bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron Microscopic Characterization of the Defectiveness of a Temperature-Sensitive Mutant of Moloney Murine Leukemia Virus Restricted in Assembly
Authors: Yuen P. H. , Wong P. K. Y.
Published: January 2020

The effect of temperature shiftdown on the assembly of ts3 virions was investigated by both scanning (SEM) and transmission (TEM) electron microscopy. Ts3 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (Mo-MuLV) which previous studies indicated to be defective in assembly or release of the virions. In the present study, both SEM and TEM revealed the following: (i) there were more cell-associated virions in ts3-infected cells grown at the nonpermissive temperature (39°C) than either in cells grown at the permissive temperature (34°C) or in wild-type MuLV-infected cells grown at 39°C; (ii) there were more normal single particles than multiploids (virions with two or more pieces of genomic RNA) in ts3-infected cells grown at the nonpermissive temperature; (iii) there were more multiploids in ts3-infected cells grown at the nonpermissive temperature than either in cells grown at the permissive temperature or in wild-type MuLV-infected cells grown at the nonpermissive temperature; (iv) upon temperature shift from 39 to 34°C, about 90% of the cell-associated virions dissociated from the cell surface. TEM studies also indicated that upon temperature shiftdown, virion assembly rapidly occurred. The above observations suggest that faulty assembly, which results in the production of multiploids, may not be the reason why ts3 virions accumulate on the cell surface at the nonpermissive temperature. The relatively higher proportion of multiploids found in ts3-infected cells grown at 39°C compared with those grown at 34°C may be due to the higher density of budding virions at the cell surface at the nonpermissive temperature, which increases the possibility of two or more particles assembling close to one another. The accumulation of ts3 virions in all stages of assembly at the nonpermissive temperature, together with the fact that rapid assembly and release of ts3 virions occurred on temperature shiftdown, indicates that virion assembly is restricted after it has been initiated. The probable role of altered glycoprotein(s) in restricting virion assembly is discussed.
Morphogenesis of Bacteriophage φ29 of
Bacillus subtilis
: Mapping and Functional Analysis of the Head Fiber Gene
Authors: Reilly Bernard E. , Nelson Rodger A. , Anderson Dwight L.
Published: January 2020

A set of mutants of
Bacillus subtilis
bacteriophage φ29 unable to synthesize the head fiber protein has been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Infectious phage are produced during restrictive infection. We have focused on mutant
sus
8.5(900) because the mutation is suppressible by both the
su
+3
and
su
+44
hosts, and it can be mapped by three- and four-factor crosses. After restrictive infection with mutant
sus
8.5(900), a fragment about 70% of the size of the normal fiber is produced as well as particles that are fast-sedimenting in sucrose gradients relative to φ29
+
. These particles have the buoyant density of particles with the fibers removed and have the absolute plating efficiency of φ29
+
. Fiber protein is absent from prohead as well as virion. A second set of mutants produces fiber protein with a slightly altered electrophoretic mobility. This type of fiber protein is either present or absent on both prohead and virion. A third class of mutants, typified by 914, produces a “normal” fiber, but a major head protein of altered electrophoretic mobility. After infection by this mutant, the fiber is absent from both prohead and virion, and the biological and physical properties of the 914
−
particle are similar to those of particles produced after infection of the
su
−
host by
sus
8.5(900). These observations suggest that the head fiber is not an essential component of the prohead or virion and that the assembly process is efficient in the absence of fiber protein. Three- and four-factor genetic crosses have established the order
sus
8(769)-8(914)-
sus
8.5(900)-
sus
9(756) and indicate that cistrons 8 and 8.5 code for the major head protein and head fiber protein, respectively.
Characterization of RNA from equine infectious anemia virus
Authors: Cheevers W P , Archer B G , Crawford T B
Published: January 2020

The genome of equine infectious anemia virus, a nononcogenic retrovirus, has been characterized by velocity sedimentation, electrophoresis in polyacrylamide gels, buoyant density in CS2SO4, and susceptibility to nuclease digestion. The nucleic acid of purified virus was resolved by sedimentation analysis into a fast-sedimenting genome component, which comprises about two-thirds of the virion RNA, and a slow-sedimenting RNA, which is probably comprised of host-derived tRNA and a trace amount of 5S RNA. The fast-sedimenting RNA had a sedimentation coefficient of 62S and a molecular weight of 5.4 X 10(6) to 5.6 X 10(6), as determined by sedimentation velocity and electrophoretic mobility. Upon heat denaturation, [3H]uridine-labeled 62S RNA dissociated into material comprised of 90 to 95% single-stranded species, sedimenting predominantly at 34S, with a molecular weight of 2.7 X 10(6) to 2.9 X 10(6) and 5 to 10% 4S RNA. The 62S RNA was predominantly single-stranded but contained double-stranded regions, as indicated by partial resistance to RNase IA and SI nuclease and by a lower buoyant density in CS2SO4 than that of the single-stranded 34S RNA derived by heat denaturation. These data indicated that the viral genome consisted of two 34S subunits of single-stranded RNA held in a high-molecular-weight complex with 4S RNA by a mechanism involving a small degree of base pairing. Thus, the structure of equine infectious anemia virus RNA is similar to that of other retroviruses.
Persistent infection of cultured mammalian cells by Japanese encephalitis virus
Authors: Schmaljohn C , Blair C D
Published: January 2020

