bluuebunny/crossref_metadata_2025 huggingface dataset:
Response of Simian Virus 40-Transformed Cell Lines and Cell Hybrids to Superinfection with Simian Virus 40 and Its Deoxyribonucleic Acid
Authors: Swetly Peter , Brodano Giuseppe Barbanti , Knowles Barbara , Koprowski Hilary
Published: January 2020

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.
Entry and Release of Poliovirus as Observed by Electron Microscopy of Cultured Cells
Authors: Dunnebacke T. H. , Levinthal J. D. , Williams Robley C.
Published: January 2020

Cells of differing culture types were inoculated with poliovirus at 37 C, sampled at intervals during the replicative cycle, and examined in thin sections by electron microscopy. The earliest samples, taken at 2 and 5 min postinoculation, showed virus particles adjacent to the exterior of the plasma membrane and others that had apparently penetrated it directly; later samples showed fewer such particles or none. Particles lying in the peripheral cytoplasm frequently appeared swollen and distorted in shape. No sign of virus entry by a pinocytotic process was found at any time. At 3 hr, and subsequently during the replication cycle, particles of progeny virus appeared in the cytoplasm. They were found free in the cytoplasmic matrix, aligned along the elements of filamentous complexes, and enclosed within vesicles. Some of the vesicles were found to be open to the extracellular space, indicating a likely mechanism of virus release.
Electron Microscopy of Equine Infectious Anemia Virus
Authors: Tajima Masanori , Nakajima Hideo , Ito Yasuichiro
Published: January 2020

Equine infectious anemia (EIA) virus was observed in thin sections of infected cultured horse leukocytes by electron microscopy. The virus particles had a spherical shape and were between 80 and 120 nm in diameter. Most of them contained an electron-dense nucleoid 40 to 60 nm in diameter. They were observed to form by a process of budding from the plasma membrane and appeared to have thin surface projections. The particles described were not detected in uninfected cultured cells, and their appearance could be prevented by adding EIA immune serum to the inoculum. The implications of these findings in the classification of EIA virus are discussed.
Synthesis of Vaccinia Viral Proteins in Cytoplasmic Extracts
Authors: Moss Bernard , Katz Ehud
Published: January 2020

The synthesis of vaccinia viral proteins has been studied in a cell-free system prepared from vaccinia virus-infected HeLa cells. The radioactively labeled proteins were identified as viral proteins by immunodiffusion, disc gel electrophoresis, and disc gel-immunoelectrophoresis. The cytoplasmic extracts, obtained from infected cells at different times during viral replication, synthesized the corresponding “early” or “late” viral proteins.
Variants of Defective Simian Papovavirus 40 (PARA) Characterized by Cytoplasmic Localization of Simian Papovavirus 40 Tumor Antigen
Authors: Butel Janet S. , Guentzel Martha J. , Rapp Fred
Published: January 2020

Three isolates of PARA (particle aiding replication of adenovirus)-adenovirus 7 out of a total of 112 clonal progeny derived by two successive plaque purifications in green monkey kidney cells (GMK) were found to induce the synthesis of simian papovavirus40 (SV 40) tumor (T) antigen in the cytoplasm of infected cells. The variant viruses induced plaque formation in human embryonic kidney cells which followed one-hit kinetics. In GMK cells, plaque formation followed two-hit kinetics which converted to first-order kinetics in the presence of additional helper adenovirus type 7. Analysis of plaque progeny from human cells showed that the progeny could replicate only in human cells, whereas progeny from monkey cells could multiply in both human and monkey cells. Heterologous human adenoviruses were able to enhance plaque formation by the variant viruses in monkey kidney cells. Neutralization tests indicated that both components of the populations had a type 7 adenovirus capsid. All three viruses were capable of inducing SV40 transplantation immunity in weanling hamsters. These results indicate the three variants are PARA-adenovirus 7 populations. Response of the induction of the synthesis of the cytoplasmic antigen to metabolic inhibitors was the same as for the synthesis of the nuclear SV40 T antigen. Different pools of sera which reacted with the intranuclear SV40 T antigen also detected the cytoplasmic antigen induced by the variant viruses. An adsorption experiment with cells containing either nuclear or cytoplasmic T antigen to remove tumor antibody from hamster sera also indicated that it is probably SV40 T antigen which is responsible for the cytoplasmic reaction. The species of the host cell—human, simian, or rabbit—appeared to play no role in the altered localization of this antigen. It is postulated that these PARA variants are further defective in some virus-mediated transport mechanism which shifts the T antigen from the cytoplasm to the nucleus.
Nonsense Mutants in the rII A Cistron of Bacteriophage T4
Authors: Schwartz Robert D. , Bryson Vernon
Published: January 2020

