bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanisms Involved in the Differential Expression of
gag
Gene Products by Clonal Isolates of a Primate Sarcoma Virus
Authors: Robbins Keith C. , Okabe Hiromi , Tronick Steven R. , Gilden Raymond V. , Aaronson Stuart A.
Published: January 2020

Clonal isolates of an early passage stock of woolly monkey sarcoma virus (WSV) have been shown to code for different numbers of woolly monkey helper leukemia virus
gag
gene products. In the present report, the molecular mechanisms responsible for their differential expression of
gag
gene products have been analyzed. Three WSV RNA genomes were shown to possess sedimentation coefficients consistent with the differences demonstrated in their allotments of helper viral sequences. The WSV variant (WSV clone 9) that expressed no detectable proteins was shown to contain the largest amount of helper viral information. Moreover, there was no additive hybridization of the WLV complementary DNA probe by RNA of this WSV clone and that of a WSV clone coding for several
gag
gene products. These results suggest that the lack of expression of
gag
gene products by WSV clone 9 is not due to a major deletion of helper viral
gag
gene sequences. Similar levels of WLV-specific RNA were demonstrated in cells nonproductively transformed by each WSV clone, arguing that the ability to express
gag
gene proteins was not related to the magnitude of viral RNA transcription. Taken together, the results are most consistent with a mechanism by which small deletions or point mutations in the genomes of some WSV variants result in premature termination of translation or synthesis of immunologically nonreactive
gag
gene proteins. The present findings have implications concerning the effects of evolutionary selective pressures on helper viral genetic information in mammalian transforming viruses.
Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus
Authors: van de Ven W J , van Zaane D , Onnekink C , Bloemers H P
Published: January 2020

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.
Segments of Influenza Virus Complementary RNA Synthesized In Vitro
Authors: Plotch Stephen J. , Krug Robert M.
Published: January 2020

In the presence of Mg
2+
and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol.,
21
:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3′ end of vRNA.
Identification of hemagglutinin and neuraminidase genes of influenza B virus
Authors: Ueda M , Tobita K , Sugiura A , Enomoto C
Published: January 2020

The genome of influenza B viruses was shown by electrophoresis to consist of eight RNA segments. The fifth largest segment coded for hemagglutinin and the sixth coded for neuraminidase.
Endogenous mink (Mustela vison) type C virus isolated from sarcoma virus-transformed mink cells
Authors: Sherr C J , Benveniste R E , Todaro G J
Published: January 2020

A previously described type virus stock (designated PP-1R), isolated by cocultivating baboon cells with mink cells transformed by Kirsten sarcoma virus (64J1), has been further cloned and characterized. End point-diluted stocks of PP-1R have been obtained that are free of focus-forming activity and lack both Kirsten sarcoma and primate type C viral sequences. Nucleic acid hybridization experiments show that the cloned virus (MiLV) is an endogenous, genetically transmitted virus of the mink (Mustela vison). MiLV replicates in canine, feline, and 64J1 mink cells but not in an untransformed mink cell line. Multiple viral gene copies can be detected in the DNA of normal mink cells in culture and in normal mink tissues; related endogenous viral genes are also detected in several related Mustela species. The virus codes for a p30 protein very closely related antigenically to that of feline leukemia virus but contains p15 and p12 proteins that are antigenically distinct. The mink cell line, Mv1Lu, and its Kirsten sarcoma-transformed derivatives, 64J1, express relatively low levels of type C viral RNA related to MiLV and normally do not produce detectable levels of MiLV p30 protein or complete, infectious viral particles. Infection of sarcoma virus-transformed mink cells with baboon type C virus, however, can augment the level of expression of endogenous mink viral RNA and can result in the synthesis and packaging of mink viral RNA and p30 antigen in extracellular virions. Since the Mv1Lu cell line and its tranformed derivatives have become widely used in studies of retroviruses, the possibility of activating endogenous mink viral genes should be considered by investigators working with these cells.
Evidence from UV transcription mapping that late adenovirus type 2 mRNA is derived from a large precursor molecule
Authors: Goldberg S , Nevins J , Darnell J E
Published: January 2020

