RD114 virus receptor gene to human chromosome 19
Authors: Schnitzer T J , Weiss R A , Juricek D K , Ruddle F H
Published: January 2020

Vesicular stomatitis virus pseudotypes bearing envelope glycoproteins of the endogenous feline type C retrovirus, RD114, were used to assay the expression of receptors specific to RD114 on the surfaces of mouse-human hybrid cells carrying different human chromosomes. These studies show that the gene encoding the RD114 receptor is located on human chromosome 19.
Physical mapping of adeno-associated virus serotype 4 DNA
Authors: Muster C J , Lee Y S , Newbold J E , Leis J
Published: January 2020

The organization of adeno-associated virus serotype 4 (AAV 4) DNA was probed by using restriction enzymes. The cleavage sites of the following enzymes were mapped: BglII, BamI, HincII, KpnI, PstI, SalI, SstI, and XhoI. The orientation of transcription on the physical cleavage map was determined by locating the fragments which contain the 3' end of the mRNA. Strand separation gels were run for HinII fragments of AAV 4 DNA. By hybridizing AAV 4 mRNA to the resolved strands, the polarity of the DNA strands was identified. Restriction digestion of AAV 4 DNA sometimes produced terminal fragments which migrated in agarose gels as doublets. However, when AAV 4 DNA was prepared devoid of any single-stranded nicks, these terminal doublet bands were not observed upon subsequent restriction analysis. During these studies, the molecular weights of both AAV 4 and AAV 2 duplex DNA were measured and were found to be somewhat larger than previously reported (3.18 x 10(6) and 3.10 x 10(6), respectively).
Binding of wheat germ ribosomes to fragmented viral mRNA
Authors: Kozak M
Published: January 2020

The specificity of binding of wheat germ ribosomes to mRNA was greatly altered by cleavage of the message. Fragmentation of reovirus mRNA allowed wheat germ ribosomes to bind and protect a variety of internal sequences which were not accessible to ribosomes in the intact message. In experiments using the polycistronic mRNA from bacteriophage R17, wheat germ ribosomes bound preferentially at the beginning of the lysis peptide and synthetase cistrons, and at a third site which may be derived from the C-terminal region of the A protein cistron. This result is similar to that reported previously in a mammalian translational system (J.F. Atkins et al., Cell 18:246-256, 1979) except that, in the present study, limited cleavage of the phage RNA was necessary to activate these sites. More extensive fragmentation of R17 RNA permitted wheat germ ribosomes to bind and protect a great many additional sites. Thus, presence of an (exposed) 5'-terminus on an RNA molecule appears to be necessary and sufficient for attachment of eucaryotic ribosomes.
Phenotypic transformation of the host cell enhances polyoma pseudovirion formation
Authors: Cashdollar L W , Yelton D B
Published: January 2020

Phenotypic transformation of the host cell affected the formation of polyoma pseuodovirions. Polyoma virus infection of various transformed derivatives of mouse 3T3 cells resulted in the formation of predominantly pseudovirions, whereas infection of mouse 3T3 cells produced mainly polyoma virus. The effect that transformation of the host cell had on polyoma pseudovirus formation was further demonstrated by using phenotypic revertants isolated from some of the transformed cell lines. The revertants were characterized by their morphology, saturation densities, and colony-forming ability in methylcellulose suspension. By these criteria they were distinct from their transformed parents and similar to 3T3 cells. After infection, the revertants produced predominantly polyoma virus and few pseudovirus. Thus, for the cell lines used in this study, phenotypic transformation enhanced the formationof polyoma pseudovirions.
Characteristics of a new bacteriophage, Psp231a, infecting Pseudomonas phaseolicola HB10Y
Authors: Taylor W D , DeLuca N , Vollherbst K , Doman T , d'Amato T , Snipes W
Published: January 2020

Bacteriophage Psp231a infects Pseudomonas phaseolicola, strain HB10Y, which is the host cell for the enveloped bacteriophage phi 6. This paper describes the biophysical characteristics of Psp231a and the physical properties of its nucleic acid. In electron micrographs the virion appears as an icosahedral structure, approximately 55 nm in diameter, with a short tail. The virion density is 1.48 g/cm3 in CsCl, and the sedimentation coefficient is approximately 407S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 12 polypeptides ranging in molecular weight from 5,000 to 117,000. The nucleic acid of Psp231a is linear, double-stranded DNA of molecular weight 28 X 10(6). Its density in CsCl is 1.716 g/cm3, and its sedimentation coefficient in 3 M CsCl is 20.0S, corresponding to an S020,W of 34S.
Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns
Authors: Eick Dirk , Stabel Silvia , Doerfler Walter
Published: January 2020

Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Ad12 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.
Molecular cloning and characterization of avian sarcoma virus circular DNA molecules
Authors: DeLorbe W J , Luciw P A , Goodman H M , Varmus H E , Bishop J M
Published: January 2020

Supercoiled DNA molecules were used for the molecular cloning of full-length avian sarcoma virus (ASV) DNA. Viral DNA produced by the Schmidt-Ruppin A (SR-A) strain of ASV was isolated from acutely infected transformed quail cells. Supercoiled DNA was separated from linear and open circular DNA by acid phenol extraction, opened into a full-length linear form by cleavage with the restriction endonuclease SacI, and cloned into lambda gtWES x lambda B. Four different cloned viral DNA molecules were isolated: SRA-1 contains two copies of the 330-base pair terminal redundancy normally found at each end of the linear DNA molecules, but harbors a 63-base pair deletion that spans the site at which the two copies of the terminal redundancy are joined in circular DNA molecules; SRA-2 contains two complete copies of the terminal redundancy; SRA-3 probably contains only one copy of the terminal redundancy but in all other respects appears to be similar to SRA-2; SRA-4 contains a 2,500-base pair deletion that removes all of the src gene (the gene responsible for transformation by ASVs) plus additional nucleotides adjacent to the src gene whose precise locations have not been determined. Transfection of chicken embryo fibroblasts by either SRA-1 or SRA-2 resulted both in the appearance of transformed cells and in the production of infectious virus. These results demonstrate that the cloned DNA molecules are functionally identical to viral DNA produced in vivo; therefore, molecular cloning did not cause any major alterations of the DNA. The infectivity of SRA-1 DNA indicates that the 63 base pairs missing from that molecule are not required for the initiation of viral RNA synthesis, even though the deletion is located in a copy of the terminal redundancy thought to carry a promoter for RNA synthesis. This suggests that the deletion does not remove any sequences required for the initiation of transcription.
Intracellular forms of simian virus 40 nucleoprotein complexes. III. Study of histone modifications
Authors: Coca-Prados M , Vidali G , Hsu M T
Published: January 2020

The modification patterns of histones present in various forms of intracellular simian virus 40 nucleoprotein complexes were analyzed by acetic acid-urea-polyacrylamide gel electrophoresis. The results showed that different viral nucleoprotein complexes contain different histone patterns. Simian virus 40 chromatin, which contains the activities for the synthesis of viral RNA and DNA, exhibits a histone modification pattern similar to that of the host chromatin. However, virion assembly intermediates and mature virions contain highly modified histones. Pulse-chase experiments with [3H]lysine showed that the newly incorporated histones in the virion assembly intermediates were already highly modified. The majority of in vivo acetylation activity of histones occurred on the 70S simian virus 40 chromatin as analyzed by pulse-labeling with [3H]acetate. These results and our previous analysis of the virion assembly pathway suggest that three stages are involved in the packaging of simian virus 40 chromatin into the mature virion: (i) modification of histones, (ii) accumulation of capsid protein around the chromatin with highly modified histones, and (iii) organization of capsid proteins into salt-resistant shells. The role of histone modification in virion assembly is discussed.
Herpes simplex virus thymidine kinase transcripts are absent from both nucleus and cytoplasm during infection in the presence of cycloheximide
Authors: Leung W C , Dimock K , Smiley J R , Bacchetti S
Published: January 2020

Nick-translated DNA from a recombinant plasmid containing the herpes simplex virus type 1 thymidine kinase gene was used as a probe for the synthesis of thymidine kinase RNA. The recombinant plasmid was generated by inserting the 3.5-kilobase fragment derived by BamHI digestion of herpes simplex virus type 1 DNA into plasmid pBR322. At 8 h after infection, cytoplasmic and nuclear RNA hybridized to 14% and 19% of the recombinant DNA probe, respectively. However, no significant hybridization was found with either nuclear or cytoplasmic RNA extracted from cells infected and maintained in the presence of cycloheximide. This suggests that no thymidine kinase-related RNA was synthesized in the absence of alpha polypeptides, and supports the hypothesis that the alpha polypeptides effect new thymidine kinase RNA synthesis rather than being involved in processing or transport of thymidine kinase RNA. In cells infected and maintained in the presence of the arginine analog canavanine, about 2 to 3% of the plasmid DNA was found to hybridize with cytoplasmic and nuclear RNA. However, when a recombinant plasmid DNA containing only thymidine kinase coding sequences was used, no significant hybridization was found. The inhibition of thymidine kinase transcription by canavanine suggests that thymidine kinase belongs to the beta 2 kinetic class.
Avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: characterization of a deletion in the provirus
Authors: Shank P R , Linial M
Published: January 2020