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of
Escherichia coli
, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.
Deletion analysis of the cloned replication origin region from bacteriophage M13
Authors: Cleary J M , Ray D S
Published: January 2020

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.
Regulation of herpesvirus macromolecular synthesis: temporal order of transcription of alpha genes is not dependent on the stringency of inhibition of protein synthesis
Authors: Mackem S , Roizman B
Published: January 2020

Operationally, alpha genes of herpes simplex virus 1 were defined on the basis of the observations that they are the earliest genes expressed in the infected cell and that the transcription, processing, accumulation of the mRNA's in the infected cell cytoplasm can take place in the presence of inhibitors of protein synthesis, such as cycloheximide. In these studies, we translated in vitro the viral mRNA's extracted from cells infected maintained in the presence of cycloheximide, emetine, or anisomycin. Inasmuch as all the major alpha proteins (no. 0, 4, 22, and 27) were translated, we conclude that the transcription of all previously defined alpha genes is independent of the stringency of inhibition of protein synthesis and that pre-alpha genes cannot be detected in such experiments.
Chemical Methylation of RNA and DNA Viral Genomes as a Probe of In Situ Structure
Authors: Yamakawa Minoru , Shatkin Aaron J. , Furuichi Yasuhiro
Published: January 2020

We used [
methyl
-
3
H] dimethyl sulfate to probe the genome structures of several RNA and DNA viruses. We compared sites of modification in nucleic acids that were methylated chemically before and after extraction from purified virions. With both single-stranded and double-stranded substrates alkylation occurred mainly at the N7 position of guanine. However, adenine N1 atoms were differentially accessible in single-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirus mRNA and deproteinized genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae methylated in situ yielded RNAs with ratios of 0.04 to 0.08, indicating that the intravirion genomes were double stranded. We obtained ratios of 0.26 and 0.35 for the RNAs of dimethyl sulfate-treated brome mosaic and avian sarcoma virions, respectively, which was consistent with partial protection of adenine N1 sites by structural proteins or genome conformation or both. The ratios of 1-methyladenosine to 7-methylguanosine for vaccinia virus DNAs methylated in situ (0.10) and after phenol extraction (0.14) were less than the ratios for φX174 and M13 DNAs (0.39 to 0.64) but considerably greater than the ratio observed with adenovirus DNA (0.002 to 0.02). The presence of a single-stranded region(s) in the vaccinia virus genome was confirmed by S1 nuclease digestion of [
methyl
-
3
H] DNA; the released radiolabeled fraction had a ratio of 0.41, compared with 0.025 for the residual duplex DNA. In addition to the structure-dependent accessibility of adenine N1, methylation of adenine N3 was severalfold lower in the intravirion genomes of vaccinia virus, φX174, and adenovirus than in the corresponding extracted DNAs. Chemical methylation of virions and subviral particles should be useful for in situ analyses of specific regions of RNA and DNA genomes, such as the sites of protein binding during virus maturation.
Late nonstructural 100,000- and 33,000-dalton proteins of adenovirus type 2. I. Subcellular localization during the course of infection
Authors: Gambke C , Deppert W
Published: January 2020