The UV inactivation of labeling of various late adenovirus type 2 mRNA's is as great or greater than the inactivation of labeling of the same sequences that are present in a large primary RNA transcript in the cell nucleus. Thus, the large nuclear adenovirus type 2-specific RNA appears to be the obligatory precursor of cytoplasmic mRNA. The majority of the mRNA's derived from the large precursor apparently require the synthesis of the whole nuclear molecule. A UV-sensitive target necessary for proper processing of all the mRNA's may exist near the end of the transcription unit.
Characterization of DNA-protein complexes from simian adenovirus SA7
Authors: Estes M K
Published: January 2020

DNA-protein complexes prepared from purified simian adenovirus SA7 virions and from lytically infected monkey kidney cells exhibited similar properties when compared with respect to size by sucrose gradient centrifugation, to configuration by electron microscopy, and to susceptibility to a variety of treatments by electron microscopy and electrophoresis in agarose gels.
Infectious primate type C virus proviral DNA in productively infected cells
Authors: Markham P D , Gallo R C
Published: January 2020

Cell lines competent to infection by DNA from cultures chronically infected by type C viruses of the simian sarcoma virus and baboon endogenous virus groups were identified. Significant differences were observed in the relative susceptibility of some cell lines to infection by a given proviral DNA. Practical applications of transfection techniques for the separation of viruses from dually infected cultures and to free virus stocks from mycoplasmal contamination are described.
Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells
Authors: Marcus S L
Published: January 2020

The microsomal supernatant fraction obtained from a murine cell line chronically infected with and producing Rauscher leukemia virus (JLSV-10) was found to contain two forms of RNA-directed DNA polymerase (reverse transcriptase). The two enzyme forms, neither of which is detectable in uninfected cells (JLSV-9), were initially partially purified by poly(C)-agarose chromatography, and their separation was achieved by phosphocellulose chromatography. The enzyme form eluting first from phosphocellulose (0.3 M KCl), designated PC I, was found to be identical in all parameters tested to that form isolated directly from purified virions. The second enzyme peak, designated PC II, eluted from phosphocellulose at 0.5 M KCl and was not detectable in purified virions. The PC II enzyme has a molecular weight, determined by velocity sedimentation, of approximately 109,000, as compared with 70,000 for the PC I enzyme, and could not be further dissociated by exposure to high salt or nonionic detergent. Mixing purified virion or PC I DNA polymerase with uninfected cells followed by fractionation did not produce the PC II form, suggesting that it is neither an artifact of purification nor the result of fortuitous complexing of reverse transcriptase with normal cellular component(s). Both PC I and PC II enzyme forms appeared antigenically similar to virion DNA polymerase, demonstrated identical divalent cation requirements for various template-primers, and were capable of copying heteropolymeric regions of rabbit globin mRNA. However, kinetic studies of heat inactivation revealed that the PC II enzyme was far more heat labile than the PC I form, which appeared identical to the virion enzyme in this respect. Furthermore, whereas the PC I and virion-derived reverse transcriptase copied poly(C).(dG)12-18 most efficiently at a template-to-primer molar nucleotide ratio of 25:1, the PC II enzyme preferred a ratio of 5:1 for optimal rates of poly(dG) synthesis. Therefore, by these criteria, there appear to exist two intracellular forms of reverse transcriptase in the JLSV-10 Rauscher leukemia virus-producing murine cell line.
In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species
Authors: Franze-Fernandez M T , Banerjee A K
Published: January 2020

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.
In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. II. Characterization of the leader RNA
Authors: Colonno R J , Banerjee A K
Published: January 2020