Persistent infections were established by serial undiluted passage of flavivirus Japanese encephalitis virus in a line of rabbit kidney cells (MA-111). The persistently infected cells resembled uninfected cells in most respects. Low levels of infectious virions were released from a small percentage of cells, and a larger and more variable percentage was shown to possess viral antigen by fluorescent-antibody staining. Released viruses were shown to interfere with replication of wild-type Japanese encephalitis virus. Persistently infected MA-111 cells could not be superinfected with homologous wild-type Japanese encephalitis virus but could be superinfected with two heterologous viruses. Transfer of cell culture medium from persistently infected MA-111 cells to a line of African green monkey kidney cells (Vero) resulted in similar persistent infections in the latter cells. Temperature sensitivity and host-cell interferon production were not involved in establishment or maintenance of persistence. Determination of ratios of physical particles to infectious particles revealed that many defective, noninfectious viruses were present, suggesting that defective interfering particles may be responsible for persistency.
Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase
Authors: Powell K L , Purifoy D J
Published: January 2020

Herpes simplex virus-induced DNA polymerase purified by published methods was found to be contaminated with many others proteins, including virus structural proteins. Thus, DEAE-cellulose and phosphocellulose chromatography were used in combination with affinity chromatography to purify DNA polymerase from herpes simplex virus type 1- and type 2-infected cells. The purified enzyme retained unique features of the herpesvirus-induced DNA polymerase, including a requirement for high salt concentrations for maximal activity, a sensitivity to low phosphonoacetate concentrations, and the capacity to be neutralized by rabbit antiserum to herpesvirus-infected cells. By polyacrylamide gel electrophoresis, the purified DNA polymerase was associated with a virus-induced polypeptide of about 150,000 molecular weight.
Electron microscopic studies of bacteriophage M13 DNA replication
Authors: Allison D P , Ganesan A T , Olson A C , Snyder C M , Mitra S
Published: January 2020

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (theta, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in theta molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of theta to other RF molecules does not change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.
RNase T1-resistant oligonucleotides of Akv-1 and Akv-2 type C viruses of AKR mice
Authors: Rommelaere J , Faller D V , Hopkins N
Published: January 2020

We used two-dimensional gel electrophoresis to obtain fingerprints of RNase T1-resistant oligonucleotides derived from the genomes of Akv-1 and Akv-2 type C viruses of AKR mice. The fingerprints of these two viruses look identical. The products of pancreatic RNase digestion of corresponding oligonucleotides of the two viruses were indistinguishable. These observations are consistent with, but not proof of, the possible identity of the genomes of the Akv-1 and Akv-2 viruses and, thus, of the viral genetic material believed to comprise the Akv-1 and Akv-2 loci of AKR mice.
DNA injection and genetic recombination of alkylated bacteriophage T7 in the presence of nalidixic acid
Authors: Karska-Wysocki B , Mamet-Bratley M D , Przewlocki G
Published: January 2020

Marker rescue experiments with alkylated T7 bacteriophage carried out in the presence and in the absence of nalidixic acid suggest that the gradient in rescue is due to two alkylation-induced causes: a DNA injection defect and an interference with DNA synthesis.
Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid
Authors: Tigges M A , Bursch C J , Snustad D P
Published: January 2020

Most, if not all, host RNA synthesis was shut off after infection of Escherichia coli strain B/5 with a bacteriophage T4 multiple mutant defective in the abilities to induce (i) unfolding of the host nucleoid (unf-), (ii) nuclear disruption (ndd-), and (iii) host DNA degradation (denA-, denB-). The shutoff of host RNA synthesis and turn-on of phage RNA synthesis were slower after infection of E. coli with unf- phage than after infection with unf+ phage. This delay in the switchover from host RNA synthesis to phage RNA synthesis in unf- infections did not result in a measurable delay in the onset of nuclear disruption, deoxyribonucleoside monophosphate kinase synthesis, or DNA synthesis. unf39 did not complement alc (allows late transcription on cytosine-containing DNA) mutants, supporting the proposal of Sirotkin et al. [Nature (London) 265:28-32, 1977] that alc and unf are possibly the same gene.
Polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus
Authors: Macnaughton M R , Madge M H , Davies H A , Dourmashkin R R
Published: January 2020

Purified avian infectious bronchitis virus was digested with bromelain (0.7 mg/ml), and the surface projections were removed. Polyacrylamide gel electrophoresis of the polypeptides from these bromelain-treated particles showed that VP1, VP2, and VP5 were missing from the seven polypeptides. VP1 to VP7, that were present in untreated virus preparations. Milder bromelain treatment (0.07 mg/ml) left visible surface projections and polypeptides comprising VP1 and VP2 intact, but removed VP5. Thus, there are apparently two types of surface projections on the virus particle. The ribonucleoprotein complex was released from virus particles disrupted with 1% Nonidet P-40. The proportion of VP6 in such preparations was greatly reduced, implying that VP6 is the structural polypeptide of the ribonucleoprotein. Polypeptides VP1, VP2, VP4, and VP5 are glycosylated, but none of the polypeptides contains lipid.
Transcriptase activity associated with rabies virion
Authors: Kawai A
Published: January 2020