We analyzed the subcellular locations of the late adenovirus type 2 nonstructural 100,000-dalton (100K) and 33K proteins in adenovirus type 2-infected HeLa cells both by biochemical cell fractionation and by immunofluorescence microscopy, using specific antisera against purified sodium dodecyl sulfate-denatured 100K and 33K polypeptides. Both methods showed that the 100K protein was present in the cytoplasm as well as in the nuclei of infected cells and that it accumulated in the nuclei during the course of infection. Phosphorylated 100K protein also was found both in the cytoplasm and in nuclei. However, the nuclear 100K protein pool was phosphorylated to a higher degree than the cytoplasmic pool. In all experiments the 33K protein, which also is a phosphoprotein, was present exclusively in the nuclei of infected cells. The 100K and 33K proteins were associated with different nuclear substructures; this was demonstrated serologically by an analysis of infected cells in which double color immunofluorescence microscopy was used. In these experiments antibodies against the 100K protein decorated different nuclear structures than antibodies against the 33K protein.
Metabolism of viral RNA in murine leukemia virus-infected cells; evidence for differential stability of viral message and virion precursor RNA
Authors: Messer L I , Levin J G , Chattopadhyay S K
Published: January 2020

Molecular hybridization techniques were used to examine the stability of viral message and virion precursor RNA in murine leukemia virus-infected cells treated with actinomycin D. Under the conditions used, viral RNA synthesis was inhibited, but viral protein synthesis continued, and the cells produced noninfectious particles (actinomycin D virions) lacking genomic RNA (J. G. Levin and M. J. Rosenak, Proc. Natl. Acad. Sci. U.S.A. 73:1154-1158, 1976). Analysis of total RNA in virions revealed that the amount of hybridizable viral RNA decreased steadily after the addition of actinomycin D and by 8 h was 10% of the control value. Studies on fractionated viral RNA showed that this low level of hybridization is due to residual 70S RNA in the virion population. The results indicated that viral RNA which is destined to be encapsidated into virions has a half-life of approximately 3 to 4 h. In contrast, other intracellular virus-specific RNA molecules appeared to be quite stable and persisted for a long period of time, with a half-life of at least 12 h. These observations support the idea that two independent functional pools of 35S viral RNA exist within the infected cell: one serving as message and the other as precursor to virion RNA. The existence of two viral RNA pools was further documented by the finding that 12 h after the addition of actinomycin D, when virion precursor RNA was depleted, 35S and 21S viral nRNA species could be identified in polyribosomal RNA as well as in total polyadenylated cell RNA. Surprisingly, 35S and mRNA declined more rapidly than did 21S mRNA, which appeared to be increased in amount.
Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system
Authors: Radany E H , Friedberg E C
Published: January 2020

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.
Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus
Authors: Pesonen Marja , Kuismanen Esa , Pettersson Ralf F.
Published: January 2020

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [
3
H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [
3
H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)
3
(GlcNAc)
2
peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two
N
-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in
N
-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [
3
H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [
35
S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.
Mapping of the 5' termini of two adeno-associated virus 2 RNAs in the left half of the genome
Authors: Lusby E W , Berns K I
Published: January 2020

The left 45% of the adeno-associated virus 2 genome was sequenced. The 5' termini of two adeno-associated virus-specific RNAs were mapped at the nucleotide level within this region of the genome (nucleotides 286 to 288 and 871 to 874). Both of these 5' termini map 31 +/- 2 nucleotides downstream from the start of "TATA' boxes.
Use of fluoresceinated Epstein-Barr virus to study Epstein-Barr virus-lymphoid cell interactions
Authors: Khelifa R , Menezes J
Published: January 2020

As a direct approach to visualize Epstein-Barr virus (EBV) binding to its cellular receptors and to learn more on the nature of this binding, virus preparations were conjugated to fluorescein isothiocyanate and used to detect EBV receptors on lymphoid cells. Different enzymatic and chemical treatments were also applied either to the virus or to target cells or to both, and the effect of these treatments on virus binding was then examined. The results obtained show that: (i) EBV can be fluoresceinated without affecting its infectivity or cell binding ability, and the fluoresceinated virus represents an important tool to investigate the biology and nature of EBV interactions with its cellular receptors; (ii) the two virus strains (P3HR-1 and B95-8) share common receptors on Raji cells; (iii) protease treatment of EBV or target cells abrogates virus binding; (iv) EBV receptors regenerate after removal of the protease, and this regeneration is inhibited by cycloheximide or sucrose; (v) EBV particles bear concanavalin A receptors, and this lectin hinders the interaction of the virus with its cellular receptors; (vi) neuraminidase treatment, various monosaccharides, ovalbumin, and glycopeptides derived from EBV or cell surface do not inhibit virus binding. Taken together, the above data also demonstrate that some cellular and viral surface (glyco-) proteins are required for EBV binding to its targets.
Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. I. Arrest at the level of DNA packaging
Authors: Kuhn A H , Moncany M L , Kellenberger E , Hausmann R
Published: January 2020