The New Jersey serotype of vesicular stomatitis virus (VSV) was able to synthesize a small RNA (leader RNA) approximately 70 bases in length similar to the leader RNA synthesized in vitro by the genetically distinct Indiana serotype of VSV. Also, the New Jersey leader RNA contained the same 5'-terminal sequence, ppA-C-G, as the Indiana leader RNA and had a very similar base composition, with 42% AMP, 16% CMP, 18.6% GMP, and 23.4% UMP. The 3'-terminal sequence of the VSV New Jersey genome RNA was detemined and found to contain the sequence- Py-G-UOH, again the same as that of the Indiana serotype of VSV. Evidence that the New Jersey leader RNA is transcribed from the 3' end of the genome RNA was obtained from the fact that it can protect the 3'-terminal base of [3H]borohydride-labeled New Jersey genome RNA from RNase digestion. Although the New Jersey and Indiana leader RNAs were similar in many respects, they were unable to form RNase-resistant hybrids when annealed to heterologous genome RNA.
Characterization and classification of virus particles associated with hepatitis A. II. Type and configuration of nucleic acid
Authors: Siegl G , Frösner G G
Published: January 2020

Virus particles banding at 1.34 g/ml in CsCl and sedimenting at 160S in sucrose gradients were isolated from fecal specimens of patients suffering from hepatitis. In the presence of 4 M urea and about 90% formamide, these particles released linear nucleic acid molecules of the kinked appearance characteristic of single-stranded RNA or single-stranded DNA. They could be distinguished from the nucleic acid of phage lambda added to the preparation as a marker for double-stranded configuration. Experiments in which the virus particles under investigation were incubated at pH 12.9 at 50 degrees C for 30 min revealed that their nucleic acid molecules were hydrolyzed as readily as the RNA genome of poliovirus type 2 analyzed in parallel. Both the single-stranded DNA of phage phiX174 and that of parvovirus LuIII, however, proved unaffected by this treatment, and the double-stranded DNA of phage lambda was denatured to single-stranded molecules. It was concluded, therefore, that the virus of human hepatitis A contains a linear genome of single-stranded RNA and has to be classified with the picornaviruses.
Production of unintegrated mouse mammary tumor virus DNA in infected rat hepatoma cells is a secondary action of dexamethasone
Authors: Ringold G M , Shank P R , Yamamoto K R
Published: January 2020

Dexamethasone, a synthetic glucocorticoid, selectively increased the rate of synthesis of mouse mammary tumor virus (MTV) RNA in clonal isolates of chronically infected rat hepatoma tissue culture cells. This hormonal effect occurred extremely rapidly and appeared to be mediated directly by the glucocorticoid-specific receptor protein. In addition to the viral RNA, unintegrated MTV DNA was also detected in these cells. Several lines of evidence are consistent with the idea that the unintegrated viral DNA is synthesized by reverse transcription of MTV RNA. (i) Unintegrated viral DNA accumulated only in the presence of dexamethasone and was produced with a time course that closely paralleled the increased accumulation of viral RNA. (ii) Density labeling of the viral DNA revealed that both strands were newly synthesized, implying a non-semiconservative mode of replication. (iii) Inhibitors of viral RNA synthesis prevented the appearance of unintegrated viral DNA. These data suggest that the production of unintegrated MTV DNA after dexamethasone treatment occurs as a secondary consequence of the hormonal induction of synthesis of viral RNA. In contrast to infected rat hepatoma cells, no unintegrated MTV DNA was detected in mouse mammary tumor cells or mouse lymphoma cells, despite the presence of high levels of viral RNA.
Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants
Authors: Devare S G , Hanson R E , Stephenson J R
Published: January 2020

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.
Biosynthesis, Immunological Specificity, and Intracellular Distribution of the Simian Virus 40-Specific Protein Induced by the Nondefective Hybrid Ad2
+
ND
1
Authors: Jay Gilbert , Jay Francis T. , Friedman Robert M. , Levine Arthur S.
Published: January 2020