We have previously described a nonconditional mutant of avian sarcoma virus (SE21Q1b) which fails to package viral RNA (Gallis et al., Virology 94:146-161, 1979; Linial et al., Cell 15:1371-1381, 1978). Quail cells transformed by SE21Q1b contain normal amounts of intracellular viral mRNA's for src, env, and gag-pol and release particles with the density of normal virus containing a typical complement of virion proteins, including reverse transcriptase. These virions are noninfectious for both chicken and quail cells and contain primarily cellular rather than viral RNA. Analysis by gel electrophoresis of the cellular DNA of quail cells transformed by SE21Q1b after restriction endonuclease digestion indicated the presence of a single provirus. The provirus was located at one site in the genome of the host cell and was flanked by the characteristic terminally repeated sequences derived from the 3' and 5' ends of viral RNA. The only defect detected in the SE21Q1b provirus was a deletion of ca. 150 base pairs of DNA somewhere between 300 and 600 bases from the left (gag-pol) end of the provirus. Analyses of the proviral DNA of cells transformed by wild-type recombinants between SE21Q1b and leukosis viruses reveal that the recombinants no longer contain this deletion. The deletion, therefore, defines a region on the viral RNA which is required for correct packaging of the virion RNA.
Arrangement of the genome of the human papovavirus RF virus
Authors: Pater A , Pater M M , di Mayorca G
Published: January 2020

DNA from plaque-purified RF virus, a variant of BK virus, was found to contain two species of molecules. Hybridization of each DNA species to the fragments of BK virus DNA revealed that one species had a deletion corresponding to at least 50% of the late region and the other had a deletion corresponding to at least 40% of the early region of BK virus DNA. Analysis by cleavage of each RF virus DNA species with restriction endonucleases EcoRI, HindIII, AvaII, and PvuII, when compared with BK virus DNA, revealed that the size and number of fragments were different. These results suggest the loss of some restriction sites and the appearance of new sites, probably as a result of base changes in each RF virus DNA species. Furthermore, analysis of the restriction map of each DNA molecule revealed in insertion(s) in both DNA species.
BALB/c myeloma retroviruses: peptide mapping and immunological analysis of the pp12 structural protein
Authors: Spriggs D R , Krueger R G
Published: January 2020

We have compared the pp12 structural protein of the MO-21 and FL-1 BALB/c myeloma retroviruses with the pp12 of several prototype retroviruses. Chymotryptic peptide maps of 125I-labeled, immune-precipitated pp12 proteins revealed that the MO-21 and FL-1 proteins can be distinguished from one another. The MO-21 pp12 most closely resembled the NIH-xenotrophic virus pp12, and the FL-1 pp12 most closely resembled the pp12 of BV-2 and WN 1802 B. Competition radioimmunoassay studies showed that the MO-21 and FL-1 pp12 proteins are also antigenically distinct from one another and that both contain pp12 antigenic determinants of a xenotropic virus. These data support our proposal that these two BALB/c viruses contain a gag gene that was generated by recombination between endogenous eco- and xenotropic viral sequences.
Partial phosphorylation in vivo of the avian retrovirus pp32 DNA endonuclease
Authors: Schiff R D , Grandgenett D P
Published: January 2020

Avian retrovirus pp32, a DNA endonuclease which is structurally related to the avian retrovirus DNA polymerase beta polypeptide, has been demonstrated to be partially phosphorylated in vivo. Unlabeled or [35S]methionine-labeled pp32 from avian sarcoma virus or avian myeloblastosis virus migrated as an electrophoretic doublet on discontinuous sodium dodecyl sulfate-polyacrylamide slab gels. However, pp32 immunoprecipitated from avian sarcoma virus labeled in vivo with [32P]orthophosphoric acid migrated as a single band, which co-electrophoresed with the slower-moving band of the doublet represented by unlabeled or 35S-labeled pp32. The presence of a slower-migrating phosphorylated band in pp32 suggests that the observed electrophoretic heterogeneity of purified pp32 is due to partial phosphorylation. Tryptic peptide analysis of 32P-labeled avian sarcoma virus beta and pp32 demonstrated that all the three labeled peptides in the beta polypeptide were also present in pp32. However, pp32 had one tryptic peptide which was preferentially labeled in comparison to the comigrating peptide found in beta digests, suggesting that phosphorylation may play a role in the processing of pp32 from beta or in the regulation of its associated DNA endonuclease activity.
Isolation of recombinant DNA clones carrying complete integrated proviruses of Moloney murine leukemia virus
Authors: Bacheler L , Fan H
Published: January 2020