The multiplication of bacteriophage T7 is blocked in Escherichia coli M. The genetic determinant of this ability (groM) to inhibit T7 growth was transferred to an E. coli K-12 recipient by means of conjugation. We determined at which precise step T7 maturation is blocked. Phage-directed protein and DNA synthesis as well as degradation of host DNA were not qualitatively affected. Instead of infective phages, only preheads were produced. These, however, were maturable in vitro. The newly synthesized phage DNA accumulated in a concatemeric form and matured from its tetrameric or longer forms (very fast sedimenting DNA) only into its dimeric form (fast-sedimenting DNA) or longer forms. The following step, i.e., the maturation of the dimeric to unit-length DNA, was not observed. Since the concatemeric form of T7 DNA accumulated in spite of the presence of maturable preheads, it is likely that the maturation process was blocked at the level of DNA packaging. As intermediates in the packaging process, we found some prehead-DNA complexes. We interpreted these as true assembly intermediates (or breakdown products thereof), since the attached DNA was still in its concatemeric form. This shows that the very first DNA packaging step, the binding of the progeny DNA to the preheads, was obviously not blocked. Rather, a later step, such as the filling of the preheads with T7 DNA or the stabilization of completely packaged particles (i.e., the final cutting of the concatemers into unit-size length), was inhibited.
Genetic structure and transforming sequence of avian sarcoma virus UR2
Authors: Wang L H , Hanafusa H , Notter M F , Balduzzi P C
Published: January 2020

We have recently shown that a newly isolated avian sarcoma virus, UR2, is defective in replication and contains no sequences homologous to the src gene of Rous sarcoma virus. In this study, we analyzed the genetic structure and transforming sequence of UR2 by oligonucleotide fingerprinting. The sizes of the genomic RNAs of UR2 and its associated helper virus, UR2AV, were determined to be 24S and 35S, respectively, by sucrose gradient sedimentation. The molecular weight of the 24S UR2 genomic RNA was estimated to be 1.1 x 10(6), corresponding to 3,300 nucleotides, by gel electrophoresis under the native and denatured conditions. RNase T1 oligonucleotide mapping indicated that UR2 RNA contains seven unique oligonucleotides in the middle of the genome and shares eight 5'- and six 3'-terminal oligonucleotides with UR2AV RNA. From these data, we estimated that UR2 RNA contains a unique sequence of about 12 kilobases in the middle of the genome, and contains 1.4 and 0.7 kilobases of sequences shared with UR2AV RNA at the 5' and 3' ends, respectively. Partial sequence analysis of the UR2-specific oligonucleotides by RNase A digestion revealed that there are no homologous counterparts to these oligonucleotides in the RNAs of other avian sarcoma and acute leukemia viruses studied to date. UR2-transformed non-virus-producing cells contain a single 24S viral RNA which is most likely the message coding for the transforming protein of UR2. On the basis of the uniqueness of the transforming sequence, we concluded that UR2 is a new member of the defective avian sarcoma viruses.
Mutations in the herpes simplex virus DNA polymerase gene can confer resistance to 9-beta-D-arabinofuranosyladenine
Authors: Coen D M , Furman P A , Gelep P T , Schaffer P A
Published: January 2020

Mutants of herpes simplex virus type 1 resistant to the antiviral drug 9-beta-D-arabinofuranosyladenine (araA) have been isolated and characterized. AraA-resistant mutants can be isolated readily and appear at an appreciable frequency in low-passage stocks of wild-type virus. Of 13 newly isolated mutants, at least 11 were also resistant to phosphonoacetic acid (PAA). Of four previously described PAA-resistant mutants, two exhibited substantial araA resistance. The araA resistance phenotype of one of these mutants, PAAr5, has been mapped to the HpaI-B fragment of herpes simplex virus DNA by marker transfer, and araA resistance behaved in marker transfer experiments as if it were closely linked to PAA resistance, a recognized marker for the viral DNA polymerase locus. PAAr5 induced viral DNA polymerase activity which was much less susceptible to inhibition by the triphosphate derivative of araA than was wild-type DNA polymerase. These genetic and biochemical data indicate that the herpes simplex virus DNA polymerase gene is a locus which, when mutated, can confer resistance to araA and thus that the herpes simplex virus DNA polymerase is a target for this antiviral drug.
Candidate product of the FBJ murine osteosarcoma virus oncogene: characterization of a 55,000-dalton phosphoprotein
Authors: Curran T , Teich N M
Published: January 2020