Rabies virion-associated transcriptase activity was investigated in vitro and compared with that of the New Jersey serotype of vesicular stomatitis virus. The concentration of detergent that affected [3H]GMP incoporation into acid-insoluble material was significantly different for both viruses. Vesicular stomatitis virus New Jersey required 0.05 to 0.1% nonionic detergent, whereas rabies virion could not be fully activated unless 4 to 5% detergent was used. Other optimal conditions were as follows: 40 mM NaCl, 5 mM Mg2+, 40 mM Tris-hydrochloride (pH 7.4), 5 mM dithiothreitol, and 30 degrees C. The reaction required four nucleoside triphosphates. The initial rate of RNA synthesis by rabies virion enzyme was 140 pmol of GMP incorporated/mg of viral protein per h and linearly increased until about 8 h, with a slight initial lag phase. The enzyme activity that correlated with the content of L protein was highest when rabies virions were grown at 33 degrees C. The product was single-stranded RNA, which was complementary in base sequences to rabies viral RNA. Most of the RNA synthesized sedimented at 6-16S.
Formation of recombinants between snowshoe hare and La Crosse bunyaviruses
Authors: Gentsch J , Wynne L R , Clewley J P , Shope R E , Bishop D H
Published: January 2020

Wild-type recombinants were obtained at high frequency from coinfections of BHK cells involving temperature-sensitive, conditional-lethal mutants of snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses. Analyses of two of the recombinants indicated that they have the genome compositions SSH/LAC/SSH and SSH/LAC/LAC for their respective L, M, and S virion RNA species. This evidence, together with that for the genetic stability of the recombinants, indicates that they were derived by segment reassortment of the competent genome pieces of the parental viruses. The SSH/LAC/SSH recombinant appears, from polypeptide analysis, to have the SSH type of nucleocapsid protein (N), whereas the SSH/LAC/LAC recombinant has the LAC nucleocapsid protein, suggesting that the viral S RNA codes for the N protein.
Identification of a 30S RNA with properties of a defective type C virus in murine cells
Authors: Howk R S , Troxler D H , Lowy D , Duesberg P H , Scolnick E M
Published: January 2020

A novel species of 30S RNA has been detected in a variety of mouse cell lines. The 30S RNA is specifically packaged by helper-independent type C viruses propagated in such cells. Nucleic acid hybridization detects no homology between the 30SRNA and the genomic RNA of helper-independent mouse type C viruses. The properties of the 30S RNA suggest that it is a defective endogenous mouse type C virus and that it is analogous to a previously described class of defective endogenous rat type C virus, which has been shown previously to be the progenitor of Kirsten and Harvey murine sarcoma viruses.
Adsorption of cyanophage AS-1 to unicellular cyanobacteria and isolation of receptor material from Anacystis nidulans
Authors: Samimi B , Drews G
Published: January 2020

Cells of unicellular cyanobacteria of typological group Ia, containing approximately 50 mol% guanine + cytosine (G+C) in their DNA (R. Y. Stanier, R. Kunisawa, M. Mandel, and G. Cohen-Bazire, Bacteriol. Rev. 35:171-205, 1971), were susceptible to infection by the cyanophage AS-1. Cyanobacteria of the same typological group, containing approximately 65 mol% G+C in their DNA, did not adsorb the cyanophage AS-1 or adsorbed it at a low rate. AS-1 was not propagated by any of the investigated strains with a high G+C content in their DNA. However, cells of strains 6907 and 6911 were lysed by cyanophage AS-1. A comparison of the host range of this phage with the lipopolysaccharide composition of host and non-host cell walls suggests that lipopolysaccharides are involved in the adsorption process. About 8 microgram of lipopolysaccharide per ml from host strains inactivated 50% of the particles of a solution containing 100 PFU/ml after 60 min of incubation at 30 degrees C. Material with receptor activity was extracted from the host strain Anacystis nidulans KM. The extract was purified of glycolipids and pigments, and a fraction showing receptor activity was isolated. This fraction contained three polypeptides of molecular weights between 54,000 and 64,000. Heat and protease treatment of whole cells and of isolated receptor material decreased the receptor activity. The fluorescence intensity of A. nidulans cells labeled with 1-anilino-8-naphthalene sulfonate was increased when AS-1 was adsorbed to these cells. The participation of lipopolysaccharides and proteins in the formation of the receptor complex is discussed.
Preliminary characterization of a temperature-sensitive mutant of Moloney murine leukemia virus that produces particles at the restrictive temperature
Authors: Wong P K , Gallick G E
Published: January 2020