Ad2
+
ND
1
, a nondefective hybrid virus containing a segment of the early region of simian virus 40 (SV40) DNA covalently inserted into the human adenovirus 2 genome, enhances the growth of human adenoviruses in simian cells and induces the SV40 U antigen. This hybrid previously has been shown to code for a 28,000 (28K) molecular weight protein not present in wild-type adenovirus 2-infected cells. By radioimmunoprecipitation using sera from hamsters bearing SV40-specific tumors, we have established that the Ad2
+
ND
1
-induced 28K protein is SV40-specific. This Ad2
+
ND
1
-induced protein is synthesized as a 30K molecular weight precursor, which is detectable only when infected cells are pulse-labeled in the presence of the protease inhibitor tosylamino phenylethyl chloromethyl ketone. Upon fractionation of labeled cell extracts, about 80% of the 28K protein is found in the plasma membrane fraction, whereas the remaining 20% is associated with the outer nuclear membrane. This protein is not detectable either in the nucleus or in the cytoplasm. Blockage of proteolytic cleavage by tosylamino phenylethyl chloromethyl ketone did not alter the topographic distribution of this SV40-specific protein, although the amount of the precursor protein in the outer nuclear membrane increased fourfold while that in the plasma membrane was proportionately decreased. This result suggests that the 28K protein is transferred from the outer nuclear membrane to the plasma membrane after posttranslational cleavage of the 30K precursor polypeptide. These data offer further support to the proposal that the 28K protein contains the determinants for SV40 U antigen and is responsible for SV40 enhancement of adenovirus growth in simian cells.
Intramembrane changes occurring during maturation of herpes simplex virus type 1: freeze-fracture study
Authors: Rodriguez M , Dubois-Dalcq M
Published: January 2020

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.
Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella
Authors: Tweeten K A , Bulla L A , Consigli R A
Published: January 2020

An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.
Genetic analysis of a cytomegalovirus-like agent isolated from human brain
Authors: Huang E S , Kilpatrick B , Lakeman A , Alford C A
Published: January 2020

An unusual cytomegalovirus (CMV, strain Colburn) isolated from brain biopsy of a boy with clinical encephalopathy was studied for genetic relatedness to human and simian CMV. Cross-examination of the purified viral DNA by DNA-DNA reassociation kinetics analyses showed more than 90% homology between Colburn virus and simian CMV (strain GR2757) and a lack of detectable homology between Colburn virus and human CMV (strains AD-169 and TW-87). Restriction endonuclease analysis of Colburn DNA showed some similarity of the DNA fragment pattern with that of simian CMV DNA, although the DNA fragment patterns were not identical, and showed no similarity to that of human CMV DNA. The molecular size and density of viral DNA were close to those of simian CMV DNA. The antigenic study, as performed by complement fixation and neutralization tests, showed strong cross-reactivity of Colburn virus to simian GR2757 virus. One-way cross-reaction of Colburn virus to several human CMV isolates (AD-169, Davis, and Town) was detected by complement fixation; this one-way cross-reaction was not obvious in a plaque neutralization test. It was concluded that Colburn is a simian CMV-related virus.
Adenovirus type 2 early proteins synthesized in vitro and in vivo: identification in infected cells of the 38,000- to 50,000- molecular-weight protein encoded by the left end of the adenovirus type 2 genome
Authors: Harter M L , Lewis J B
Published: January 2020

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein synthesized early after injection of human cells with adenovirus type 2 (Ad2) showed that polypeptides of 11,000 (11K), 12K, 14K, 15K, 19K, 21K, 24K, and 72k molecular weight were present in infected but not in mock-infected cells. These polypeptides corresponded in electrophoretic mobility to the following polypeptides synthesized in vitro by using mRNA complementary to specific regions of the Ad2 genome: 11K, 19K, 21K (91.5 to 96.8 map units), 14K (78.2 to 83.4 map units), 72K (62.4 to 67.9 map units), and 15K (4.9 to 11.0 map units). Polypeptides of 25K, 17K, 15.5K, and 13K were also synthesized in vitro, but have not yet been detected in infected cells. In addition, six adeno-specific polypeptides of 38 to 50K molecular weight could be discerned in infected cells if two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis was used to compare extracts from infected and mock-infected cells. Partial protease digestion showed these 38 to 50K polypeptides to be related in sequence to each other and to the 40 to 50K polypeptide made in vitro (1.3 to 4.0 map units).
Effect of RNase III on the size of bacteriophage T7 lysozyme mRNA
Authors: Hagen F S , Young E T
Published: January 2020

