J. Thomas August

High molecular weight precursor polypeptides to structural proteins of Rauscher murine leukemia virus

The synthesis of three structural proteins of Rauscher murine leukemia virus was examined by autoradiography of radiolabeled proteins immunoprecipitated from extracts of cells pulse-labeled with [ 35 S]methionine and fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Polypeptides carrying virus-specific antigenic determinants were immunoprecipitated with monospecific antiserum directed against either the Rauscher virus envelope glyco-protein gp69/71, the major core protein p30, or a small molecular weight internal protein 15. Analysis of polypeptides immunoprecipatated by anti-gp69/71 serum revealed a 90,000mmolecular weight protein as the immediate precuror of gp69/71. Very high molecular weight proteins of 260,000 and 350,000 that appeared to carry gp69/71 antigenicity were also detected. Labeling with 3 H-labeled sugars revealed that the 90,000 molecular weight molecule was glycosylated but its carbohydrate moiety was deficient in fucose relative to the mature envelope protein. The synthesis of a 70,000 molecular weight polypeptide with gp69/71 antigenic determinants was observed when glycosylation was inhibited. Tryptic digest analysis confirmed the procursor-product relationships between the 70,000 molecular weight polypeptide, the 90,000 molecular weight protein and the mature envelope glycoprotein. Analysis of the polypeptides immunoprecipitated by anti-p30 serum and anti-p15 serum indicated that these virion proteins were both cleavage products of a 65,000 molecular weight precursor molecule. Tryptic digest analysis showed that the 65,000 molecular weight molecules precipitated by anti-p30 serum and anti-p15 serum were the same molecule and confirmed the precursor-product relationship of the 65,000 molecular weight precursor molecule to the virion protein p30. Possible very large precursors to the p30 and p15 proteins with molecular weights of 260,000 and 350,000 were also observed. These proteins migrated in electrophoretic gels parallel with the very large polypeptides immunoprecipitated by anti-gp69/71 serum. The 65,000 molecular weight precursor molecule was not detectable when viral-specific protein was synthesized in the presence of amino acid analogs which can inhibit precursor protein cleavage. However, a slightly larger polypeptide with a molecular weight of about 70,000 was observed. The concentrations of the very large possible precursors were not enhanced by amino acid analog incorporation. Released virions were found to contain precursor protein and intermediatesized cleavage products carrying antigenic determinants of virion proteins p30 and p15 in addition to the mature sized proteins. Precursor to gp69/71 was not detected in released virus particles.

Oncornavirus gene expression during embryonal development of the mouse.

Abstract BALB/c, C3H, and AKR strains of mice were analyzed for the concentrations of the major internal protein of about 30,000 daltons and the major envelope glycoprotein of about 70,000 daltons of endogenous type-C oncornaviruses in embryos isolated during the second half of gestation, in newborn mice, and in specific organs of adult animals. Each strain showed a different pattern of protein expression. BALB/c tissues contained very low concentrations of both proteins (

Replication of RNA bacteriophage R23: I. Quantitative aspects of phage RNA and protein synthesis☆

Abstract Infection of Escherichia coli by R23, a newly isolated strain of RNA bacteriophage, has been found markedly to suppress synthesis of bacterial RNA. Approximately 60% of total RNA synthesized after infection was encapsulated in progeny phage particles. In contrast, after infection with the RNA phages f2 or Qβ, only 25 to 30% of newly synthesized RNA was phage RNA. Thirty to 40% of the RNA synthesized after B23 infection was characterized as a second form of phage-specific RNA by its resistance to RNase. Synthesis of bacterial proteins was also markedly inhibited after infection by R23 and ultimately replaced by phage-directed protein synthesis. B23 “early” protein synthesis, detected as phage RNA polymerase activity, was evident five to ten minutes after infection and increased until 20 to 30 minutes. In contrast, in the normally infected cell, synthesis of coat protein was predominant at a later time, beginning 30 to 45 minutes after infection and reaching a maximum between 45 and 75 minutes of infection, when coat protein constituted almost all of the newly synthesized protein. Coat protein synthesis was not detectable in the absence of RNA synthesis, whereas phage RNA polymerase induction required little or no RNA synthesis.