The isolation and preliminary characterization of a new temperature-sensitive mutant of Moloney murine leukemia virus, designated ts7, are reported. The infectivity of ts7, determined by a focus-forming unit assay, was reduced at least 100-fold when the virus was assayed at the nonpermissive temperature (39 degrees C) as compared with assay at the permissive temperature (34 degrees C). However, several lines of evidence indicated that the diminution of ts7 titer at 39 degrees C is not due to its inability to form virus particles at that temperature. The supernatant from ts7-infected cells grown at 39 degrees C showed significant infectivity when assayed at 34 degrees C; only small reductions in reverse transcriptase activity and fusion ability were observed when compared with supernatant from 34 degrees C ts7-infected cultures. That particles are produced at the nonpermissive temperature was confirmed by transmission electron microscopy of the supernatant from ts7-infected cells at 39 degrees C and by transmission electron microscope observations of mature particles trapped in the intercellular spaces of pelleted thin cell section. A possible explanation for the productivity at 39 degrees C of particles that are infectious at 34 degrees C but not at 39 degrees C is that the virus is heat labile at the nonpermissive temperature. Consistent with this hypothesis is the extreme heat lability of virus harvested at 34 degrees C. Such virus, when incubated at 39 degrees C, has a half-life one-sixth that of identical virus incubated at 34 degrees C, or that of wild-type virus at either temperature.
Electron microscope study of the base sequence homology between simian virus 40 and human papovavirus BK
Authors: Newell N , Lai C J , Khoury G , Kelly T J
Published: January 2020

The base sequence homology between the genomes of simian virus 40 (SV40) and human papovavirus BK (BKV) was studied by the heteroduplex method of Ferguson and Davis (J. Mol. Biol. 94:135-149, 1975). When mounted for microscopy in 30% formamide (Tm-35 degrees C), BKV/SV40 heteroduplexes were an average of 92% double-stranded and contained only two small nonhomologous regions that mapped near the junctions between the early and late regions of the SV40 Genome. At higher formamide concentrations, the fraction of duplex DNA in the BKV/SV40 heteroduplexes decreased, indicating significant base mismatching in the homologous regions. The strongest regions of homology were located in the late region.
Polyacrylamide gel electrophoresis of visna virus polypeptides isolated by agarose gel chromatography
Authors: Lin F H
Published: January 2020

The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS-PAGE show that the excluded material from the GuHCl column contains an aggregate of 10 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gp115, were isolated from the column eluate. The major glycoprotein gp115 was coeluted with P90, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (100 microgram) by SDS-PAGE shows that 20 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins.
Synthesis and processing of precursor polypeptides to murine mammary tumor virus structural proteins
Authors: Racevskis J , Sarkar N H
Published: January 2020

Biosynthesis of murine mammary tumor virus (MuMTV) proteins was studied in the chronically MuMTV-infected epithelial cell line MuMT-73 by using monospecific antisera to the major MuMTV core protein p27 and the major envelope glycoprotein gp47. In pulse-labeling experiments using [35S]methionine, monospecific antisera to p27 precipitated a 75,000-molecular-weight protein as the major intracellular component. Analysis of the same cellular extracts with monospecific antisera to gp47 revealed that the gp47 precursor was a 70,000-dalton protein. After chase periods, there was a loss of label from the precursors and a concomitant increase of labeled extracellular mature viral proteins. The glycoprotein precursor incorporated labeled glucosamine and seemed to be processed more rapidly than the p27 precursor. Considerable amounts of apparently nonvirion-associated gp47 and glycoprotein precursor could be detected in the extracellular culture fluid.
Inhibition of maturation of Rauscher leukemia virus by amino acid analogs
Authors: Jamjoom G A , Ng V L , Arlinghaus R B
Published: January 2020

Synthesis of primary precursor polyproteins of Rauscher leukemia virus (RLV) core and envelope proteins occurs in the presence of amino acid analogs canavanine and p-fluorophenylalanine, but cleavage of these precursors is severely inhibited or slowed down. After treatment with these agents, the release of characteristic virus or stable virus-like particles is greatly depressed.
Glycoproteins of avian tumor virus recombinants: evidence for intragenic crossing-over
Authors: Galehouse D M , Duesberg P H
Published: January 2020

The envelope glycoproteins of several avian tumor virus recombinants selected for the host range of a leukosis virus and the transforming function of a sarcoma virus were compared with each other and with those of their parents. It was found that the glycoproteins of different recombinant viruses, derived from the same parents, differed in their electrophoretic mobilities measured in polyacrylmide gels. The glycoproteins that had lower electrophoretic mobilities had higher precentages of carbohydrate. The carbohydrate of viral glycoproteins was estimated to range between 8 and 18% from their buoyant densities in CsCl, using known glycoproteins as standards. After exhaustive Pronase digestion, the carbohydrate was recovered from viral glycoproteins as a mixture of glycopeptides with molecular weights ranging from 2,500 to 5,000. It was estimated that distinct viral glycoproteins contained between two and five such oligosaccharide chains and that the glycoproteins of different recombinants expressing the same host range marker may differ in the number of oligosaccharide chains and consequently also in their polypeptide structure. Those with lower electrophoretic mobility contain more oligosaccharide chains per molecule than those with higher electrophoretic mobilities. It is suggested that not oligosaccharide chains define the viral host range.
Late effect of bacteriophage T4D on the permeability barrier of Escherichia coli
Authors: Thompson S , Wiberg J S
Published: January 2020