Sera from rat bearing tumors induced by inoculation of FBJ murine osteogenic sarcoma virus (FBJ-MSV) nonproducer rat cells precipitate two proteins with molecular weights of 55,000 (p55) and 39,000 (p39) from FBJ-MSV-transformed cells. These proteins cannot be precipitated from uninfected cells or cells transformed by other strains of murine sarcoma virus, nor can they be precipitated by sera specific for the viral structural proteins. A methionine tryptic peptide mapping analysis showed that p55 and p39 have little or no homology and that they are not related to the helper virus gag and env gene products. p55 could also be detected among the in vitro translation products of 70S RNA from FBJ murine leukemia virus plus FBJ-MSV virions but not among those from FBJ murine leukemia virus alone. This suggests that p55 is encoded by the FBJ-MSV genome, whereas p39, which was not detected among the in vitro translation products, may not be virus encoded. Another difference between p55 and p39 is that p55 is phosphorylated, with most of the phosphate on a serine residue(s), whereas p39 is phosphorylated to a much lesser extent, if at all. No protein kinase activity was associated with p55 and p39 immune complexes under standard conditions. Our data suggest that p55 is a strong candidate for the FBJ-MSV oncogene product.
Posttranslational modifications of Sindbis virus glycoproteins: electrophoretic analysis of pulse-chase-labeled infected cells
Authors: Bonatti S , Cancedda F D
Published: January 2020

Cytoplasmic extracts prepared from Sindbis virus-infected chicken embryo fibroblasts pulse-chase-labeled with [35S]methionine 6 h postinfection were analyzed on a highly resolving sodium dodecyl sulfate-gel either directly or after various treatments. The results we obtained suggest that (i) the proteolytic cleavage which converts PE2 to E2 glycoprotein takes place intracellularly, before or at least during the formation of complex-type oligosaccharide side chains; and (ii) E1 glycoprotein undergoes a complex maturation pattern. Newly synthesized E1 has a molecular weight of 53,000: shortly thereafter, this 53,000 (53K) form was converted to a 50K form. Subsequently, the 50K form decreased its apparent molecular weight progressively and eventually comigrated with E1 glycoprotein present in the extracellular virus, which displays a molecular weight of 51,000 to 52,000. The conversion of the 53K to the 50K form was not the result of a proteolytic processing and did not depend on glycosylation or disulfide bridge formation and exchange. The possible mechanisms of this conversion are discussed. The second conversion step (from the 50K to the 51-52K form) was due to the formation of complex-type oligosaccharide and was reversed by incubating the cellular extracts with neuraminidase before electrophoretic analysis.
Transfer of mouse anti-xenotropic virus neutralizing factor to human lipoproteins
Authors: Levy J A , Dimpfl J , Hardman D , Kane J P
Published: January 2020

We examined the migratory properties of the mouse serum protein responsible for specific neutralization of the endogenous mouse xenotropic type C virus and demonstrated that this neutralizing factor can be transferred from mouse lipoproteins to human and to nonreactive mouse lipoproteins. After acceptance of this activity by these lipoproteins, it could be back-transferred to human and to mouse lipoproteins. This result was compatible with equilibrium binding of the responsible proteins by several classes of lipoproteins. The best expression of the transferred activity occurred with human chylomicrons. Small quantities of neutralizing factor which exist in free dispersion in serum were also detected by mixing the p greater than 1.21 g/ml density fraction of mouse serum with human chylomicrons; antiviral activity was subsequently found associated with the human lipoproteins. This result reflects the potentiation of antiviral activity of the protein in a lipoprotein environment. These observations offer a useful approach for the eventual purification and characterization of the neutralizing factor.
Cleavage maps for human cytomegalovirus DNA strain AD169 for restriction endonucleases EcoRI, BglII, and HindIII
Authors: Spector D H , Hock L , Tamashiro J C
Published: January 2020