EcoRI DNA fragments from a Moloney murine leukemia virus (M-MuLV)-infected mouse fibroblast line (M-MuLV clone A9) were cloned in lambda phage Charon 4A cloning vector to derive clones containing integrated M-MuLV proviral DNA. A 10- to 16-megadalton class of EcoRI fragments was chosen for cloning, based on (i) its ability to induce XC-positive virus upon transfection of NIH/3T3 cells, and (ii) its content of a 0.8-megadalton viral KpnI fragment diagnostic for M-MuLV. Six recombinant DNA clones were isolated which contain a complete M-MuLV provirus, as judged by (i) restriction endonuclease mapping and (ii) the fact that all of the clones gave rise to XC-positive, NB-tropic virus upon DNA infection in NIH/3T3 cells. The sizes of the inserts were 12.0 (for three clones) or 12.5 megadaltons (for three clones). Restriction mapping indicated that these six clones represent five different M-MuLV proviral integrations into different cellular DNA sites.
Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies
Authors: Locarnini S A , Coulepis A G , Westaway E G , Gust I D
Published: January 2020

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.
Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy
Authors: Murti K G , Bondurant M , Tereba A
Published: January 2020

The secondary structural features in the 70S RNAs of the Prague strain of avian Rous sarcoma virus, subgroup A (PR-RSV-A), and Moloney murine leukemia virus (M-MuLV) were compared by electron microscopy. The PR-RSV-A genome contained two subunits joined by a linkage structure as in the genomes of M-MuLV and other mammalian retroviruses. In both viral genomes, a highly reproducible hairpin occurred at about 70 nucleotides from the 5' end of each subunit and contained 320 +/- 8 nucleotides. The stable point of linkage between the subunits in both viral genomes involved fewer than 50 nucleotides and occurred at 466 +/- 9 nucleotides from the 5' end. This places the linkage about 350 nucleotides further toward the 3' end of the subunit than the binding site of primer tRNA. Another structural feature common to both genomes was a loop in each subunit. In M-MuLV, the loop contained 3.9 +/- 0.10 kilobases (kb) and occurred at a distance of 2.2 +/- 0.05 kb from the 5' end. In PR-RSV-A, the loop was smaller (2.3 +/- 0.10 kb) and further (3.3 +/- 0.10 kb) from the 5' end. When M-MuLV RNA was heated to 70, 85, or 90 degrees C and cooled, the hairpin consistently reformed at the 5' end. No other structures typical of the native molecules reappeared. In RNA samples heated to 70 degrees C, a new loop reproducibly occurred near the 5' end of each subunit, but this loop was not found in samples heated to higher temperatures. Based on all of these findings, we conclude that the genome of PR-RSV-A shares several features with M-MuLV and other mammalian retroviruses and that the primer tRNA molecules are not involved in the linkage of the two subunits in either genome. We also conclude that the dimer linkage and the loops in subunits are typical of the native molecules and that their formation requires a special environment.
Resistance of cultures of normal T cells to infection with murine type C viruses
Authors: Horak I , Enjuanes L , Lee J C , Ihle J N
Published: January 2020

Long-term continuous cultures of normal T cells were established from C57BL/6 and BALB/c mice by using conditioned medium from concanavalin A-stimulated lymphocytes. The ability of various murine type C viruses to infect these normal T cell cultures was examined and compared with their ability to infect transformed T cells. All of the viruses examined, including a thymotropic radiation leukemia virus, were unable to infect and replicate in normal T cells but readily did so in transformed T cells.
In Vitro Translation of Uukuniemi Virus-Specific RNAs: Identification of a Nonstructural Protein and a Precursor to the Membrane Glycoproteins
Authors: Ulmanen Ismo , Seppälä Päivi , Pettersson Ralf F.
Published: January 2020