The size of lysozyme mRNA from T7-infected E. coli RNase III+ and RNase III- strains was analyzed by sucrose gradient sedimentation, dimethylsulfoxide (Me2SO) sucorse gradient sedimentation, and preparative gel electrophoresis. Each technique revealed a similar size distribution of multiple lysozyme mRNA's. Analysis by preparative gel electrophoresis of RNA extracted after infection of Escherichia coli Bst (RNase III+) separated lysozyme mRNA into six peaks of activity ranging in size from 0.2 x 10(6) to 1.9 x 10(6) daltons. Four well-resolved major peaks of activity were detected, having apparent molecular weights of approximately 0.61 x 10(6), 0.76 x 10(6), 0.92 x 10(6), and 1.3 x 10(6). A broad band of activity, with a molecular weight range from 0.2 x 10(6) to 0.37 x 10(6), was also present, and a sixth peak of activity was sometimes observed that migrates with a mobility corresponding to a molecular weight of 1.9 x 10(6). Judging from their molecular weight as estimated by electrophoresis, most, if not all, of the lysozyme mRNA's were polycistronic. The RNA extracted after infection of an RNase III- host contained a more heterogeneous collection of lysozyme mRNA's. In addition to lysozyme mRNA activity on RNAs with molecular weights between 0.2 x 10(6) and 1.9 x 10(6), RNA species with molecular weights estimated at 4 x 10(6) to 5 x 10(6) were also detected. The data indicate that RNase III processes at least some of the primary lysozyme transcripts. The multiple lysozyme mRNA's represent discrete RNA species rather than aggregates because analysis of the size of lysozyme mRNA under completely denaturing conditions, in Me2SO, produced a similar size distribution of lysozyme mRNAs. Also, treatment of RNA with 90% Me2SO, which separates the strands of a completely double-stranded RNA, did not significantly alter the electrophoretic mobility of the lysozyme mRNA.
Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells
Authors: Nowinski R C , Emery S , Ledbetter J
Published: January 2020

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.
Type-Common CP-1 Antigen of Herpes Simplex Virus Is Associated with a 59,000-Molecular-Weight Envelope Glycoprotein
Authors: Cohen Gary H. , Katze Michael , Hydrean-Stern Cassandra , Eisenberg Roselyn J.
Published: January 2020

The CP-1 antigen of herpes simplex virus type 1 (HSV-1) is a glycoprotein found in the soluble portion of infected cells, in detergent extracts of infected cell membranes, and in the envelope of purified virus. Antisera were prepared against a further purified form of CP-1 prepared from HSV soluble antigen mix; a glycoprotein, gp52, isolated from detergent-treated infected cells; and detergent extracts of purified virus. Each of the antisera reacted with CP-1 to give a single immunoprecipitin band of identity, and each antiserum neutralized the infectivity of HSV-1 and HSV-2. Our results suggested that the type-common determinants involved in the stimulation of neutralizing antibody resided on a 52,000-molecular-weight (52K) glycoprotein. The envelope of HSV contains several glycoproteins: one component at 59K and a complex of two or three components at 130K, none of which corresponds in molecular weight to gp52. Using the antisera as immunological probes, we performed pulse-chase experiments with [
35
S]methionine-labeled HSV-1-infected cells and followed the disposition of the glycoproteins during the infectious cycle. Each antiserum immunoprecipitated a
35
S-labeled 52K protein from lysates of cells pulse-labeled at 5 h after infection. By 10 h, the label was chased into a 59K protein also precipitable by each of the three antisera. The results suggest that gp52 is a precursor of gp59 and that the latter corresponds in molecular weight to one of the major glycoproteins of the virion envelope.
Levels of cellular DNA polymerases in synchronized bovine paravovirus-infected cells
Authors: Pritchard C , Bates R C , Stout E R
Published: January 2020