Cold centrifugation of lysis-inhibited Escherichia coli B infected with wild-type T4D results in extensive lysis beginning around 20 min after infection at 37 degrees C. Infection with an e mutant, which fails to make lysozyme, prevents lysis, but does not prevent a marked loss of K+ and Mg3+. The t gene product, thought to disrupt the cytoplasmic membrane in natural lysis, is not required for this handling-induced cation loss or lysis. Three lines of evidence argue that late protein synthesis is required to develop this potential for cation loss; the potential does not develop in infections by: (i) mutants defective in DNA synthesis, (ii) mutants defective in gene 55, and (iii) wild-type T4 when chloramphenicol is added at 6 min after infection. All late mutants examined, which are blocked in the major pathways of morphogenesis, do not prevent development of the potential. The evidence argues for a new, late effect of T4 infection on the cytoplasmic membrane.
Quantitation and Localization of Rous Sarcoma Virus-Specific RNA in Transformed and Revertant Field Vole Cells
Authors: Krzyzek Richard A. , Lau Alan F. , Vogt Peter K. , Faras Anthony J.
Published: January 2020

Hybridization analysis of RNA from transformed clones of Rous sarcoma virus (RSV)-infected field vole cells and revertant subclones indicated the presence of similar amounts of viral-specific RNA in both cell types. Employing both a relatively uniform and representative complementary DNA probe and genomelength complementary DNA, we have demonstrated that the majority of RSV proviral DNA is transcribed into viral-specific RNA in both transformed and revertant clones. The viral-specific RNA is present in several size classes, the largest of which is genome-length 35S RNA. Studies on the intracellular distribution of viral-specific RNA indicate that both transformed and revertant cells contain viral RNA in their cytoplasm. Furthermore, the bulk of viral-specific nucleotide sequences are also associated with polyribosomes in both cell types. These data indicate that, contrary to previously reported studies with other retrovirus-revertant cell systems, the entire RSV provirus DNA is transcribed into viral RNA similarly in both transformed and revertant vole cells. Since we have previously demonstrated similarities in sarcoma-specific viral RNA in both revertant and transformed vole cells, these data collectively indicate that the loss of the transformed phenotype does not reflect changes in transcription of all or part of the RSV provirus, or the processing, transport, or polyribosome association of viral-specific RNA representing the entire RSV genome.
Translation of RNAs Synthesized In Vivo and In Vitro from Bacteriophage SP82 DNA
Authors: Hiatt William R. , Whiteley H. R.
Published: January 2020

The synthesis of 69 phage-specific polypeptides during the infection of
Bacillus subtilis
with bacteriophage SP82 was detected by pulse-labeling, one-dimensional electrophoresis, and autoradiography. SP82 virions were found to contain approximately 22 polypeptides, most of which were synthesized late in infection; evidence was obtained for the processing of the major virion protein. RNAs extracted at different times during infection were translated by using an
Escherichia coli
cell-free extract. Only smaller-molecular-weight peptides were produced efficiently in vitro; in the 9,000- to 60,000-molecular-weight range, 50 to 60% of the peptides synthesized in vivo were produced by translation of RNAs extracted from infected cells. Eight of the virion peptides were produced by in vitro translation of RNAs extracted from infected cells. RNAs were synthesized under defined conditions by RNA polymerase extracted from uninfected
B. subtilis
and by polymerases isolated from cells 8 and 20 min after infection with SP82. Translation of these RNAs yielded characteristic and different patterns of polypeptides. Nine of the 12 polypeptides produced by translation of RNAs synthesized by the host polymerase corresponded in mobility to peptides appearing in vivo in the 0 to 3 and 3 to 6 min intervals of pulse-labeling after infection; 12 of the 25 peptides synthesized from RNAs produced by polymerase extracted 8 min after infection corresponded in mobility to peptides detected in vivo 8 min after infection, and 15 of the 22 peptides directed by RNAs made by the polymerase isolated 20 min after infection corresponded to peptides present in vivo late in infection. Five of the peptides produced in vitro from the latter RNA corresponded to virion peptides.
Synthesis of Alphavirus-Specified RNA
Authors: Brzeski Henry , Kennedy S. Ian T.
Published: January 2020

UV irradiation of chicken fibroblasts infected with Semliki Forest or Sindbis virus has been used to investigate the mechanism of synthesis of 42S and 26S RNA, the major plus-strand virus-specified RNAs formed during the multiplication of standard virus particles. From an analysis of the kinetics of UV inactivation of the synthesis of these two RNAs, we conclude (i) that 26S RNA is formed by internal transcriptive initiation from a point about two-thirds of the way from the 3′ end of the 42S negative-strand template; (ii) that there exists a population of plus-strand synthesizing complexes whose members are each capable of synthesizing both 42S and 26S RNA; and (iii) that, on a time-averaged basis, each complex in wild-type virus-infected cells contains one virus polymerase mediating 42S RNA synthesis and three mediating 26S RNA synthesis. The RNA phenotypes of 15 RNA
−
ts
mutants of Sindbis virus have been examined after temperature shift to the restrictive temperature. Under these conditions, cells infected with three mutants, N2, N7, and E268, synthesized four to six times as much 42S RNA (relative to 26S RNA) as wild-type virus-infected cells. These studies were extended by examining, in detail, the RNA and polypeptide phenotypes of mutants N2 and E268. These experiments showed that, in N2- and E268-infected cells, one of the virus-specified nonstructural (NS) polypeptides (NS p89; H. Brzeski and S. I. T. Kennedy, J. Virol.
22
:420-429, 1977) is thermolabile after shift up to restrictive temperature. This finding, together with the observation that, after shift, the 26S/42S RNA ratio in N2-infected cells changes markedly in favor of 42S RNA synthesis, leads us to conclude that, of the three NS polypeptides, NS p89 modulates 26S RNA synthesis.
Transcription of BK virus DNA by Escherichia coli RNA polymerase: size and sequence analysis of RNA
Authors: Meneguzzi G , Milanesi G
Published: January 2020