8 - Methods for Selecting RNA Bacteriophage*

Publisher Summary This chapter focuses on the methods used for selecting RNA bacteriophage. The RNA phages infect only male strains (Hfr and F + ) of Escherichia coli ( E. coli )or other bacteria, which have received the E. coli F + episome. These phages are small and circular and contain a single-stranded, linear RNA of molecular weight approximately 1 × 10 6 . RNA-containing bacteriophages have also been reported for Pseudomonas aeruginosa and for Caulobacter . Phage titer and host range are determined by plaque formation with the agar-layer method. Bacteria used for plating are harvested during early log-phase growth. Male-specific RNA bacteriophages do not produce plaques on organisms harvested during stationary growth, presumably because they become F - phenocopies. Sewage samples may be treated by centrifugation or filtration to remove particulate material, and the clear solution is stored over chloroform (1%, v/v) at 2–4°C. Soil samples may be mixed with water, allowed to stand at room temperature, filtered, and stored in a similar manner. These samples are used for selection of RNA bacteriophages. Phage stocks are prepared by picking single plaques from samples plated on appropriate bacteria. Plaques are suspended in phage diluent and replated several times to ensure homogeneity. An early log-phase culture of bacteria, 25–50 ml, is then infected with a single phage plaque. Infected cultures are incubated at 37°C with vigorous aeration for 3–6 hours. Lysing solution, 0.1 volumes, and chloroform (1%, v/v) are then added, and the culture is shaken vigorously. Bacterial debris is removed by centrifugation or filtration and then, the lysate is stored over chloroform.

The use of immunoprecipitation to study the synthesis and cleavage processing of viral proteins.

Several factors have been considered in developing an immunoprecipitation procedure which minimizes the background of non-immunospecifically precipitated protein. The procedure evolved utilizes the technique of direct immunoprecipitation (specific antibody plus sufficient unlabeled antigen to form a precipitable complex). High speed centrifugation of the cell extract immediately prior to immunoprecipitation and the use of test tubes coated with silicone greatly reduced background precipitation.

Replication of RNA bacteriophage R23: II. Inhibition of phage-specific RNA synthesis by phleomycin☆

Abstract The multiplication in Escherichia coli of both DNA and RNA bacteriophages was found to be inhibited by the antibiotic phleomycin. The cessation of RNA phage replication appeared to result from inhibition of phage-directed RNA synthesis. After addition of phleomycin, there was simultaneous cessation of synthesis of both single-stranded phage RNA and RNase-resistant RNA. Synthesis of coat protein, phage assembly and the formation of infective particles were unaffected and phleomycin treatment did not result in defective particles. This drug also inhibited the synthesis of bacterial RNA, and secondarily, of bacterial protein in E. coli. Inhibition by phleomycin of viral RNA polymerase and DNA-dependent RNA polymerase activities was evident in in vitro studies and there was evidence of phleomycin binding to RNA.

Characterization of a defective pseudotype particle of Kirsten sarcoma virus

Abstract A cell line has been isolated that produces a defective pseudotype particle of Kirsten sarcoma virus without detectable infectious helper virus. Normal rat kidney cells were infected at low multiplicity with a virus stock obtained by the rescue of Kirsten sarcoma virus with woolly monkey leukemia virus. The individual transformed cell foci which resulted were propagated. One was found to produce noninfectious oncovirus particles containing high reverse transcriptase activity. These defective particles contained an active sarcoma genome but no detectable helper virus genome as cells fused to the particles became transformed but did not show evidence of helper woolly monkey leukemia virus expression. Nevertheless, the rat kidney cells producing the defective particles contained the complete woolly monkey leukemia virus genome; after prolonged culture, infectious woolly monkey leukemia viruses were released and rapidly spread through the culture. The defect of the noninfectious particle could be attributed to the absence of the viral envelope glycoprotein. Helper virus env gene products were not detected in cells producing the defective particle, nor did these cells exhibit interference to superinfection by woolly monkey leukemia virus. The protein composition of the defective particle was unusual. Major proteins were a woolly monkey virus p28 and a novel 55,000-dalton protein of rodent origin. The 55,000-dalton protein was not immunologically related to the known rodent type-C virus gag or env gene products; nevertheless, it was immunoprecipitated by several anti-murine leukemia virus sera, suggesting that a related protein is commonly present in preparations of purified mouse type-C viruses.