We isolated the virus-specific RNA species from Uukuniemi virus-infected chicken embryo cells and fractionated them by sucrose gradient centrifugation. In addition to three RNA species cosedimenting with the three viral RNA segments L (29S), M (23S), and S (17S), a fourth major RNA species, sedimenting at about 12S (S2), was found early in the infection. Annealing experiments indicated that the cytoplasmic L and M RNA species consisted of both plus and minus strands, with the plus strands in slight excess. Most of the S1 RNA was of negative polarity, whereas S2 was of positive polarity. The S2 RNA specifically annealed to the virion S RNA segment, indicating that it is transcribed from this segment. In vitro translation of the individual RNA species in micrococcal nuclease-treated cell-free reticulocyte extracts showed that an mRNA cosedimenting with the virion M RNA directed the synthesis of a virus-specific 110,000-dalton polypeptide (p110). This polypeptide could be immunoprecipitated with antiserum prepared against purified virions. When translation was carried out in the presence of dog pancreas microsomes, p110 was absent. Instead, an immunoprecipitable polypeptide band, with a molecular weight of about 70,000 and migrating between the virion surface glycoproteins G1 and G2, was observed. It is thus likely that the glycoproteins are synthesized as a precursor (p110), which during translation is cleaved roughly in the middle to yield G1 and G2. The 12S RNA species directed the synthesis of the nucleocapsid protein and a novel polypeptide with an apparent molecular weight of about 30,000. The latter was not precipitated with antivirion serum and was absent from lysates programmed with the corresponding RNA fraction from a mock-infected extract. Since, in addition, it was not found in purified virions and was present in the cytoplasm of infected cells but not in uninfected cells, it probably represents a nonstructural polypeptide.
Procoat, the precursor of M13 coat protein, inserts post-translationally into the membrane of cells infected by wild-type virus
Authors: Date T , Wickner W T
Published: January 2020

In growing cells infected by wild-type coliphage M13, the synthesis of procoat protein is completed before it inserts into the plasma membrane ane is converted to coat protein.
Characterization and classification of virus particles associated with hepatitis A. III. Structural proteins
Authors: Tratschin J D , Siegl G , Frösner G G , Deinhardt F
Published: January 2020

Hepatitis A virus was purified from fecal samples collected at various times in the incubation period of patients with naturally acquired hepatitis A. The proteins of particles banding at around 1.34 g/ml in CsCl and sedimenting at about 160S were radioiodinated in vitro and separated by electrophoresis on polyacrylamide gels in the presence of 0.1% sodium dodecyl sulfate and 8 M urea. Under these conditions, the capsid proteins resolved into four polypeptides with molecular weights of approximately 31,000, 24,500, 21,000, and 9,000, respectively. A fifth protein of about 40,000 daltons in size and assumed to be equivalent to the precursor polypeptide VP0 of the picornaviruses was present in particles sedimenting at only 150 to 155S and banding at around 1.33 g/ml in CsCl. The physicochemical characteristics of these particles are consistent with those of the provirion structures of picornaviruses. In several of the fecal samples, these particles represented a considerable fraction of all particles present. The significance of this finding with respect to the antigenicity of hepatitis A antigen extracted from stool specimens is discussed.
Measles virus polypeptides in infected cells studied by immune precipitation and one-dimensional peptide mapping
Authors: Graves M C
Published: January 2020

Measles virus does not turn off host cell polypeptide synthesis, making it difficult to precisely identify the polypeptides specified by the virus during the infectious cycle. By using the technique of immune precipitation with measles-specific antisera, the host cell background has been eliminated, and new observations have been made concerning measles virus polypeptides H, P, NP, F, and M. The H polypeptide is first synthesized as a monomer which is processed by further glycosylation and by the formation of disulfide-bonded dimers. Polypeptide P (70,000 daltons) has been found to occur also as a 65,000-dalton molecule, P2, and both forms of the molecule are equally phosphorylated. Polypeptide NP is processed from a cleavage-sensitive form (which undergoes cleavage during the process of isolation to form polypeptide 6 [41,000 daltons]) to a form which is resistant to this cleavage. The fusion and hemolysin polypeptide is first found in the cells as a 55,000-dalton precursor, F0, which is clearly resolved from the NP polypeptide on gel electrophoresis. The measles virus F0 protein identified in previous reports had not been resolved from the 60,000-dalton NP polypeptide. The M protein occurs in the infected cells as two distinct bands, and, as in the case of Sendai virus, one of these two M protein bands represents a phosphorylated form of the other.
Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus
Authors: Kettmann R , Couez D , Burny A
Published: January 2020

A detailed restriction map was deduced for the genome of the exogenous bovine leukemia virus. The cleavage sites for nine restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with bovine leukemia virus was isolated. The linear viral DNA had a unique restriction map, indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-enriched probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. Restriction enzyme analysis of integrated bovine leukemia virus information present in animals with enzootic bovine leukosis provided evidence for the existence of genetic variants of the virus.
Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA
Authors: Siddiqui A , Marion P L , Robinson W S
Published: January 2020

Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group. Both viruses contain small circular DNA molecules which are partially single stranded. Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600. Two cloned recombinants were characterized. One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA. A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined. The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA. Hybridization with an HBV [32P]DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA. The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs. These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.
Method for induction of mutations in physically defined regions of the herpes simplex virus genome
Authors: Sandri-Goldin R M , Levine M , Glorioso J C
Published: January 2020

A procedure was developed for inducing mutations in isolated restriction enzyme fragments of herpes simplex virus type 1 (HSV-1) DNA with nitrous acid. The mutations were then transferred to the viral genome by genetic recombination during cotransfection of rabbit kidney cells with the mutagenized fragments and intact HSV-1 DNA. The HpaI restriction enzyme fragments LD, B, LG, I, and J were mutagenized. Temperature-sensitive mutants were found at frequencies of 1 to 5% among the progeny of the transfections. Syncytial mutants also were found at high frequency when fragment B or LD was used for mutagenesis. Fifteen of these mutants, 11 temperature sensitive and 4 syncytial, were used for further studies, including complementation analysis, DNA synthesis, and marker rescue. Marker rescue data presented here and in the accompanying publication (A. L. Goldin, R. M. Sandri-Goldin, M. Levine, and J. C. Glorioso, J. Virol. 38: 50-58, 1981) confirm the map position of some of the newly isolated mutants.
Virus-receptor interaction in the adenovirus system I. Identification of virion attachment proteins of the HeLa cell plasma membrane
Authors: Svensson U , Persson R , Everitt E
Published: January 2020

Plasma membranes from HeLa cells were isolated in a two-phase polymer system. To compare the efficiency of attachment protein extraction, a normalized assay for the assessment of adenovirus type 2 (Ad2) receptor-active components interfering with the attachment of Ad2 to HeLa cells was developed. An optimized detergent extraction procedure, 0.5% Triton X-100, was used, and solubilized membrane proteins were radioisotope labeled in vitro. Proteins with affinity for Ad2 virions were quantified and identified in a sucrose gradient sedimentation assay and by affinity chromatography with cross-linked Ad2 virions immobilized to AH-Sepharose 4B. From virions recovered in the sucrose gradient system, one major membrane component of high affinity was identified with a polypeptide molecular weight of around 40,000. Glycosylated proteins isolated by wheat germ lectin chromatography with high affinity for immobilized virus particles were isolated, and two major components with apparent molecular weights of 40,000 and 42,000 were identified. We suggest that a glycosylated protein with high affinity for Ad2 virions and a polypeptide molecular weight of 40,000 to 42,000 is one component of the Ad2 attachment site on HeLa cells.
Temporal regulation of human cytomegalovirus transcription at immediate early and early times after infection
Authors: Wathen M W , Thomsen D R , Stinski M F
Published: January 2020

The immediate early transcripts of human cytomegalovirus originated from restricted regions of the viral genome. In contrast, transcription at early times was complementary to all regions of the viral genome that were fractionated by restriction endonuclease treatment followed by agarose gel electrophoresis. The viral genome was also extensively transcribed when 2 h of protein synthesis or longer was permitted after infection in permissive cells treated with an inhibitor of viral DNA replication or in nonpermissive cells of animal origin that permit little or no viral DNA replication. The size and in vitro translation products of the cytomegalovirus-specified mRNA's at immediate early and early times after infection were determined. Discrete size classes of virus-specified polyadenylated RNA accumulated on the polyribosomes of cells infected in the presence of an inhibitor of protein synthesis. When 2 or 24 h of protein synthesis occurred after infection, there were changes in the relative abundance of the virus-specified RNAs that accumulated on polyribosomes. Treatment of nonpermissive cells had little effect on the size classes of viral RNA found associated with the polyribosomes at early times after infection. These viral mRNA's were assumed to represent early viral gene expression. In vitro translation of the viral mRNA isolated from polyribosomes at immediate early and early times after infection identified many of the virus-specified gene products and demonstrated (i) a switch from immediate early to early viral gene expression and (ii) a prolonged phase of early viral gene expression. The data also indicated that the initiation of viral RNA synthesis does not depend on the formation of viral protein, but that de novo viral protein synthesis may influence the extent of transcription of the viral genome.
Effect of tunicamycin on cell fusion induced by Mason-Pfizer monkey virus
Authors: Chatterjee S , Bradac J , Hunter E
Published: January 2020

Mason-Pfizer monkey virus, a D-type retrovirus, has been shown to induce multinucleate cell (syncytium) formation or cell fusion in several normal primate cells. A series of experiments has been carried out to examine whether a glycosylated "fusion-inducing" product is responsible for this biological property of Mason-Pfizer monkey virus. Treatment of rhesus monkey fetal lung cells with different concentrations of tunicamycin, a potent inhibitor of glycosylation, during infection with Mason-Pfizer monkey virus had no effect on cell fusion even though up to 5 micrograms of the drug per ml was tested. Furthermore, no significant effect on the extent of syncytium formation in rhesus monkey fetal lung cells was observed when the time of addition or duration of treatment with this inhibitor was varied. Nevertheless, tunicamycin was very effective in blocking glycosylation in rhesus cells since virions produced in the presence of this drug completely lacked gp70 and gp20, the two structural glycoproteins of Mason-Pfizer monkey virus. These non-glycosylated virus particles produced in the presence of tunicamycin were noninfectious as determined by a protein A binding assay and were unable to induce syncytium formation when assayed on rhesus cells. These results indicate that glycosylation of the fusion-inducing product is not required for multinucleate cell formation induced by Mason Pfizer monkey virus.
Molecular Cloning of B- and N-Tropic Endogenous BALB/c Murine Leukemia Virus Circular DNA Intermediates: Isolation and Characterization of Infectious Recombinant Clones
Authors: Rassart Eric , DesGroseillers Luc , Jolicoeur Paul
Published: January 2020

The unintegrated closed circular DNA intermediates from B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were isolated from acutely infected mouse fibroblasts and cleaved at their unique
Hin
dIII sites. The linearized B- and N-tropic MuLV DNAs were then inserted in λ vector Charon 21A and cloned. Seventeen B-tropic and ten N-tropic recombinants were analyzed. The viral insert of these 27 MuLV recombinants could be grouped into 3 classes. The first class included permuted full-length molecules of 8.8 kilobase pairs (kbp) having two long terminal repeats (LTR) (0.55 kbp) mixed with 8.25-kbp molecules. The second class included 8.25-kbp molecules which harbored only one LTR copy. The third class of inserts was made of molecules with deletions of various lengths (0.1 to 3 kbp). These deletions were localized by restriction enzyme analysis. Significantly more deletions were observed in N-tropic than in B-tropic recombinants, although these two genomes isolated from acutely infected cells seemed to differ only by the absence of a 70-base-pair (bp) sequence in the N-tropic LTR. The biological activity of each of the recombinants of the first and second classes was assayed by transfection on NIH/3T3 cells. Two recombinants, one B-tropic (λ B-16) and one N-tropic (λN-20) were found to be infectious. Both were able to give rise to replication-competent MuLV which could form large XC plaques and had kept the tropism of its progenitor. Like its progenitor in vivo DNA, λN-20-cloned DNA lacked a 70-bp sequence in its unique LTR. The emerging N-tropic MuLV after transfection with λN-20 was used to acutely infect NIH/3T3 cells. The viral DNA intermediates isolated were now found to have two LTR copies. They had also acquired a 70-bp sequence in each LTR. The acquisition or loss of this 70-bp sequence did not seem to affect the replication, the XC plaque formation, or the tropism of the virus.
Epstein-Barr virus membrane antigens: characterization, distribution, and strain differences
Authors: Edson C M , Thorley-Lawson D A
Published: January 2020

The Epstein-Barr virus (EBV)-associated membrane antigen polypeptides (350,000, 220,000, 140,000, and 85,000 daltons) are recognized by a rabbit anti-EBV serum and are present on the plasma membranes of producer cell lines, as we demonstrated previously. In this report, we show that these polypeptides are present on intact virus particles. Subcellular fractionation revealed that these antigens are distributed throughout the cell, except for the 85,000-dalton protein, which was poorly represented in the nuclear fraction. In addition, an EBV-associated protein of 160,000 daltons, which comigrates with a major component of the viral capsid, was detected in the cytoplasmic and nuclear fractions. The immunoprecipitation patterns of 13 different EBV isolates were similar, with two exceptions. First, the 350,000- and 220,000-dalton polypeptides from marmoset cell lines had slightly larger molecular sizes than the corresponding polypeptides from human cell lines. Second, B95-8 virus and B95-8-derived human and marmoset cell lines contained little of the 220,000-dalton protein; however, 883L, the human parent line of B95-8, has a normal amount of the 220,000-dalton protein. Thus, the B95-8 strain of EBV appears to be a structurally defective variant. We have not observed any variation in protein patterns associated with different EBV disease states. The 350,000-, 220,000-, and 85,000-dalton polypeptides were shown to be glycoproteins by incorporation of [3H]mannose and [3H]glucosamine and to contain N-asparagine-linked glycosyl groups by their sensitivity to tunicamycin. To simplify future work, the following nomenclature for these EBV-associated polypeptides is suggested: 350,000 (gp350), 220,000 (gp220), 160,000 (p160), 140,000 (p140), and 85,000 (gp85).
Transformation-defective mutants of Snyder-Theilen feline sarcoma virus lack tyrosine-specific protein kinase activity
Authors: Barbacid M , Donner L , Ruscetti S K , Sherr C J
Published: January 2020