Transcription of human papovarirus BK superhelical DNA by Escherichia coli RNA polymerase yielded symmetric RNA with an average chain length of 1,3000 nucleotides. All regions of human papovavirus BK DNA were equally transcribed. At least four initiation sites were available to the procaryotic enzyme.
Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. III. Cytochemical localization of lysosomal enzymes in infected cells
Authors: Smith J D , de Harven E
Published: January 2020

Cytochemical localization of the lysosomal enzymes acid phosphatase and arylsulfatase in cells infected by herpes simplex virus (HSV) or human cytomegalovirus (CMV) showed the following interactions between viruses and host cell lysosomes: (i) many enveloped progeny viruses were located within cytoplasmic vacuoles containing lysosomal enzyme activity; (ii) naked cytoplasmic capsids appeared to acquire an envelope by budding directly into lysosomes; and (iii) many of the cytoplasmic dense bodies that are characteristic of CMV-infected cells and are thought to represent noninfectious aggregates of CMV structural proteins (I. Sarov and I. Abady, Virology 66:464-473, 1975) also acquired a limiting membrane by budding into lysosomes. Autophagy of other cytoplasmic elements was not observed, suggesting that there is some specificity involved in the association of viral particles and CMV dense bodies with lysosomes. Despite the presence of potentially destructive hydrolases, there was little evidence of significant morphological damage to intralysosomal viruses, and high titers of infectious particles were released into the medium. It would therefore appear that significant levels of HSV and CMV infectivity normally persist even though many progeny particles are directly exposed to lysosomal enzymes.
Effects of 2'-deoxy-2'-azidocytidine on polyoma virus DNA replication: evidence for rolling circle-type mechanism
Authors: Bjursell G
Published: January 2020

Rolling circle-type molecules were found in polyoma virus-infected cells after inhibition of DNA synthesis with 2'-deoxy-2'-azidocytidine. The circular DNA molecules were always relaxed and of polyoma length. Most of the attached tails were less than two times the length of the polyoma genome, but tails with a length of up to 4.75 times the genome were also found. After cleavage of the total pool of replicating molecules with either endo R.EcoRI or endo R.BamI, Y-shaped molecules with replicated portions of various lengths were generated from rolling circle-type molecules. Moreover, after cleavage, Y-shaped molecules with three unequal arms were found, which could be explained as derived from the tail in rolling circle-type molecules starting from the normal origin, i.e., 29% from the endo R.EcoRI cleavage site. Rolling circle-type molecules were also found during a normal, noninhibited infection cycle. In such cells, a relatively higher frequency of rolling circle-type molecules was observed late during infection. Compared with control cultures, cultures inhibited with 2'-deoxy-2'-azidocytidine showed a greater amount of rolling circle-type molecules relative to normal replicative intermediates. 2'-Deoxy-2'-azidocytidine has previously been shown to inhibit the initiation of new rounds of replication; thus, the result obtained here indicates that a rolling circle-type mechanism is independent of the reinitiation of DNA synthesis.
T1 oligonucleotide maps of N-, B-, and B leads to NB-tropic murine leukemia viruses derived from BALB/c
Authors: Faller D V , Hopkins N
Published: January 2020

We previously described and characterized RNase T1 RNA fingerprints of an N-, a B-, and five B leads to NB-tropic murine leukemia viruses derived from BALB/c mice (Faller and Hopkins, J. Virol. 23:188-195, 1977, and J. Virol. 24:609-617, 1977). These viruses share the majority of their large RNase T1-resistant oligonucleotides, but each possesses some "unique" oligonucleotides relative to the others. We have ordered the large T1-resistant oligonucleotides of the N-, the B-, and one NB-tropic virus relative to the 3' end of their genomes to obtain oligonucleotide maps. These maps indicate that (i) the large T1 oligonucleotides shared by the N-, B-, and NB-tropic viruses probably occupy the same relative positions on their genomes; (ii) the 14 T1 oligonucleotides that differ between the N- and B-tropic viruses are derived from regions scattered along the genomes; and (iii) an oligonucleotide that is present in five NB-tropic viruses but not in their B-tropic virus progenitors lies toward the 5' end of the NB-tropic virus oligonucleotide map.
Multiple RNase H activities in mammalian type C retravirus lysates
Authors: Gerard G F
Published: January 2020