Infection of synchronized bovine fetal spleen cells with bovine parvovirus results in changes in the levels and patterns of DNA polymerases alpha and gamma during the cell cycle. The pattern of DNA polymerase alpha activity closely paralled viral DNA synthesis and the production of progeny virus, and levels, of this enzyme were threefold greater than in mock-infected cells during the period of maximal viral DNA synthesis. DNA polymerase gamma activity remained slightly elevated during viral DNA replication. Levels and patterns of DNA polymerase beta were similar in mock- and virus-infected cells.
Origin and biological properties of a new BALB/c mouse sarcoma virus
Authors: Aaronson S A , Barbacid M
Published: January 2020

A focus-forming virus previously isolated from a BALB/c mouse hemangiosarcoma has been shown to be replication defective. Analysis of individual BALB/c mouse sarcoma virus (BALB-MSV) nonproducer transformants for expression of helper virus-coded proteins revealed genetically stable variants that expressed two, three, or all four gag gene products in the absence of detectable helper viral env gene expression. The type-specific antigenic determinants of helper viral proteins encoded by the BALB-MSV genome and by the B-tropic virus isolated from the BALB-MSV stock were demonstrated to be indistinguishable from those of BALB:virus-1, a known endogenous virus of BALB/c cells. These findings imply that a BALB/c endogenous virus was involved in the generation of BALB-MSV. By the same immunological approach, the presence of at least a portion of the Moloney-MuLV gag gene has been identified in two other transforming viruses--Moloney-MSV and Abelson lymphosarcoma virus--previously isolated from the BALB/c strain. The tissue culture properties of cells transformed by these defective viruses were also shown to be distinguishable. These findings indicate that transforming virus isolates of the same inbred strain differ in their transforming activities as well as in the helper viral sequences stably associated with their genomes.
Reverse transcriptase-associated RNase H activity. II. Inhibition by natural and synthetic RNA
Authors: Marcus S L , Smith S W , Modak M J
Published: January 2020

The RNase H activity associated with purified avian myeloblastosis virus and Rauscher murine leukemia virus DNA polymerases is inhibited by homopolymeric RNA molecules, although the efficiency of inhibition by each homopolymer appears enzyme specific. Formation of duplex RNA molecules abolished the inhibitory activity. In contrast to these results, DNA polymerase-independent RNase H activities, such as the RNase H-II from Rauscher murine leukemia virus and calf thymus RNase H, were unaffected by the addition of exogenous RNA molecules to reaction mixtures. These results support the concept (M. J. Modak and S. L. Marcus, J. Virol. 22:253--256, 1977) that the catalytic site of DNA polymerase-associated RNase H activity may be that which is also involved in template binding. Naturally occurring RNA molecules of oncornaviral, procaryotic, or eucaryotic origin also proved to be efficient inhibitors of avian myeloblastosis virus DNA polymerase-associated RNase H activity. In contrast to this result, naturally occurring RNA molecules, at concentrations which inhibited the avian myeloblastosis virus enzyme, did not inhibit Rauscher murine leukemia virus DNA polymerase-catalyzed RNase H activity. This finding represents a new biochemical distinction between the two reverse transcriptases, and may be indicative of differences in the relative affinities of these enzymes for natural RNA molecules.
Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes
Authors: van de Ven W J , Vermorken A J , Onnekink C , Bloemers H P , Bloemendal H
Published: January 2020

A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction.
Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2
Authors: Johansson K , Persson H , Lewis A M , Pettersson U , Tibbetts C , Philipson L
Published: January 2020

Complementary strand-specific adenovirus DNA of full length or from endonuclease BamHI fragments was used as a probe to estimate the fractional representation and abundance of viral sequences in five hamster cell lines (Ad2HE1-5) transformed with UV-inactivated adenovirus type 2. The fraction of the viral genome present in the five transformed cell lines varied from 44% in the Ad2HE5 cell line to 84% in the Ad2HE3 cell line. The number of viral DNA copies per diploid cell equivalent ranged from 1.8 in the Ad2HE1 line to 7.1 in the Ad2HE4 line. In vivo labeling with [35S]methionine followed by immunoprecipitation with an antiserum against adenovirus type 2 early proteins revealed virus-specific polypeptides with molecular weights of 42,000 to 58,000 in extracts from all five hamster cell lines. Several other early viral polypeptides were detected in some of the adenovirus type 2-transformed hamster cell lines.
Viral gene expression in murine sarcoma virus(murine leukemia virus)-infected cells
Authors: Dina D , Penhoet E E
Published: January 2020