We have used cloned EcoRI fragments of the human CMV (HCMV) genome, strain AD169, to prepare restriction endonuclease maps of the DNA. Individual 32P-labeled cloned fragments were hybridized to Southern blots of HCMV DNA cleaved to completion with the restriction endonucleases BglII and HindIII and cleaved partially with EcoRI. By determining which EcoRI fragments hybridized to the same band on a Southern blot, we were able to establish linkage groups. This information coupled with the data derived from digestion of the cloned fragments with the enzymes BglII and HindIII (Tamashiro et al., J. Virol. 42:547-557, 1982) provided the basis for the construction of detailed maps for the enzymes EcoRI, BglII, and HindIII. We also identified the EcoRI fragments derived from the termini of this genome and mapped them with respect to the BglII and HindIII terminal fragments. From our mapping data, we conclude that the genome of HCMV is approximately 240 kilobases in length and is divided into long (198 kilobases) and short (42 kilobases) regions. Both regions consist of a unique sequence bounded by inverted repeats (11 to 12 kilobases for the long region and 2 to 3 kilobases for the short region). Furthermore, the long and short regions can invert relative to each other.
Polyoma viral middle T-antigen is required for transformation
Authors: Mes A M , Hassell J A
Published: January 2020

To determine whether small or middle T-antigen (or both) of polyoma virus is required for transformation, we constructed mutants of recombinant plasmids which bear the viral oncogene and measured the capacity of these mutants to transform rat cells in culture. Insertion and deletion mutations in sequences encoding small and middle T-antigens (79.7, 81.3, and 82.9 map units) rendered the DNA incapable of causing transformation by the focus assay. Similar mutations in sequences that encoded middle but not small T-antigen (89.7, 92.1, and 96.5 map units) generally abolished the transforming activity of the DNA. However, two mutants (pPdl1-4 and PPd12-7) that carried deletions at 92.1 map units retained the capacity to transform cells; pPdl1-4 did so at frequencies equal to those of the parental plasmid, whereas pPdl2-7 transformed at 10% the frequency of its antecedent. From these studies we conclude that small T-antigen alone is insufficient to cause transformation and that middle T-antigen is required for transformation, either in combination with small T-antigen or by itself.
Molecular biology of rotaviruses. III. Isolation and characterization of temperature-sensitive mutants of bovine rotavirus
Authors: Faulkner-Valle G P , Clayton A V , McCrae M A
Published: January 2020

Twenty-six temperature-sensitive (ts) mutants of United Kingdom tissue culture-adapted bovine rotavirus were isolated and characterized. Fourteen of these mutants were determined to be ts both by efficiency of plating and by virus yield at the nonpermissive temperature of 39.5 degrees C as compared with that at the permissive temperature of 32 degrees C. The remaining mutants were only ts by the criterion of efficiency of plating. High-frequency recombination (gene reassortment) was observed when some pairs of mutants were crossed, and this allowed the classification of the mutants into five separate recombination groups. Groups III and V have prototype ts mutants (ts34 and ts115, respectively) that do not synthesize RNA or polypeptides at 39.5 degrees C. The other groups, I, II, and IV, have prototype mutants (ts17, ts7, and ts6, respectively) that synthesize both RNA and polypeptides at 39.5 degrees C, although ts17 does so only at a reduced level.
Specificity and localization of the hepatitis B virus-associated protein kinase
Authors: Gerlich W H , Goldmann U , Müller R , Stibbe W , Wolff W
Published: January 2020