Lysates of Moloney murine sarcoma-leukemia virus [M-MSV(MLV)], a virus complex grown in the rat cell line 78A-1, were found to contain three RNase H species separable by polycytidylic acid[poly(C)]-agarose chromatography. RNase H activity (RNase H I) associated with RNA-directed DNA polymerase eluted at 0.23 M KCI from poly(C)-agarose. RNase H II, which eluted from poly(C)-agarose at 0.12 M KCI and was not associated with DNA polymerase activity, was shown to be identical to an RNase H species (designated RNase H II) previously isolated from M-MSV(MLV) by a different procedure (G. F. Gerard and D. P. Grandgenett, J. Virol. 15:785-797, 1975). M-MSV(MLV) RNase H II was established to be a random exohybridase that requires free-chain termini in its hybrid substrate for activity. Lysates of Rickard feline leukemia virus also contained RNase H activity not associated with DNA polymerase activity that eluted from poly(C)-agarose at 0.12 M KCl. A third species of enzyme from M-MSV(MLV) lysates, called RNase H III, did not bind to poly(C)-agarose in 0.06 M KCl. RNase H III was purified from lysates of M-MSV(MLV) and M-MLV (grown in mouse cells) by sequential chromatography on poly(C)-agarose, DEAE-cellulose, phosphocellulose, and polyuridylic acid-Sepharose. Purified RNase H III (i) was free of any associated DNA polymerase activity, (ii) had an apparent molecular weight of 30,000 determined by Sephadex G-100 gel filtration, (iii) had an absolute requirement for Mn2+ (1 mM optimum) for the degradation of [3H](A)n.(dT)n, (iv) was inhibited by the presence of any salt in reaction mixtures, and (v) was endoribonucleolytic in its mode of action as indicated by the size distribution of limited degradation products of [3H](A)n.(dT)n. RNase H III was inhibited by antisera prepared against Rauscher MLV and simian sarcoma virus reverse transcriptase, and the quantity of RNase H III and RNase H I present in lysates of M-MLV were reduced and increased proportionately if virus was lysed in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. These results indicate that RNase H III is a proteolytic cleavage product of DNA polymerase-RNase H. Substantial RNase H activity that did not bind to poly(C)-agarose in 0.06 M KCl was also found in lysates of Harvey MSV(MLV), Rauscher MLV, and Rickard feline leukemia virus, but not in lysates of avian myeloblastosis virus.
Simian virus 40 (SV40)-specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U-antigen determinants
Authors: Deppert W
Published: January 2020

The distribution of simian virus 40 (SV40)-specific proteins in nuclear subfractions of pulse-chase-labeled HeLa cells infected with nondefective adenovirus type 2 (Ad2)-SV40 hybrid viruses was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND5 specifically associate with the nuclear matrix and are virtually absent from the high-salt nuclear extract. In Ad2+ND4-infected HeLa cells, the SV40-specific proteins with molecular weights of 64,000 (64K) and lower also specifically associate with the nuclear matrix. The SV40-specific 72K, 74K, and 95K proteins were found both in the nuclear matrix and in the high-salt nuclear extract. Analyses of the nuclear matrices isolated from hybrid virus-infected cells by immunofluorescence microscopy showed that SV40 U-antigen-positive sera from SV40 tumor-bearing hamsters react with SV40-specific proteins integrated into nuclear matrices of HeLa cells infected by Ad2+ND1, Ad2+ND2, and Ad2+ND4, but not with nuclear matrices of HeLa cells infected by Ad2+ND5. This suggests that SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND4 integrated into the nuclear matrix carry SV40 U-antigen determinants. The apparent discrepancy in the subcellular localization of SV40-specific proteins in hybrid virus-infected cells when analyzed by biochemical cell fractionation procedures and when analyzed by immunofluorescence staining is discussed.
Biochemical characterization of the amphotropic group of murine leukemia viruses
Authors: Chattopadhyay S K , Hartley J W , Lander M R , Kramer B S , Rowe W P
Published: January 2020

The recently described amphotropic group of murine leukemia viruses constitutes a distinct biological group, differing from the ecotropic and xenotropic groups in host range, cross interference, and serological reactivity. Viruses of this group have been detected only in wild mice from certain areas in California. By using a [3H]DNA probe synthesized in an endogenous reaction from detergent-lysed amphotropic virus (strain 1504-A), it was demonstrated that the amphotropic murine leukemia viruses are distinct biochemically, in that 20% of the viral genome sequences are not shared by AKR-type ecotropic or nay of three types of xenotropic murine leukemia virus tested. A subset of these amphotropic unique sequences, comprising one half of them, is present in the genome of wild mouse ecotropic viruses and in Moloney and Rauscher viruses as well. Sequences homologous to the entire genome of 1504-A amphotropic virus are present in the cellular DNA of all eight inbred mouse strains tested, as well as in wild Mus in Asia, in amounts varying from three to six complete viral genomes per haploid cell genome. Evidence is presented that at least 20% of the DNA sequences in both mouse- and mink-grown murine leukemia virus probes are of host-cell origin.
Characterization and classification of virus particles associated with hepatitis A. I. Size, density, and sedimentation
Authors: Siegl G , Frösner G G
Published: January 2020