NIH 3T3 cells infected with Moloney murine sarcoma virus (murine leukemia virus) produce virions which contain about 99% murine sarcoma virus RNA and 1% murine leukemia virus RNA. This report describes experiments which measured intracellular concentrations of proviral DNA and RNA transcripts for each of the viruses. We found that three to four copies of proviral DNA from each virus were integrated into the cellular DNA. Measurements of RNA specific for each of the genomes by hybridization to specific cDNA reagents revealed a 10- to 15-fold difference in concentration in both nuclear and polysomal RNA fractions, with murine sarcoma virus RNA predominating in both cases. Unless there are major differences in stability between the two viral RNAs, our results suggest that transcriptional control is responsible for much of the difference in final levels of virus synthesis.
In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome
Authors: Salzman L A , Fabisch P , Parr R , Garon C , Wali T
Published: January 2020

Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.
Extraction and fingerprint analysis of simian virus 40 large and small T-antigens
Authors: Smith A E , Smith R , Paucha E
Published: January 2020

A study of simian virus 40 (SV40) T-antigens isolated from productively infected CV1 cells using a variety of different extraction procedures showed that under some conditions the highest molecular weight form of T-Ag (large-T) isolated comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with large-T from SV40-transformed H65-90B cells. Other faster-migrating forms of large-T are probably generated during the extraction procedure by a protease which is active at low pH, and such forms are probably experimental artifacts. After extraction under conditions which minimize proteolytic degradation of large-T, a further form of T-antigen was isolated; this has an apparent molecular weight in the range 15,000 to 20,000 and is referred to as small-t. Fingerprint analysis of [35S]methionine-labeled SV40 proteins showed that small-t has 10 to 12 methionine peptides whereas large-T has 15 to 18 methionine peptides. All but two of the methionine tryptic peptides present in small-t are also present in large-T. The fingerprint data also showed that T-antigens have no peptides in common with SV40 VP1. Experiments using reagents which inhibit posttranslational cleavage of encephalomyocarditis virus polyproteins showed that these reagents do not affect the synthesis of small-t and suggest that it is not made by proteolytic cleavage of large-T in vivo. An alternative model, which proposes that large-T and small-t are synthesized independently, is discussed in terms of the fingerprint data and the number of methionine tryptic peptides predicted from the primary sequence of SV40 DNA.
Replication of Nondefective Parvoviruses: Lack of a Virion-Associated DNA Polymerase
Authors: Pritchard Cynthia , Patton John T. , Bates Robert C. , Stout Ernest R.
Published: January 2020

We have examined four of the nondefective parvoviruses for an associated DNA polymerase. Virions were purified from neuraminidase-treated infected-cell lysates by isopycnic centrifugation in CsCl or from infected cell material by CaCl
2
precipitation and centrifugation through sucrose into CsCl. Preparations of bovine parvovirus or Kilham rat virus obtained by the former procedure contained DNA polymerase activity but were not free of contaminating cellular proteins. The latter method produced viral preparations free of contaminating cellular proteins, and no DNA polymerase activity was detected in light infectious particles of H-1, LuIII, bovine parvovirus, or Kilham rat virus. Examination of levels of each cellular DNA polymerase in these preparations from each step of both purification procedures revealed that DNA polymerase β had a greater tendency to copurify with bovine parvovirus and Kilham rat virus than did DNA polymerases α or γ. Disruption of infectious virions obtained by the second purification method with detergents and sonic treatment did not result in the detection of a DNA polymerase activity. The biological activity and purity of each of the four different viruses obtained by the latter procedure were determined by hemagglutination and infectivity assays, polyacrylamide gel electrophoresis, and electron microscopy. In each case, the virions banding at a density of 1.39 to 1.41 g/cm
2
in CsCl were infectious and contained only the virion structural proteins. DNA polymerase activity was not detected in any of these preparations, and we have concluded that a virion-associated DNA polymerase is not required for productive infection with the nondefective parvoviruses.
Biochemical studies on bovine adenovirus type 3. III. Cleavage maps of viral DNA by restriction endoncleases EcoRI, BamHI, and HindIII
Authors: Kurokawa T , Igarashi K , Sugino Y
Published: January 2020