Four phenotypically normal mink cell clones, each containing a transformation-defective provirus of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV), synthesized an 85,000-dalton viral polyprotein (P85) indistinguishable in size and antigenic complexity from that encoded by wild-type transforming ST-FeSV. An additional transformation-defective, ST-FeSV-containing flat cell clone produced a polyprotein of 88,000 daltons (P88). The viral polyproteins immunoprecipitated from cytoplasmic extracts of these cells lacked the tyrosine-specific protein kinase activity associated with the wild-type ST-FeSV gene product. In addition, the products encoded by representative transformation-defective ST-FeSV genomes were poorly phosphorylated in vivo and lacked detectable phosphotyrosine residues. Whereas proteins of ST-FeSV transformants contained elevated levels of phosphotyrosine, those of mink cells containing transformation-defective ST-FeSV exhibited phosphotyrosine levels no higher than those found in uninfected cells. These findings provide genetic evidence that the tyrosine-specific protein kinase activity associated with ST-FeSV P85 is required for virus-induced transformation.
Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus type 1 Angelotti DNA
Authors: Kaerner H C , Ott-Hartmann A , Schatten R , Schröder C H , Gray C P
Published: January 2020

It has been shown earlier that the reiterated regions TRS and IRS bracketing the Us segment of herpes simplex virus type 1 Angelotti DNA are heterogeneous in size by stepwise insertion of one to six copies of a 550-base-pair nucleotide sequence. Considerably higher amplification of this sequence was observed in defective viral DNA: up to 14 copies were detected to be inserted in the repeat units of a major class of defective herpes simplex virus type 1 Angelotti DNA, dDNA1, which originated from noncontiguous sites located in UL and the inverted repeats of the S component of the parental genome. Physical maps were established for the cleavage sites of KpnI, PstI, XhoI, and BamHI restriction endonucleases on the repeats of dDNA1. The map position of the insertion sequence was determined. It was demonstrated that the amplified inserts were not distributed at random among or within the repeats. A given total population of dDNA1 molecules consisted of different homopolymers, each of which contained a constant number of inserts in all of its repeats. Assuming that a rolling-circle mechanism is involved in the generation of full-length defective herpes simplex virus type 1 Angelotti DNA from single repeat units, these data suggest that the 550-base-pair sequence is amplified in the repeats before the replication process.
Transcription of vesicular stomatitis virus activated by pardaxin, a fish toxin that permeabilizes the virion membrane
Authors: Pal R , Barenholz Y , Wagner R R
Published: January 2020

The toxic protein, Pardaxin, of the Red Sea flatfish Pardachirus marmoratus readily induced transcription of vesicular stomatitis virus by making the virion membrane permeable to nucleoside triphosphates in the absence of nonionic detergents. Virion transcription was activated over a wide range of Pardaxin concentrations, but at optimal concentrations, the rate of transcription exceeded that induced by Triton X-100. The inhibitory effect of M protein was manifested for both Pardaxin-induced and Triton-induced transcription at high concentrations of vesicular stomatitis virions; however, unlike the Triton-induced reaction, the inhibitory effect of M protein was not reversed by polyglutamic acid added to the Pardaxin-induced transcription reaction. We propose that activation of virion transcription by Pardaxin resembles more closely intracellular transcription initiated by virion penetration than does detergent-activated transcription of vesicular stomatitis virus.
Structure of replicating herpes simplex virus DNA
Authors: Jongeneel C V , Bachenheimer S L
Published: January 2020

We have investigated the molecular anatomy of the herpes simplex virus replicative intermediates by cleavage with the restriction endonuclease BglII. We find that in populations of multiply infected cells, pulse-labeled replicating herpes simplex virus DNA contains at least two and probably all four sequence isomers. Also, it contains no detectable termini. In pulse-chase experiments, we show that endless replicative intermediates are the precursors to virion DNA and that maturation is a relatively slow process. The results are discussed in terms of their significance to possible models of herpes simplex virus DNA replication.