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Types 1, 2, and 5 of structural proteins of adenovirus as well as human albumin, pepsin, trypsin, and ribonuclease tend to exhibit characteristic substance patterns when exposed to pyrolysis-gas chromatography. Those patterns may be helpful in differentiating between those proteins. The presence of a distributional pattern is assumed to depend, in most cases, not only on the given amino acid composition, but also on the amino acid sequence of the protein proper. Samples of complex composition, such as complete viruses or allantois fluids, were much less suitable for differentiation by means of pyrolysis-gas chromatography.
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The processing of virus polypeptides synthesized in cells infected with HVJ (haemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) was studied. Maturation of a temperature-sensitive (ts) mutant (HVJ-pB) derived from an HVJ carrier culture was inhibited at 38 degrees C incubation. A considerable amount of viral components were made at the restrictive temperature. They were, with the exception of the polypeptide HN, well preserved without a great loss of their function and successfully incorporated into virus particles released after lowering the incubation temperature. The membrane (M) protein seems to be essential for virus morphogenesis.
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The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholestetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1(-14C)]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examinatin of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.
The localization of the avian sarcoma virus src gene product (termed p60src) was examined by indirect immunofluorescence in cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D (SR-RSV-D). Antiserum to p60src was obtained from rabbits bearing SR-RSV-D-induced tumors, and immunofluorescence was performed on chicken embryo fibroblasts (CEF) transformed with SR-RSV-D, as well as normal rat kidney (NRK) cells transformed by the same virus (termed SR-RK cells). Both acetone and formaldehyde fixation were used for the immunofluorescence tests. The specificity of the anti-tumor serum was first demonstrated in both cell systems by gel electrophoresis of immunoprecipitates prepared from 35S--methionine-labeled cells. Anti-tumor serum precipitated p60src from SR-RSV-D-transformed CEF but not from CEF infected with a transformation-defective mutant of SR-RSV-D. All viral structural proteins and precursors contained in these immunoprecipitates could be eliminated by competition with unlabeled virus. Similar experiments on SR-RK cells indicated that no viral proteins other than p60src were expressed in these cells, and this observation was supported by immunofluorescence tests using antiserum to whole virus. For immunofluorescence localization of p60src, reactions with viral structural proteins were blocked with unlabeled virus. This presaturation step, obligatory for p60src detection in the SR-RSV-D-transformed CEF, was unnecessary when antitumor serum was tested on SR-RK cells, since p60src was the only viral protein detectable in these cells. With acetone-fixed cells, p60src-specific immunofluorescence revealed a characteristic fluorescence pattern which was similar in both cell systems. The principal pattern was diffuse and situated in the cytoplasm. A clear nuclear fluorescence was never observed. Immunofluorescence on formaldehyde-fixed cells also indicated the cytoplasmic location of p60src and revealed a specific subcytoplasmic concentration of the fluorescence. With both fixation methods, an additional fluorescence pattern was seen between cells in contact, and was also found in both SR-RK cells and SR-RSV-D-transformed CEF. Immunofluorescence on viable cells suggested that p60src was not on the surface of these transformed cells. The fluorescence patterns were specific for avian sarcoma virus-transformed cells and were not found in uninfected cells, cells infected with a transformation-defective mutant of SR-RSV-D or cells transformed by an antigenically unrelated murine sarcoma virus. Furthermore, anti-tumor serum did not contain antibodies to proteins of the microtubules or intermediate filaments.
From the same batch of virus, the four major avian viral structural proteins p27, p19, p15, and p12, the reverse transcriptase, the envelope glycoprotein gp85, and the high molecular weight 70 S RNA have been recovered. All proteins, except for gp85, have been purified by use of column chromatography procedures to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gels and isoelectric focusing. A new isolation procedure for p12 by affinity column chromatography takes advantage of its nucleic acid binding properties. The recovery of nondenatured viral structural proteins is demonstrated by the proteolytic activity revealed by p15. The purified proteins were used for the production of monospecific antibodies. The 70 S RNA served as source for the isolation of 35 S RNA subunits.
The polypeptide precursor pr76 to the internal viral group specific (gs) antigen proteins of Rous sarcoma virus, synthesized in a cell-free system of ascites cells, has been processed in vitro into the viral proteins by purified viral protein p15 as well as by disrupted Rous sarcoma virus. Disrupted Rauscher murine leukemia virus does not stimulate the cleavage process in vitro. Autocatalytic cleavage of the polypeptide precursor pr76 or Rous sarcoma virus, which contains the peptide sequence of p15, is not observed.
Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.
Expression of antigenous determinants of structural proteins G-MuLV (p10, p12, p30, gp14, gp17) and R-MuLV (gp69/71, p15) on thymocytes of normal and leukemic AKR mice was studied by membrane immunofluorescence. From this sign sharp difference between normal and malignant thymocytes was shown. A possible role in the antitumour immunity of antigens to structural viral proteins MuLV expressed on the leukemic cell membrane is discussed.
SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.
The retrovirus designated RPMI8226V (isolated from human myeloma cells RPMI8226) has been characterized with respect to its morphological, biochemical and immunological properties as well as its propagation in various animal and human cells. The myeloma cells RPMI8226 produce intracytoplasmatic A-type particles and extracellular particles. The extracellular particles have been classified as immature particles with translucent core center, typical mammalian C-type virus particles and C-type particles with intermediate membrane. However, the budded particles in secondarily infected human neoplastic cells contained complete doughnut-shaped nucleoids. This type of budding is rather characteristic for B-type particles. The 3H-uridine labeled RPMI8226 viral particles have a buoyant density 1.17 g/ml in sucrose gradient containing high molecular weight RNA and the distribution of viral structural proteins in SDS-PAGE is characteristic for oncornaviruses. The internal structural proteins according to MW are ranged from 13 000 to 30 000 daltons. The virus contains a magnesium-dependent reverse transcriptase. The cellular homogenate and viral concentrate from RPMI8226 cultures do not react with antibodies against ALSV, MuLV, FeLV, RD114, MP-MV and SiSLV. The only reaction was scored with anti BLV antibodies. However, anti BLV serum inhibiting the reverse transcriptase activity of BLV to 60% does not cross-react with the reverse transcriptase of RPMI8226V. In contrast to BLV concentrates, neither XC nor KC cells show syncytia formation by RPMI8226V. The RPMI8226V replication is restricted to human tumor and normal human glia-like cells. The possible origin of the virus is discussed.
Increasing exposure of vesicular stomatitis virus particles to ultraviolet radiation caused differential inhibition of the synthesis in vitro of individual mRNA species which code for the viral structural proteins L, G, M, NS, and N.
Somatic cells of vertebrates contain gene sequences which are an integral part of chromosomal DNA and which code for the production of complete type C RNA viruses. These virogenes are genetically transmitted from parent to progeny along with other cellular genes (virogene-oncogene hypothesis). Activation of this endogenous virogene information from a normally repressed state, rather than infection by exogenous oncogenic viruses, has been proposed as the most common mechanism of cancer causation in animals, including man. Recent isolates of baboon type C RNA viruses, while related morphologically and biochemically to other mammalian type C RNA viruses, can be distinguished by nucleic acid hybridization and immunologic criteria. Within primates, type C virogenes have evolved as the species have evolved; virogenes from closely related genera and families have the closest gene sequence homology. Endogenous viruses from one species may infect animals of a distantly related species and become incorporated into their germ line. Genomes of exogenous viruses, such as the murine leukemia viruses, which are infectious from animal to animal within the same species, evolve more rapidly than the endogenous virogenes which replicate solely as cellular genes. A major viral structural protein of baboon type C RNA viruses, p30 was detected by radioimmunoassay in normal primate tissues. Radioimmunoassays have also detected p30 antigen in human tissues which appear to be immunologically related to primate viral p30. Hybridization experiments have confirmed that type C viral sequences are also present in the human genome.
When cells are infected by C-type viruses such as Moloney sarcoma/leukaemia virus, new antigens appear on the cell membrane. Mice and rats will respond immunologically to the antigen(s). It was uncertain whether the antigens were related to the viral structural proteins or to non-virion, tumour-specific surface antigens (TSSA) or both. In an 125I-antiglobulin binding assay, Moloney virus completely blocked the binding of mouse and rat sera to virus shedding target cells, thus suggesting that mice and rats recognise only viral proteins. Mice responded to type-specific and rats mainly to group-specific determinants on the virus. Individual Moloney viral proteins were prepared using the guanidine HCl method and were used to block the binding of the rat anti-Moloney immune serum to Moloney virus shedding target cells. By this method, it was demonstrated that the rat serum contains specificities for the viral proteins gp70 and p30, but it was not possible to detect any antibodies directed towards non-virion or TSSA-like molecules.
Production of infectious Mason-Pfizer monkey virus (M-PMV) was enhanced after treatment of the CMMT cell line with 2.5 x 10(-5) M dexamethasone phosphate (DXM). The reverse transcriptase (RT) activity and infectivity titers of treated culture fluids were enhanced by five- and tenfold, respectively. Along with stimulation of M-PMV synthesis, a simian type C virus (SCV) was also detected by electron microscopic and RT analyses. The SCV was serologically related to the endogenous baboon type C virus. 5-iododeoxyuridine (IUDR) also activated the SCV in the CMMT cell line while significantly inhibiting the production of infectious M-PMV. The activation of endogenous SCV by IUDR or DXM was transitory, since removal of these compounds from the growth medium resulted in the disappearance of SCV buds and the related RT activity; however, low levels of specific viral structural proteins continued to be synthesized intracellularly. Similarly, the enhancement of M-PMV production seen with DXM was lost when the treated cells were subcultured for 2 weeks in the absence of the hormone.