The nature of the protein kinase (PK) which phosphorylates the core protein of hepatitis B virus in vitro was studied. The PK copurified with the core particles during rate zonal centrifugation and gel chromatography. It showed the same size heterogeneity as the core particles, which consisted of a main fraction of 28-nm particles and a subfraction of 22- to 26-nm particles. DNA-containing heavy core particles with a density of 1.33 to 1.35 g/ml and less endogenous PK than did the light cores. The phosphorylation reaction had a rapid initial phase (several minutes) and a slow but long-lasting second phase (many hours). The PK had a high affinity for ATP (KM = 0.5 mumol/liter). Only few of the several hundred P21.9 subunits in one core particle were phosphorylated in vitro. The only amino acid which was phosphorylated in vitro was serine. The resistance of the introduced phospho group against alkaline phosphatase showed that the PK acceptor, and probably the enzyme itself, was located inside the core particle.
The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species
Authors: Huang Y T , Wertz G W
Published: January 2020

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).
Isolation and characterization of defective simian virus 40 genomes which complement for infectivity
Authors: O'Neill F J , Maryon E B , Carroll D
Published: January 2020

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.
Epstein-Barr virus RNA. VIII. Viral RNA in permissively infected B95-8 cells
Authors: Hummel M , Kieff E
Published: January 2020

More than 50 RNAs expressed by Epstein-Barr virus late in productive infection have been identified. B95-8-infected cells were induced to a relatively high level of permissive infection with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate. Polyadenylated RNAs were extracted from the cell cytoplasm, separated by size on formaldehyde gels, transferred to nitrocellulose, and hybridized to labeled recombinant Epstein-Barr virus DNA fragments. Comparison of RNAs from induced cultures with RNAs from induced cultures also treated with phosphonoacetic acid to inhibit viral DNA synthesis identifies two RNA classes: a persistent early class of RNAs whose abundance is relatively resistant to viral DNA synthesis inhibition and a late class of RNAs whose abundance is relatively sensitive to viral DNA synthesis inhibition. The persistent early and late RNAs are not clustered but are intermixed and scattered through most of segments UL and US. The cytoplasmic polyadenylated RNAs expressed during latent infection were not detected in productively infected cells, indicating that different classes of viral RNA are associated with latent and productive infection. Non-polyadenylated small RNAs originally identified in cells latently infected with Epstein-Barr virus are expressed in greater abundance in productively infected cells and are part of the early RNA class.
Patterns of genomic distribution and sequence heterogeneity of a murine "retrovirus-like" multigene family
Authors: Keshet E , Itin A
Published: January 2020

The mouse genome contains over 100 copies of a dispersed gene family known as "virus-like" genes encoding 30S RNA (VL30). Although they do not share nucleotide sequence homology with known retroviruses, these genetic elements are distinguished by several "retrovirus-like" features, notably, the capacity of the 30S RNA transcripts of these genes to be encapsidated by c-type virions and the transmissibility of VL30 information to other cells via pseudovirion infection. Using VL30 DNA units, cloned from the BALB/c mouse embryonic gene library, we have recently shown that VL30 DNA units share basic structural features with retrovirus proviruses. To shed light on the relatedness of VL30 information to endogenous proviruses and possibly other genetic elements, we extended our previous studies concerning genomic distribution patterns of VL30 elements and patterns of sequence heterogeneity among VL30 units. The following observations were made: (i) VL30 units were distributed among different mouse chromosomes; (ii) distribution patterns of VL30 units markedly differed among mouse strains; (iii) there was constancy of VL30 restriction patterns in different tissues; (iv) a high degree of sequence divergence existed among different VL30 units cloned from the same embryo; and (v) VL30 units were heterogeneous with respect to the state of DNA methylation. The results are discussed in terms of the possible modes of evolution of this multigene family.
Internal organization of endogenous proviral DNAs of xenotropic murine leukemia viruses
Authors: Hoggan M D , Buckler C E , Sears J F , Chan H W , Rowe W P , Martin M A
Published: January 2020

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.
mRNA- and DNA-directed synthesis of herpes simplex virus-coded exonuclease in Xenopus laevis oocytes
Authors: Preston C M , Cordingley M G
Published: January 2020