Cleavage of bovine adenovirus type 3 (BAV3) DNA by restriction endonucleases EcoRI, BamHI, and HindIII yielded 7 (A to G), 5 (A to E), and 12 (A to L) fragments, respectively. The order of these fragments has been determined to be GDACBFE for EcoRI fragments, AEBDC for BamHI fragments, and JEBKACDHFGIL for HindIII fragments, and cleavage sites of these enzymes have been mapped on the genome of BAV3. BAV3 preparation contains incomplete virus whose genome has a deletion of about 13% of complete virus genome. Restriction endonuclease digestion of the incomplete virus DNA revealed that EcoRI E and F, BamHI C and HindIII G, I, and L fragments were deleted. Therefore, the deleted region of incomplete virus DNA is located near the right-hand end of the BAV3 DNA molecule, a result consistent with our previous electron-microscopic observations on heteroduplex molecules formed between complete and incomplete BAV3 DNA.
Biochemical Studies on Bovine Adenovirus Type 3 IV. Transformation by Viral DNA and DNA Fragments
Authors: Igarashi K. , Sasada R. , Kurokawa T. , Niiyama Y. , Tsukamoto K. , Sugino Y.
Published: January 2020

By the calcium technique, intact DNA of bovine adenovirus type 3 (BAV3) was found to transform A31 cells, a clone of BALB/3T3. Transforming activity was resistant to RNase and Pronase but sensitive to DNase. The efficiency of transformation was approximately 5 to 10 foci per μg of DNA. Attempts were also made to test for transforming activity of BAV3 DNA fragments prepared with restriction endonucleases
Eco
RI and
Hin
dIII. The activity was found to associate exclusively with the
Eco
RI D fragment mapped in the region of 3.6 and 19.7 units (molecular weight, 3.9 × 10
6
). No transformation could be obtained with three
Hin
dIII fragments, J, E, and B, located at the left-hand end of the BAV3 genome. However, the enzymatic joining of J and E fragments (0 to 11.9 map units) with a ligase restored the transforming activity. These results suggest that all the genetic information of BAV3 required for transformation is located in the region between 3.6 and 11.9 units on the viral genome. Some properties of A31 cells transformed by BAV3 DNA
Eco
RI D fragment (TrD) and the ligated DNA of
Hin
dIII J and E fragments (TrJE), as well as those transformed by whole BAV3 DNA (Tr), were examined. As compared to untransformed A31 cells, all the transformed cell lines tested showed rapid growth, high saturation densities, and anchorage-independent growth. Moreover, they contained BAV3-specific T antigen and induced tumors in adult nude and BALB/c mice. These properties of Tr, TrD, and TrJE lines were similar to those of BAV3-transformed cells.
Papain solubilization of the Epstein-Barr virus-induced membrane antigen
Authors: Pearson G R , Qualtiere L F
Published: January 2020

The Epstein-Barr virus (EBV)-induced membrane antigen (MA) was successfully solubilized from the membranes of viable EBV-infected Raji cells by treatment with papain (5 to 6 U per 1 X 10(7) to 2 X 10(7) cells). The loss of MA from viable cells was monitored by membrane immunofluorescence and antibody-dependent cellular cytotoxicity. Soluble MA was demonstrated in papain digests through inhibition of antibody-dependent cellular cytotoxicity and by inhibition of the binding of anti-MA antibodies to cells as detected by use of 125I-labeled staphylococcal protein A. Approximately 75% of the MA activity in the extracts was not sedimentable at 100,000 X g,, indicating that the majority of EBV MA activity that was released by this procedure was associated with small-molecular-weight material. Antiserum prepared from an owl monkey immunized with these papain extracts contained antibody to MA and neutralizing antibodies, but lacked detectable antibodies against viral capsid antigens and EBV-induced early antigens.