Virus-like particles were purified from stools of patients in an epidemic of hepatitis A in Germany. When reference MS-1 chimpanzee pre-inoculation and convalescent sera were used, the close serological relationship of the purified particles to well-known isolates of hepatitis A could be established. On the other hand, the physicochemical characteristics of the particles were determined in parallel to the characteristics of a marker parvovirus (LuIII) and a marker picornavirus (poliovirus type 2). It could be shown that the majority of the hepatitis A-associated particles band at 1.34 g/ml in CsCl and, like poliovirus, sediment at about 160S. In addition, a distinct hepatitis A antigen was observed, which banded at 1.305 g/ml and sedimented between 50 and 90S. A further component accumulated in the density range of between 1.38 and 1.44 g/ml. However, it seemed to be rather labile. Upon reisolation from CsCl and sedimentation in sucrose, it resolved into a 160S, a 90 to 100S, and a 50S form. The size of the 160S particles (27 to 29 nm) could be readily distinguished from that of the parvovirus (22 to 24 nm). It is concluded, therefore, that hepatitis A-associated virus particles are more likely to be classified with the picornaviruses than with the parvoviruses.
Synthesis of defective viral DNA in HeLa cells infected with adenovirus type 3
Authors: Daniell E , Mullenbach T
Published: January 2020

Virus-specific DNA fragments that are shorter than the full-length viral genomes have been isolated from HeLa cells productively infected with adenovirus type 3. A number of predominant size classes could be detected by gel electrophoresis and hybridization, and the array of sizes was similar or identical to the selection in DNA purified from incomplete particles of this serotype (E. Daniell, J. Virol. 19:685-708, 1976). A large fraction of these short DNA molecules contained long inverted terminal repetitions, as did DNA molecules from incomplete particles. Restriction analysis showed that these subgenomic molecules consist of sequences from the two molecular ends of the normal genome. These results suggest that the predominance of left-hand end fragments seen in packaged incomplete DNAs results from selective packaging, whereas the predominance of certain size classes of intracellular viral DNA is a function of prepackaging events. The incomplete DNAs were generated at all times during viral DNA replication, and the yield relative to complete DNA did not seem to vary significantly with time or multiplicity of infection or when the virus was propagated on different human cell types.
RNA of mouse hepatitis virus
Authors: Lai M M , Stohlman S A
Published: January 2020

The RNA of mouse hepatitis virus, a coronavirus, was isolated from the virus released early in the infection and analyzed by sucrose gradient sedimentation and electrophoresis. It was found to consist of a piece of single-stranded RNA of about 60S. Its molecular weight was estimated to be 5.4 X 10(6) by electrophoresis in methylmercury-agarose gels. At least one third of the RNA contained polyadenylated sequences. It is, therefore, probably positive stranded. The virus harvested late in the infection contained, in addition to 60S, some 30 to 50S RNA that are possibly degradation products of the 60S RNA. No difference in the electrophoretic behavior could be detected between the RNA isolated from a pathogenic (JHM) and a nonpathogenic (A59) strain.
Proteins of Rous-associated virus 61, an avian retrovirus: common precursor for glycoproteins gp85 and gp35 and use of pactamycin to map translational order of proteins in the gag, pol, and env genes
Authors: Shealy D J , Rueckert R R
Published: January 2020

Cells infected by Rous-associated virus 61 (RAV-61) contained a precursor-like protein, pr90, that was specifically precipitated by antiserum directed against envelope glycoproteins, gp85 and gp35. Tryptic peptide mapping showed that pr90 contained tryptic sequences of both gp85 and gp35. Pactamycin mapping experiments indicated that the two glycoproteins are translated from the env-mRNA in the order (5') gp85--gp35. The pactamycin mapping experiments also indicated a translational order of p10--(p27, p12)--p15 for the gag proteins; this agreement with the order previously reported from tryptic mapping studies on precursor pr76 of avian myeloblastosis virus implied that the stoichiometry of the core proteins was unchanged when virions were assembled in the presence of pactamycin. The reverse transcriptase proteins, unlike those of the env and gag genes, fell on the right side of the pactamycin map. This result is in accord with the idea that most, if not all, of the reverse transcriptase protein is translated by read-through of the gag(pol) message rather than by translation of a hypothetical pol-mRNA devoted solely to synthesis of that protein.
Structural components of mouse mammary tumor virus. II. Isolation and purification of virion polypeptides
Authors: Yagi M J , Stutzman R E , Robertson B H , Compans R W
Published: January 2020

Mouse mammary tumor virus (MMTV) glycoproteins and nonglycosylated polypeptides were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary amino groups were labeled with fluorescamine to enable visualization of MMTV polypeptides in the gels. Protein bands were sliced from the gels and eluted with 90 to 95% recovery. Eight MMTV polypeptides, including three of the major viral components as well as five minor proteins, were routinely obtained. Double diffusion assays and immunoelectrophoresis confirmed the retention of antigenicity identical to that of untreated MMTV virions. Antisera obtained from MMTV-free BALB/c mice immunized with these purified proteins reacted with the polypeptide immunogen as well as with detergent-disrupted MMTV virions from mouse milk or cell culture. Double diffusion assays using the specific mouse antisera failed to detect any cross-reactivity among the isolated polypeptides. A hemagglutination-inhibition assay demonstrated that the ability of MMTV virions to inhibit the hemagglutinating properties of influenza virus resides in the glycosylated polypeptides gp52, gp37.7, and gp33.