Microinjection of herpes simplex virus (HSV)-infected cell mRNA into Xenopus laevis oocytes resulted in the production of a new exonuclease activity. This enzyme strongly resembled the HSV alkaline exonuclease in many biochemical properties, and hybrid-arrested translation studies showed that it was virus coded, mapping at 0.080 to 0.185 genome map units. Exonuclease mRNA had a size and genome location equivalent to the mRNA encoding V185 in reticulocyte lysates, suggesting that V185 is the exonuclease. The enzyme synthesized in oocytes was found to act as an exonuclease in vivo. Two plasmids containing HSV DNA fragments directed the synthesis of exonuclease when microinjected into oocyte nuclei, and this finding enabled the coding and control sequences for this gene to be localized to 0.155 to 0.185 genome map units.
Frog Virus 3 DNA Replication Occurs in Two Stages
Authors: Goorha R.
Published: January 2020

Viral DNA synthesis in frog virus 3 (FV3)-infected cells occurs both in the nucleus and in the cytoplasm (Goorha et al., Virology
84
:32-51, 1978). Relationships between viral DNA molecules synthesized in these two compartments and their role in the virus replication were examined. The data presented here suggest that (i) FV3 DNA replicated in two stages and (ii) nucleus and cytoplasm were the sites of stages 1 and 2 of DNA replication, respectively. Stages 1 and 2 were further distinguished by their temporal appearance during infection and by the sizes of the replicating DNA as determined by sedimentation in neutral sucrose gradients. In stage 1, replicating molecules, between the size of unit and twice the unit length, were produced early in infection (2 h postinfection). In contrast, stage 2 of DNA replication occurred only after 3 h postinfection, and replicating molecules were large concatemers. Results of pulse-chase experiments showed that the concatemeric DNA served as the precursor for the production of mature FV3 DNA. Denaturation of concatemeric DNA with alkali or digestion with S1 nuclease reduced it to less than genome size molecules, indicating the presence of extensive single-stranded regions. Analysis of replicating DNA by equilibrium centrifugation in CsCl gradients after a pulse-chase suggested that these single-stranded regions were subsequently repaired. Based on these and previous data, a scheme of FV3 replication is presented. According to this scheme, FV3 utilizes the nucleus for early transcription and stage 1 of DNA replication. The viral DNA is then transported to the cytoplasm, where it participates in stage 2 DNA replication to form a concatemeric replication complex. The processing of concatemers to produce mature viral DNA and virus assembly also occurs in the cytoplasm. This mode of replication is strikingly different from any other known DNA virus.
Identification of a nonvirion protein of Aleutian disease virus: mink with Aleutian disease have antibody to both virion and nonvirion proteins
Authors: Bloom M E , Race R E , Wolfinbarger J B
Published: January 2020

We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK cells labeled with [35S]methionine at 60 h postinfection were studied by immunoprecipitation with sera from infected mink, the major Aleutian disease virus virion polypeptides (p85 and p75) were consistently identified, as was a 71,000-dalton nonvirion protein (p71). The peptide maps of p85 and p75 were similar, but the map of p71 was different. p85, p75, and p71 were all precipitated by sera from Aleutian disease virus-infected mink, including those with signs of progressive disease, but heterologous sera raised against purified Aleutian disease virus did not precipitate the nonvirion p71. These results indicated that the nonvirion p71 was unrelated to p85 and p75 and further suggested that mink infected with Aleutian disease virus develop antibody to nonvirion, as well as structural, viral proteins.
In situ hybridization: general infectivity assay for retroviruses
Authors: Rein A , Rice N , Simek S , Cohen M , Mural R J
Published: January 2020

We have devised a general infectivity assay for retroviruses. A virus-specific [32P]DNA probe is hybridized in situ to a monolayer culture, and foci of infected cells in the monolayer are detected by exposure of the hybridized culture to X-ray films. The method is quantitative, in that it gives the same titer for Moloney murine leukemia virus as does the standard UV-XC test. The specificity of the assay is indicated by the fact that murine leukemia virus and baboon endogenous virus do not cross hybridize under the conditions used. The assay is completed within 1 to 3 weeks and should be broadly applicable for retroviruses which replicate without altering cellular morphology: its use is demonstrated with mouse mammary tumor virus and the helper virus of the reticuloendotheliosis complex.
Expression of cloned herpesvirus genes. I. Detection of nuclear antigens from herpes simplex virus type 2 inverted repeat regions in transfected mouse cells
Authors: Middleton M H , Reyes G R , Ciufo D M , Buchan A , Macnab J C , Hayward G S
Published: January 2020

Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence.